WO2021190561A1 - 一种治疗帕金森病的方法和药物 - Google Patents

一种治疗帕金森病的方法和药物 Download PDF

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WO2021190561A1
WO2021190561A1 PCT/CN2021/082715 CN2021082715W WO2021190561A1 WO 2021190561 A1 WO2021190561 A1 WO 2021190561A1 CN 2021082715 W CN2021082715 W CN 2021082715W WO 2021190561 A1 WO2021190561 A1 WO 2021190561A1
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plasminogen
mice
group
control group
plasmin
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French (fr)
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李季男
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Talengen International Ltd
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Talengen International Ltd
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Priority to US17/914,267 priority Critical patent/US20230143354A1/en
Priority to CA3176934A priority patent/CA3176934A1/en
Priority to EP21774984.5A priority patent/EP4122490A4/en
Priority to KR1020227036798A priority patent/KR20220158036A/ko
Priority to JP2022557660A priority patent/JP2023518563A/ja
Priority to CN202180023651.0A priority patent/CN115697386A/zh
Publication of WO2021190561A1 publication Critical patent/WO2021190561A1/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • A61K38/484—Plasmin (3.4.21.7)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/49—Urokinase; Tissue plasminogen activator
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21007—Plasmin (3.4.21.7), i.e. fibrinolysin

Definitions

  • the present invention relates to a method for treating Parkinson's disease, comprising administering to a subject an effective amount of a component of the plasminogen activation pathway or its related compound, such as plasminogen, to improve clinical symptoms and signs.
  • Parkinson’s disease (Parkinson’s disease, PD) is a common neurodegenerative disease. It is more common in the elderly. The average age of onset is about 60 years old. Parkinson’s disease is less common in young people under 40 years of age. Most Parkinson's disease patients are sporadic cases, and less than 10% of patients have a family history. The most important pathological change of Parkinson's disease is the degeneration and death of dopamine (DA) neurons in the substantia nigra of the midbrain, which causes a significant decrease in DA content in the striatum and causes disease. The exact cause of this pathological change is still unclear. Genetic factors, environmental factors, aging, oxidative stress, etc. may be involved in the degeneration and death of PD dopaminergic neurons.
  • DA dopamine
  • Parkinson’s disease The prominent pathological changes of Parkinson’s disease are the degeneration and death of dopamine (DA) neurons in the substantia nigra of the brain, the significant reduction of DA content in the striatum, and the appearance of eosinophilic inclusions in the cytoplasm of the substantia nigra remnant neurons, that is, Lewy Lewy body.
  • DA dopamine
  • the non-dopaminergic system of Parkinson's disease patients is also significantly impaired.
  • cholinergic neurons of the basal nucleus of Meynert Such as the cholinergic neurons of the basal nucleus of Meynert, the noradrenergic neurons of the locus coeruleus, the serotonergic neurons of the raphe nucleus of the brainstem, and the nerves of the cerebral cortex, brainstem, spinal cord, and peripheral autonomic nervous system Yuan.
  • the significant decrease in dopamine content in the striatum is closely related to the appearance of motor symptoms of Parkinson's disease.
  • the significant reduction of dopamine concentration in the midbrain-limbic system and midbrain-cortex system is closely related to the loss of intelligence and affective disorders in patients with Parkinson's disease.
  • Parkinson's disease has an insidious onset and slow progress.
  • the first symptom is usually tremor or clumsiness in one limb, which affects the opposite limb.
  • the main clinical manifestations are static tremor, bradykinesia, muscle rigidity and postural and gait disorders.
  • non-motor symptoms such as depression, constipation and sleep disturbance are also common complaints of Parkinson's disease patients, and their impact on the quality of life of patients even exceeds motor symptoms.
  • Drug therapy is the most important treatment for Parkinson's disease. It can improve the symptoms to a certain extent, but it cannot prevent the progression of the disease. It is necessary to find other treatment methods and drugs.
  • plasminogen can promote the recovery of memory function of subjects with Parkinson's disease, improve cognitive ability, promote the expression of substantia nigra DTA, promote the recovery of striatal Nissl body, promote the expression of substantia nigra GLP-1R, and increase the expression of black matter GLP-1R.
  • TH-positive cells promote the repair of striatal myelin sheath, promote the degradation of ⁇ -synuclein in brain tissue, promote the expression of striatal NF, promote the repair of axonal damage, reduce the expression of striatal GFAP, and reduce the striatum Neuronal damage, promote the cleavage of Pro-BDNF in the brain tissue to form BDNF, improve depression or anxiety symptoms, and thus have the potential to develop into a drug for the treatment of Parkinson's disease.
  • the present invention relates to the following items:
  • this application relates to a method for preventing and treating Parkinson's disease, comprising administering a therapeutically effective amount of one or more compounds selected from the following group to a subject with Parkinson's disease: plasminogen activation pathway Components, compounds capable of directly activating plasminogen or indirectly activating plasminogen by activating the upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin Compounds, compounds capable of up-regulating the expression of plasminogen or plasminogen activator, plasminogen analogs, plasmin analogs, tPA or uPA analogs, and antagonists of fibrinolytic inhibitors.
  • plasminogen activation pathway Components compounds capable of directly activating plasminogen or indirectly activating plasminogen by activating the upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin Compounds, compounds capable of up-regulating the expression of plasminogen or plasminogen activator,
  • the present application relates to the use of one or more compounds selected from the following in the preparation of drugs for the treatment of Parkinson’s disease, the one or more compounds selected from components of the plasminogen activation pathway, Compounds that can directly activate plasminogen or indirectly activate plasminogen by activating the upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin, can be upregulated Plasminogen or plasminogen activator-expressed compounds, plasminogen analogs, plasmin analogs, tPA or uPA analogs, and antagonists of fibrinolytic inhibitors.
  • the present application relates to a medicine or a pharmaceutical composition for the treatment of Parkinson’s disease comprising one or more compounds selected from the group consisting of: plasminogen activation pathway Components, compounds that can directly activate plasminogen or indirectly activate plasminogen by activating the upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin , Compounds capable of up-regulating the expression of plasminogen or plasminogen activator, plasminogen analogs, plasmin analogs, tPA or uPA analogs and antagonists of fibrinolytic inhibitors.
  • plasminogen activation pathway Components compounds that can directly activate plasminogen or indirectly activate plasminogen by activating the upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin , Compounds capable of up-regulating the expression of plasminogen or plasminogen activator, plasminogen analogs, plasmin analogs, tPA or
  • the component of the plasminogen activation pathway is selected from the group consisting of plasminogen, recombinant human plasmin, Lys-plasmin Enzyme, Glu-plasminogen, plasmin, plasminogen and plasmin containing one or more kringle domains and protease domains of plasminogen and plasmin Variants and analogs, mini-plasminogen, mini-plasmin, micro-plasminogen, micro-plasmin, delta-plasminogen, delta-plasmin, plasminogen activator, tPA and uPA.
  • plasminogen contains at least 80%, 90%, 95%, 96%, 97%, 98%, A protein that has an amino acid sequence with 99% amino acid sequence identity and still has the proteolytic activity of plasminogen.
  • plasminogen is selected from Glu-plasminogen, Lys-plasminogen, microplasminogen, microfibrinogen Lysinogen, delta-plasminogen or their variants that retain the proteolytic activity of plasminogen.
  • the plasminogen may have at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, and the sequence 2, 6, 8, 10 or 12. %, 98%, or 99% sequence identity, and still have plasminogen activity, such as lysine binding activity or proteolytic activity.
  • the plasminogen is added, deleted and/or substituted 1-100, 1-90, 1-80, 1-70 on the basis of sequence 2, 6, 8, 10 or 12. , 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1 -3, 1-2, 1 amino acid, and still have plasminogen activity, such as lysine binding activity or proteolytic activity protein.
  • the plasminogen is a protein that contains active fragments of plasminogen and still has plasminogen activity, such as lysine binding activity or proteolytic activity.
  • the plasminogen is selected from Glu-plasminogen, Lys-plasminogen, microplasminogen, microplasminogen, delta-plasminogen or their retention Plasminogen activity, such as variants of proteolytic activity.
  • the plasminogen is natural or synthetic human plasminogen, or a variant or fragment thereof that still retains plasminogen activity, such as lysine binding activity or proteolytic activity.
  • the plasminogen is a human plasminogen ortholog from a primate or rodent or it still retains plasminogen activity, such as lysine binding activity or protein Hydrolytically active variants or fragments.
  • the amino acid of the plasminogen is shown in sequence 2, 6, 8, 10 or 12.
  • the plasminogen is natural human plasminogen.
  • the subject is a human. In some embodiments, the subject lacks or lacks plasminogen. In some embodiments, the deficiency or deletion is congenital, secondary, and/or local.
  • the pharmaceutical composition comprises a pharmaceutically acceptable carrier and plasminogen used in the aforementioned methods.
  • the kit may be a prophylactic or therapeutic kit comprising: (i) plasminogen for the aforementioned method and (ii) for delivery of the plasminogen to the The means of the subject.
  • the member is a syringe or vial.
  • the kit further includes a label or instructions for use that instructs the plasminogen to be administered to the subject to perform any of the foregoing methods.
  • the article of manufacture comprises: a container containing a label; and a pharmaceutical composition comprising (i) plasminogen or plasminogen used in the foregoing method, wherein the label indicates that the plasminogen
  • a pharmaceutical composition comprising (i) plasminogen or plasminogen used in the foregoing method, wherein the label indicates that the plasminogen
  • the prolysozyme or composition is administered to the subject to perform any of the aforementioned methods.
  • the kit or article of manufacture further includes one or more additional components or containers that contain other drugs.
  • the plasminogen is administered systemically or locally, preferably by the following routes: intravenous, intramuscular, or subcutaneous administration of plasminogen for treatment.
  • the plasminogen is administered in combination with an appropriate polypeptide carrier or stabilizer.
  • the plasminogen is at a dose of 0.0001-2000 mg/kg, 0.001-800 mg/kg, 0.01-600 mg/kg, 0.1-400 mg/kg, 1-200 mg/kg, 1-100 mg per day /kg, 10-100mg/kg (calculated per kilogram of body weight) or 0.0001-2000mg/cm2, 0.001-800mg/cm2, 0.01-600mg/cm2, 0.1-400mg/cm2, 1- 200mg/cm2, 1-100mg/ cm2, 10-100 mg/cm2 (calculated per square centimeter of body surface area) dose administration, preferably repeated at least once, preferably at least daily administration.
  • the present invention clearly covers all combinations of technical features belonging to the embodiments of the present invention, and the technical solutions after these combinations have been clearly disclosed in this application, just as the above-mentioned technical solutions have been separately and clearly disclosed.
  • the present invention also clearly covers the combinations between the various embodiments and their elements, and the technical solutions after the combination are clearly disclosed herein.
  • Figure 1 The statistics of the total distance of movement in the open field test of Parkinson's model mice 14 days after administration of plasminogen. The results showed that the mice in the blank control group exercised a certain distance during the experiment; the total movement distance of the mice in the plasminogen administration group was significantly shorter than that in the vehicle control group. The total movement distance of the mice in the zymogen group was close to that of the blank control group. It shows that plasminogen can enhance the avoidance of Parkinson's model mice and relieve their anxiety.
  • FIG. 2 The statistical results of the resting time rate in the border zone of the Parkinson's model mice 14 days after administration of plasminogen.
  • the results showed that the mice in the blank control group had a certain rate of resting time in the border zone; the resting time rate in the border zone of the mice in the plasminogen group was significantly greater than that in the vehicle control group, and the statistical difference was extremely significant (** means P ⁇ 0.01) , And the resting time rate in the border zone of the plasminogen group was close to that of the blank control group. It shows that plasminogen can enhance the avoidance of Parkinson's model mice and relieve their anxiety.
  • FIG. 3 Statistic results of the movement distance of the boundary zone of the Parkinson model mice in the open field experiment 14 days after the administration of plasminogen.
  • Figure 4 Statistic results of the percentage of movement distance in the boundary zone of the Parkinson's model mice in the open field experiment 14 days after the administration of plasminogen.
  • the results showed that the blank control group had a certain percentage of the boundary zone movement distance; the percentage of the boundary zone movement distance of the mice in the plasminogen group was significantly greater than that in the vehicle control group, and the statistical difference was extremely significant (** means P ⁇ 0.01), and the fiber was given The percentage of movement distance in the border zone of the lysinogen group was close to that of the blank control group. It shows that plasminogen can enhance the avoidance of Parkinson's model mice and relieve their anxiety.
  • Figure 5 Calculated results of the percentage of slow motion time in the boundary zone of the open field experiment of Parkinson's model mice 14 days after administration of plasminogen. The results showed that the mice in the blank control group had a certain percentage of slow-moving time in the boundary zone; the percentage of slow-moving time in the boundary of the mice in the plasminogen group was significantly lower than that in the vehicle control group, and the statistical difference was extremely significant (** means P ⁇ 0.01); and the time rate of slow border exercise in the plasminogen group was close to that of the blank control group. It shows that plasminogen can enhance the avoidance of Parkinson's model mice and relieve their anxiety.
  • Figure 6 The statistical results of the percentage of time in the border zone of the open field experiment of Parkinson's model mice 14 days after administration of plasminogen.
  • Fig. 7 Statistic results of the number of times the Parkinson model mice entered the border zone in the open field test 14 days after the administration of plasminogen. The results showed that the blank control group mice had a certain number of times to enter the border zone; the number of times the mice in the plasminogen group entered the border zone was significantly less than the vehicle group, and the statistical difference was significant (* indicates P ⁇ 0.05), and the administration of fibrinolysis The number of times the mice in the zymogen group entered the boundary was close to that of the blank control group. It shows that plasminogen can enhance the avoidance of Parkinson's model mice and relieve their anxiety.
  • Fig. 8 Statistic results of the movement distance of the central area of the Parkinson model mice in the open field experiment 14 days after the administration of plasminogen.
  • the results showed that the blank control group mice had a certain central area movement distance; the central area movement distance of the mice in the plasminogen administration group was significantly lower than that of the vehicle control group, and the statistical difference was significant (* indicates P ⁇ 0.05); and The movement distance in the central area of the mice in the plasminogen group was close to that of the blank control group. It shows that plasminogen can enhance the avoidance of Parkinson's model mice and relieve their anxiety.
  • Fig. 9 The statistical results of the percentage of movement distance in the central area of the Parkinson's model mice in the open field experiment 14 days after the administration of plasminogen.
  • the results showed that the blank control group mice had a certain percentage of the central area movement distance; the central area movement percentage of the mice in the plasminogen administration group was significantly lower than that of the vehicle control group mice, and the statistical difference was significant (* indicates P ⁇ 0.05) ; And the percentage of movement distance in the central area of the mice in the plasminogen group was close to that of the blank control group. It shows that plasminogen can enhance the avoidance of Parkinson's model mice and relieve their anxiety.
  • FIG 10 The statistical results of the maximum movement speed of the central area of the Parkinson's model mice in the open field experiment 14 days after the administration of plasminogen.
  • the results showed that the mice in the blank control group had a certain maximum movement speed in the central area; the maximum movement speed in the central area of the mice in the plasminogen administration group was significantly lower than that in the vehicle control group, and the statistical difference was extremely significant (** means P ⁇ 0.01). And the maximum movement speed of the central area of the plasminogen group was close to that of the blank control group. It shows that plasminogen can enhance the avoidance of Parkinson's model mice and relieve their anxiety.
  • FIG 11 The statistical results of the time percentage in the central area of the open field experiment in Parkinson's model mice 14 days after the administration of plasminogen.
  • Fig. 12 Statistic results of the number of times that Parkinson model mice entered the central area in an open field experiment 14 days after plasminogen administration. The results showed that the blank control group mice had a certain number of times to enter the central area; the number of times the mice in the plasminogen administration group entered the central area was significantly less than that of the vehicle control group, and the statistical difference was significant (* indicates P ⁇ 0.05); and The number of mice in the plasminogen group entering the central area was close to that of the blank control group. It shows that plasminogen can enhance the avoidance of Parkinson's model mice and relieve their anxiety.
  • Figure 13 is a representative picture of the trajectory of the Parkinson's model mice in the open field experiment 14 days after the administration of plasminogen.
  • the results showed that the mice in the blank control group had almost no activity in the central area; the mice in the plasminogen administration group showed a significantly reduced activity in the central area compared to the vehicle control group and close to the control group. It shows that plasminogen can enhance the avoidance of Parkinson's model mice and relieve their anxiety.
  • FIG. 14 A-C DTA immunohistochemical results of the substantia nigra of Parkinsonian model mice 14 days after administration of plasminogen.
  • A is the blank control group
  • B is the vehicle control group
  • C is the plasminogen administration group.
  • the results showed that the dopamine neurons in the substantia nigra of the blank control group expressed a certain amount of DTA (arrow mark), the expression level of DTA in the substantia nigra of the vehicle group was lower than that of the blank control group, while the substantia nigra dopamine neurons of the vehicle group were lower than that of the control group.
  • the expression level of DTA was higher than that of the vehicle group and close to the blank control group. This result indicates that plasminogen can promote the expression of DTA in the substantia nigra of Parkinsonian model mice.
  • FIG. 15 results of tar violet staining in the striatum of Parkinson's model mice 14 days after plasminogen administration.
  • A is the blank control group
  • B is the vehicle control group
  • C is the plasminogen administration group
  • D is the analysis result of the number of Nissl bodies in the striatum.
  • Figure 16 A-C results of tar violet staining in the substantia nigra of Parkinsonian model mice 14 days after administration of plasminogen.
  • A is the blank control group
  • B is the vehicle control group
  • C is the plasminogen administration group.
  • the results showed that there was a certain amount of Nissl bodies in the substantia nigra of the blank control group of mice (arrow mark); the number of Nissl bodies in the substantia nigra of the vehicle control group was less than that of the blank control group; The number of Nissl bodies is higher than that of the solvent group.
  • the results indicate that plasminogen can promote the recovery of Nissl body in Parkinson's model mice.
  • FIG. 17 The results of GLP-1R immunohistochemistry in the substantia nigra of Parkinsonian model mice 14 days after A-C administration of plasminogen.
  • A is the vehicle PBS group
  • B is the plasminogen administration group
  • C is the average optical density quantitative analysis result.
  • the results showed that the expression of GLP-1R in the substantia nigra of mice in the plasminogen group (marked by the arrow) was significantly higher than that in the vehicle PBS control group, and the statistical difference was significant (* indicates P ⁇ 0.05). This result indicates that plasminogen can promote the expression of GLP-1R in the substantia nigra of Parkinsonian model mice.
  • FIG. 18 The results of TH immunohistochemistry in the substantia nigra of Parkinsonian model mice 14 days after A-D administration of plasminogen.
  • A is the blank control group
  • B is the vehicle PBS group
  • C is the plasminogen administration group
  • D is the quantitative analysis result.
  • the results showed that there were a certain amount of TH-positive cells in the substantia nigra of mice in the blank control group (arrow mark), the number of TH-positive cells in the substantia nigra of mice in the solvent group decreased, and TH-positive cells in the substantia nigra of mice in the plasminogen group The number was significantly higher than that of the solvent group. This result indicates that plasminogen can restore TH-positive cells in the substantia nigra of Parkinson's model mice.
  • FIG 19 A-C immunohistochemical results of Iba-1 substantia nigra in Parkinsonian model mice 14 days after administration of plasminogen.
  • A is the blank control group
  • B is the vehicle PBS group
  • C is the plasminogen administration group.
  • the results showed that there were a certain amount of microglia in the substantia nigra of mice in the blank control group (arrow mark), and the number of microglia in the substantia nigra of mice in the solvent group was higher than that in the control group.
  • the number of glial cells was significantly lower than that of the vehicle control group and close to that of the blank control group. This result shows that plasminogen can promote the recovery of substantia nigra microglia in Parkinson's model mice.
  • FIG. 20 A-C immunohistochemical results of LFB in the striatum of Parkinsonian model mice 14 days after administration of plasminogen.
  • A is the blank control group
  • B is the vehicle PBS group
  • C is the plasminogen administration group.
  • the results showed that there was a certain amount of myelin in the striatum of the blank control group.
  • the number of myelin sheaths in the striatum of the mice in the vehicle group was less than that in the control group.
  • the number of myelin sheaths in the striatum of the mice in the plasminogen group was significantly higher. In the solvent group. This result shows that plasminogen can restore the myelin sheath of the striatum of Parkinson's model mice.
  • Figure 21 A-D immunohistochemical results of substantia nigra ⁇ -synuclein ( ⁇ -synuclein) in Parkinson's model mice 14 days after plasminogen administration.
  • A is the blank control group
  • B is the vehicle group
  • C is the plasminogen administration group
  • D is the average optical density quantitative analysis result.
  • the blank control group only had a small amount of ⁇ -synuclein in the substantia nigra; the amount of ⁇ -synuclein in the substantia nigra of the vehicle group was significantly higher than that of the blank control group (* means P ⁇ 0.05); The amount of substantia nigra ⁇ -synuclein in the plasminogen group was significantly lower than that of the vehicle group, and the statistical difference was significant (* indicates P ⁇ 0.05); to the substantia nigra ⁇ -synucleus of the plasminogen group The amount of protein is close to that of the blank control group. It shows that plasminogen can reduce the expression of alpha-synuclein in the substantia nigra of Parkinson's model mice and improve the degeneration of nerve injury.
  • FIG 22 A-D immunohistochemical results of NF in the striatum of Parkinson's model mice 14 days after administration of plasminogen.
  • A is the blank control group
  • B is the vehicle group
  • C is the plasminogen administration group
  • D is the average optical density quantitative analysis result.
  • the results showed that there was a certain amount of NF in the striatum of the blank control group (arrow mark); the amount of NF in the striatum of the vehicle group was lower than that of the blank control group; the amount of NF in the striatum of the plasminogen group was given The amount is significantly higher than that of the vehicle group, and the statistical difference is extremely significant (** means P ⁇ 0.01). It shows that plasminogen can promote the recovery of NF expression in the striatum of Parkinson's model mice, and improve the axonal injury of the Parkinson's striatum.
  • FIG. 23 A-C immunohistochemical results of GFAP in the striatum of Parkinsonian mice 14 days after administration of plasminogen.
  • A is the blank control group
  • B is the vehicle group
  • C is the plasminogen administration group.
  • the results showed that there was a small amount of GFAP expression in the striatum of the blank control group mice (arrow mark), and the expression of GFAP in the striatum of the vehicle group was significantly higher than that of the blank control group; The expression of is lower than that of the vehicle group. It shows that plasminogen can reduce the expression of GFAP in the striatum of Parkinson's model mice and reduce the damage of the striatum.
  • Figure 24 results of the effect of plasminogen on ⁇ -synuclein in mouse brain homogenate.
  • A is Tricine-page
  • B, C, and D are the quantitative analysis results of ⁇ -synuclein, polymer a, and polymer b, respectively.
  • Figure 25 results of the effect of plasminogen on recombinant human ⁇ -synuclein in mouse brain homogenate.
  • A is a Western-blotting chart
  • B and C are the results of quantitative analysis of ⁇ -synuclein and polymer band scanning, respectively.
  • Figure 26 A-B in the brain homogenate of Parkinson model mice the effect of plasminogen on recombinant human Pro-BDNF
  • A is an SDS-PAGE imaging image
  • B is a quantitative analysis result of SDS-PAGE bands.
  • the results showed that in the brain homogenate of Parkinson's model mice, the amount of Pro-BDNF in the plasminogen group was significantly lower than that in the vehicle control group, and the difference was extremely significant (*** means P ⁇ 0.001). It is suggested that plasminogen can promote the cleavage of recombinant human Pro-BDNF in the brain homogenate of Parkinson's model mice.
  • FIG. 27 A-C The effect of plasminogen on recombinant human Pro-BDNF in the brain homogenate of Parkinson's model mice.
  • A is a Western blot imaging picture
  • B is the analysis result of the Pro-BDNF band optical density (OD) value in the Western blot
  • C is the analysis result of the BDNF band optical density (OD) value in the Western blot.
  • the results showed that in the brain homogenate of Parkinson's model mice, the amount of Pro-BDNF in the plasminogen group was significantly lower than that in the vehicle control group, and the difference was significant (* means P ⁇ 0.05, *** means P ⁇ 0.001) ; The amount of BDNF in the plasminogen group was significantly higher than that in the vehicle control group, and the difference was extremely significant. It is suggested that plasminogen can promote the lysis of recombinant human Pro-BDNF and the formation of mature BDNF in the brain homogenate of Parkinson's model mice.
  • Fibrinolytic system also known as fibrinolytic system, is a system composed of a series of chemical substances involved in the process of fibrinolysis (fibrinolysis), mainly including fibrinolytic enzyme (plasminogen) and plasmin , Plasminogen activator, fibrinolysis inhibitor.
  • Plasminogen activators include tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA).
  • t-PA tissue-type plasminogen activator
  • u-PA urokinase-type plasminogen activator
  • t-PA activates plasminogen
  • fibrin urokinase-type plasminogen activator
  • u-PA urokinase-type plasminogen activator
  • PLG Plasminogen
  • Plasminase is a serine protease, which has the following functions: degrades fibrin and fibrinogen; hydrolyzes a variety of coagulation factors V, VIII, X, VII, XI, II, etc.; turns plasminogen into fibrinolysis Enzymes; hydrolysis of complement, etc.
  • Fibrinolytic inhibitors including plasminogen activator inhibitor (PAI) and ⁇ 2 antiplasmin ( ⁇ 2-AP).
  • PAI mainly has two forms, PAI-1 and PAI-2, which can specifically bind to t-PA in a ratio of 1:1 to inactivate it and activate PLG at the same time.
  • ⁇ 2-AP is synthesized by the liver and combined with PL in a ratio of 1:1 to form a complex, inhibiting PL activity; FXIII makes ⁇ 2-AP covalently bond with fibrin, reducing the sensitivity of fibrin to PL.
  • Substances that inhibit the activity of the fibrinolytic system in the body PAI-1, complement C1 inhibitor; ⁇ 2 anti-plasmin; ⁇ 2 macroglobulin.
  • component of the plasminogen activation pathway covers:
  • Plasminogen activators such as tPA and uPA, and tPA or uPA variants and analogs containing one or more domains of tPA or uPA (such as one or more kringle domains and proteolytic domains) .
  • variants of plasminogen, plasmin, tPA and uPA include all naturally occurring human genetic variants and other mammalian forms of these proteins, as well as by addition, deletion and/or substitution such as 1- 100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, Proteins of 1-10, 1-5, 1-4, 1-3, 1-2, 1 amino acid that still have plasminogen, plasmin, tPA or uPA activity.
  • variants of plasminogen, plasmin, tPA, and uPA include those by, for example, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1- 45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2, 1 conservative Mutant variants of these proteins obtained by amino acid substitutions.
  • the "plasminogen variant” of the present invention encompasses sequences 2, 6, 8, 10 or 12 that have at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99 % Sequence identity, and still have plasminogen activity, such as lysine binding activity or proteolytic activity of the protein.
  • the "plasminogen variant” of the present invention can be added, deleted and/or substituted 1-100, 1-90, 1-80, 1- on the basis of sequence 2, 6, 8, 10 or 12.
  • the plasminogen variants of the present invention include all naturally occurring human genetic variants and other mammalian forms of these proteins, as well as through conservative amino acid substitutions such as 1-100, 1-90, 1-80, 1- 70, 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, Mutant variants of these proteins obtained from 1-3, 1-2, 1 amino acids.
  • the plasminogen of the present invention can be a human plasminogen ortholog from primates or rodents or it still retains plasminogen activity, such as lysine binding activity or proteolytic activity.
  • plasminogen activity such as lysine binding activity or proteolytic activity.
  • the plasminogen shown in sequence 2, 6, 8, 10 or 12 for example, the human natural plasminogen shown in sequence 2.
  • plasminogen, plasmin, tPA, and uPA include compounds that provide substantially similar effects to plasminogen, plasmin, tPA, or uPA, respectively.
  • variants and analogs of plasminogen, plasmin, tPA and uPA encompass fibers comprising one or more domains (for example, one or more kringle domains and proteolytic domains)
  • variants and analogs encompass fibers comprising one or more domains (for example, one or more kringle domains and proteolytic domains)
  • variants and analogs encompass plasminogen comprising one or more plasminogen domains (eg, one or more kringle domains and proteolytic domains) Variants and analogs, such as mini-plasminogen.
  • Variants and “analogs” of plasmin encompass plasmin "variants” that include one or more plasmin domains (eg, one or more kringle domains and proteolytic domains) And “analogs” such as mini-plasmin and delta-plasmin.
  • plasminogen, plasmin, tPA or uPA have the activity of plasminogen, plasmin, tPA or uPA respectively, or whether they provide the same
  • the substantially similar effects of plasminogen, plasmin, tPA or uPA can be detected by methods known in the art, for example, by methods based on enzymography, ELISA (enzyme-linked immunosorbent assay) and FACS ( Fluorescence-activated cell sorting method) is measured by the level of activated plasmin activity, for example, it can be measured with reference to a method selected from the following documents: Ny, A., Leonardsson, G., Hagglund, AC, Hagglof, P.
  • the "component of the plasminogen activation pathway" of the present invention is plasminogen.
  • the plasminogen is human full-length plasminogen or a conservative substitution variant thereof that retains plasminogen activity (e.g., its lysine binding activity and proteolytic activity).
  • the plasminogen is selected from Glu-plasminogen, Lys-plasminogen, microplasminogen, microplasminogen, delta-plasminogen or their retention Variants of plasminogen activity (such as its lysine binding activity or proteolytic activity).
  • the plasminogen is natural or synthetic human plasminogen, or a conservative substitution variant thereof that still retains plasminogen activity (for example, its lysine binding activity or proteolytic activity) Or fragments thereof.
  • the plasminogen is a human plasminogen ortholog from a primate or rodent or a conservative substitution variant or a fragment thereof that still retains plasminogen activity.
  • the plasminogen comprises an amino acid sequence as shown in sequence 2, 6, 8, 10 or 12.
  • the plasminogen comprises a conservatively substituted sequence of the amino acid sequence shown in sequence 2, 6, 8, 10 or 12.
  • the amino acid of the plasminogen is shown in sequence 2, 6, 8, 10 or 12.
  • the plasminogen is a conservative substitution variant of plasminogen shown in sequence 2, 6, 8, 10 or 12. In some embodiments, the plasminogen is human natural plasminogen or a conservative mutant thereof. In some embodiments, the plasminogen is human natural plasminogen as shown in sequence 2 or a conservatively substituted variant thereof.
  • a compound capable of directly activating plasminogen or indirectly activating plasminogen by activating upstream components of the plasminogen activation pathway refers to a compound capable of directly activating plasminogen or by activating plasminogen Any compound that activates upstream components of the pathway and indirectly activates plasminogen, such as tPA, uPA, streptokinase, saruplase,reteplase, reteplase, tenecteplase, aniplase, Monteplase, Lanoteplase, Pamideplase, Staphylokinase.
  • the "antagonist of the fibrinolysis inhibitor" of the present invention is a compound that antagonizes, weakens, blocks, and prevents the action of the fibrinolysis inhibitor.
  • the fibrinolysis inhibitors are, for example, PAI-1, complement C1 inhibitor, ⁇ 2 antiplasmin, and ⁇ 2 macroglobulin.
  • the antagonist such as PAI-1, complement C1 inhibitor, ⁇ 2 anti-plasmin or ⁇ 2 macroglobulin antibody, or blocking or down-regulating such as PAI-1, complement C1 inhibitor, ⁇ 2 antiplasmin or ⁇ 2 macroglobulin Antisense RNA or small RNA expressed by globulin, or occupy the binding site of PAI-1, complement C1 inhibitor, ⁇ 2 antiplasmin or ⁇ 2 macroglobulin but without PAI-1, complement C1 inhibitor, ⁇ 2 anti-fibrosis Compounds that function as lysozyme or ⁇ 2 macroglobulin", or compounds that block the binding domain and/or active domain of PAI-1, complement C1 inhibitor, ⁇ 2 antiplasmin or ⁇ 2 macroglobulin.
  • Plasmin is a key component of the plasminogen activation system (PA system). It is a broad-spectrum protease that can hydrolyze several components of the extracellular matrix (ECM), including fibrin, gelatin, fibronectin, laminin, and proteoglycan. In addition, plasmin can activate some metalloprotease precursors (pro-MMPs) to form active metalloproteases (MMPs). Therefore, plasmin is considered to be an important upstream regulator of extracellular proteolysis. Plasmin is formed by proteolysis of plasminogen through two physiological PAs: tissue-type plasminogen activator (tPA) or urokinase-type plasminogen activator (uPA).
  • tPA tissue-type plasminogen activator
  • uPA urokinase-type plasminogen activator
  • PAs Due to the relatively high levels of plasminogen in plasma and other body fluids, it is traditionally believed that the regulation of the PA system is mainly achieved through the synthesis and activity levels of PAs.
  • the synthesis of PA system components is strictly regulated by different factors, such as hormones, growth factors and cytokines.
  • the main inhibitor of plasmin is ⁇ 2-antiplasmin ( ⁇ 2-antiplasmin).
  • the activity of PAs was inhibited by both uPA and tPA's plasminogen activator inhibitor-1 (PAI-1) and mainly inhibited uPA's lysinogen activator inhibitor-2 (PAI-2).
  • PAI-1 uPA and tPA's plasminogen activator inhibitor-1
  • PAI-2 mainly inhibited uPA's lysinogen activator inhibitor-2
  • Certain cell surfaces have uPA-specific cell surface receptors (uPAR) with direct hydrolytic activity.
  • Plasminogen is a single-chain glycoprotein consisting of 791 amino acids and a molecular weight of approximately 92kDa. Plasminogen is mainly synthesized in the liver and exists in large amounts in the extracellular fluid. Plasminogen content in plasma is about 2 ⁇ M. Therefore plasminogen is a huge potential source of proteolytic activity in tissues and body fluids. Plasminogen exists in two molecular forms: Glu-plasminogen and Lys-plasminogen. The naturally secreted and uncleaved form of plasminogen has an amino terminal (N-terminal) glutamate and is therefore called glutamate-plasminogen.
  • glutamate-plasminogen is hydrolyzed at Lys76-Lys77 to lysine-plasminogen.
  • lysine-plasminogen has a higher affinity for fibrin and can be activated by PAs at a higher rate.
  • the Arg560-Val561 peptide bond of these two forms of plasminogen can be cleaved by uPA or tPA, resulting in the formation of a disulfide bond-linked double-chain protease plasmin.
  • the amino terminal part of plasminogen contains five homologous tricyclic rings, so-called kringles, and the carboxy terminal part contains the protease domain.
  • Some kringles contain lysine binding sites that mediate the specific interaction of plasminogen with fibrin and its inhibitor ⁇ 2-AP.
  • Plasmin also has substrate specificity for several components of ECM, including laminin, fibronectin, proteoglycan and gelatin, indicating that plasmin also plays an important role in ECM reconstruction.
  • plasmin can also degrade other components of ECM by converting certain protease precursors into active proteases, including MMP-1, MMP-2, MMP-3 and MMP-9. Therefore, it has been suggested that plasmin may be an important upstream regulator of extracellular proteolysis.
  • plasmin has the ability to activate certain latent forms of growth factors. In vitro, plasmin can also hydrolyze components of the complement system and release chemotactic complement fragments.
  • Pulminin is a very important enzyme present in the blood, which can hydrolyze fibrin clots into fibrin degradation products and D-dimers.
  • “Plasminogen” is the zymogen form of plasmin. According to the sequence in swiss prot, it is composed of 810 amino acids and the molecular weight is about 90kD, a glycoprotein mainly synthesized in the liver and able to circulate in the blood. The cDNA sequence encoding this amino acid sequence is shown in sequence 3. The full-length plasminogen contains seven domains: a serine protease domain at the C-terminus, a Pan Apple (PAp) domain at the N-terminus, and five Kringle domains (Kringle1-5).
  • PAp Pan Apple
  • Kringle1 includes residues Cys103-Cys181
  • Kringle2 includes residues Glu184-Cys262
  • Kringle3 includes residues Cys275-Cys352
  • Kringle4 Including residues Cys377-Cys454
  • Kringle5 includes residues Cys481-Cys560.
  • the serine protease domain includes residues Val581-Arg804.
  • Glu-plasminogen is a human natural full-length plasminogen, composed of 791 amino acids (without the signal peptide of 19 amino acids).
  • the cDNA sequence encoding this sequence is shown in sequence 1, and its amino acid sequence is shown in sequence 2 shown.
  • Lys-plasminogen formed by hydrolysis from amino acids 76-77 of Glu-plasminogen.
  • sequence 6 the cDNA sequence encoding this amino acid sequence is as shown in sequence 5.
  • Delta-plasminogen ( ⁇ -plasminogen) is a fragment of the full-length plasminogen without the Kringle2-Kringle5 structure, and only contains Kringle1 and serine protease domain (also called protease domain (PD)).
  • Mini-plasminogen (Mini-plasminogen) is composed of Kringle5 and serine protease domain. It has been reported in the literature that it includes residues Val443-Asn791 (with the Glu residue of the Glu-plasminogen sequence without signal peptide as the starting amino acid). ), its amino acid sequence is shown in sequence 10, and the cDNA sequence encoding the amino acid sequence is shown in sequence 9.
  • Micro-plasminogen (Micro-plasminogen) only contains the serine protease domain.
  • plasminogen includes Kringle 1, 2, 3, 4, and 5 domains and serine protease domains (also called protease domains (protease domain, PD)), among which Kringles is responsible for plasmin Proto binds to low-molecular-weight and high-molecular-weight ligands (ie, lysine binding activity), which causes plasminogen to transform into a more open configuration, which is easier to be activated; the protease domain (PD) is residue Val562 -Asn791, tPA and UPA specifically cleave the Arg561-Val562 activation bond of plasminogen, so that plasminogen forms plasmin.
  • protease domain is residue Val562 -Asn791, tPA and UPA specifically cleave the Arg561-Val562 activation bond of plasminogen, so that plasminogen forms plasmin.
  • protease domain confers plasminogen proteolytic activity area.
  • plasmin can be used interchangeably and have the same meaning;
  • plasminogen is equivalent to “plasminogen” and “plasmin Original” can be used interchangeably and has the same meaning.
  • the meaning or activity of the "deficiency" of plasminogen means that the content of plasminogen in the subject is lower than that of a normal person, and is low enough to affect the normal physiological function of the subject;
  • the meaning or activity of "deletion" of plasminogen is that the content of plasminogen in the subject is significantly lower than that of normal people, even the activity or expression is minimal, and normal physiological functions can only be maintained through external sources.
  • plasminogen of the present invention covers both plasminogen and plasmin.
  • plasminogen activator PA
  • PA plasminogen activator
  • the active plasmin can further hydrolyze the fibrin clot into fibrin degradation products and D-dimers, and then dissolve the thrombus.
  • the PAp domain of plasminogen contains important determinants that maintain plasminogen in an inactive closed conformation, while the KR domain can bind to lysine residues present on the receptor and substrate.
  • a variety of enzymes that can act as plasminogen activators are known, including: tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), kallikrein, and coagulation factor XII (Hager Man factor) and so on.
  • “Plasminogen active fragment” refers to the activity of binding to lysine in the target sequence of the substrate (lysine binding activity), or the activity of exerting a proteolytic function (proteolytic activity), or proteolytic activity and lysine Amino acid-binding active fragments.
  • the technical scheme of the present invention related to plasminogen covers the technical scheme of replacing plasminogen with active fragments of plasminogen.
  • the active fragment of plasminogen according to the present invention comprises or consists of the serine protease domain of plasminogen.
  • the active fragment of plasminogen according to the present invention comprises sequence 14, or has at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with sequence 14.
  • the amino acid sequence of is either composed of sequence 14, or is composed of an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity with sequence 14.
  • the active fragment of plasminogen according to the present invention comprises a region selected from one or more of Kringle 1, Kringle 2, Kringle 3, Kringle 4, Kringle 5, or conservative substitution variants thereof, or A region selected from one or more of Kringle 1, Kringle 2, Kringle 3, Kringle 4, Kringle 5, or conservative substitution variants thereof.
  • the plasminogen of the present invention includes a protein containing the above-mentioned active fragment of plasminogen.
  • the methods for measuring plasminogen and its activity in blood include: the detection of tissue plasminogen activator activity (t-PAA), the detection of plasma tissue plasminogen activator antigen (t-PAAg), Detection of plasma tissue plasminogen activity (plgA), detection of plasma tissue plasminogen antigen (plgAg), detection of plasma tissue plasminogen activator inhibitor activity, plasma tissue plasminogen activator inhibition Detection of substance antigens, plasma plasmin-antiplasmin complex detection (PAP).
  • t-PAA tissue plasminogen activator activity
  • t-PAAg the detection of plasma tissue plasminogen activator antigen
  • plgA Detection of plasma tissue plasminogen activity
  • plgAg detection of plasma tissue plasminogen antigen
  • PAP plasma tissue plasminogen activator inhibition
  • the most commonly used detection method is the chromogenic substrate method: adding streptokinase (SK) and chromogenic substrate to the tested plasma, the PLG in the tested plasma is transformed into PLM under the action of SK, and the latter acts on The chromogenic substrate is subsequently measured with a spectrophotometer, and the increase in absorbance is proportional to the activity of plasminogen.
  • SK streptokinase
  • immunochemical methods, gel electrophoresis, immunoturbidimetry, radioimmuno-diffusion methods, etc. can also be used to determine the plasminogen activity in the blood.
  • orthologs or orthologs refer to homologs between different species, including both protein homologs and DNA homologs, and are also called orthologs and vertical homologs. It specifically refers to proteins or genes in different species that evolved from the same ancestor gene.
  • the plasminogen of the present invention includes human natural plasminogen, and also includes plasminogen orthologs or orthologs derived from different species that have plasminogen activity.
  • Constant substitution variant refers to a given amino acid residue that changes but does not change the overall conformation and function of the protein or enzyme. This includes, but is not limited to, those with similar characteristics (such as acidic, basic, hydrophobic, etc.) Amino acids replace amino acids in the amino acid sequence of the parent protein. Amino acids with similar properties are well known. For example, arginine, histidine, and lysine are hydrophilic basic amino acids and can be interchanged. Similarly, isoleucine is a hydrophobic amino acid and can be replaced by leucine, methionine or valine. Therefore, the similarity of two proteins or amino acid sequences with similar functions may be different.
  • Constant substitution variants also include polypeptides or enzymes that are determined by BLAST or FASTA algorithms to have more than 60% amino acid identity. If it can reach more than 75%, it is better, preferably more than 85%, or even more than 90%. It is the best, and has the same or substantially similar properties or functions compared with the natural or parent protein or enzyme.
  • isolated plasminogen refers to plasminogen protein separated and/or recovered from its natural environment.
  • the plasminogen will be purified (1) to a purity (by weight) greater than 90%, greater than 95%, or greater than 98%, as determined by the Lowry method, for example, greater than 99% (By weight), (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by using a rotating cup sequence analyzer, or (3) to homogeneity, which is achieved by using Coomassie blue or silver staining is determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions.
  • the isolated plasminogen also includes plasminogen prepared from recombinant cells by bioengineering technology and separated by at least one purification step.
  • polypeptide refers to polymerized forms of amino acids of any length, which can include genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derived Modified amino acids, and polypeptides with modified peptide backbones.
  • the term includes fusion proteins, including but not limited to fusion proteins with heterologous amino acid sequences, fusions with heterologous and homologous leader sequences (with or without N-terminal methionine residues); and so on.
  • the “percent amino acid sequence identity (%)" with respect to the reference polypeptide sequence is defined as when gaps are introduced when necessary to achieve the maximum percent sequence identity, and any conservative substitutions are not considered as part of the sequence identity, the candidate sequence is Refers to the percentage of amino acid residues that are identical to amino acid residues in the polypeptide sequence.
  • the comparison for the purpose of determining percent amino acid sequence identity can be achieved in a variety of ways within the technical scope of the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for the alignment of the sequences, including any algorithm that achieves the maximum alignment requirements over the entire length of the sequence being compared. However, for the purposes of the present invention, the percent amino acid sequence identity value is generated using the sequence comparison computer program ALIGN-2.
  • the% amino acid sequence identity of a given amino acid sequence A relative to a given amino acid sequence B (or can be expressed as having or containing relative to, with, or against a given amino acid sequence)
  • a given amino acid sequence A) of a certain% amino acid sequence identity of B is calculated as follows:
  • treatment refers to obtaining a desired pharmacological and/or physiological effect.
  • the effect may be to completely or partially prevent the occurrence and onset of the disease or its symptoms, partially or completely reduce the disease and/or its symptoms, and/or partially or completely cure the disease and/or its symptoms, including: (a) prevention of disease Occurs or attacks in a subject, who may have the cause of the disease but has not yet been diagnosed as having the disease; (b) inhibit the disease, that is, block its formation; and (c) reduce the disease and/or its symptoms , That is, causing the disease and/or its symptoms to subside or disappear.
  • mice rats, mice
  • non-human primates humans
  • dogs and cats
  • Hoofed animals such as horses, cows, sheep, pigs, goats
  • “Therapeutically effective amount” or “effective amount” refers to a component or component of the plasminogen activation pathway that is sufficient to achieve the prevention and/or treatment of the disease when administered to a mammal or other subject to treat the disease
  • the amount of related compounds e.g. plasminogen.
  • the “therapeutically effective amount” will depend on the components of the plasminogen activation pathway used or related compounds (such as plasminogen), the severity of the disease and/or symptoms of the subject to be treated, and the age. , Weight, etc.
  • Plasminogen can be isolated from nature and purified for further therapeutic use, or it can be synthesized by standard chemical peptide synthesis techniques. When a polypeptide is synthesized chemically, it can be synthesized via a liquid phase or a solid phase.
  • Solid phase peptide synthesis (SPPS) (where the C-terminal amino acid of the sequence is attached to an insoluble support, followed by sequential addition of the remaining amino acids in the sequence) is a suitable method for the chemical synthesis of plasminogen.
  • SPPS Solid phase peptide synthesis
  • Various forms of SPPS, such as Fmoc and Boc can be used to synthesize plasminogen.
  • the attached solid phase free N-terminal amine is coupled to a single N-protected amino acid unit. Then, the unit is deprotected, exposing a new N-terminal amine that can be attached to other amino acids.
  • the peptide remains immobilized on the solid phase, after which it is cut off.
  • Standard recombinant methods can be used to produce the plasminogen of the present invention.
  • a nucleic acid encoding plasminogen is inserted into an expression vector so that it is operably linked to the regulatory sequence in the expression vector.
  • Expression control sequences include, but are not limited to, promoters (such as naturally associated or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences.
  • Expression control can be a eukaryotic promoter system in a vector that can transform or transfect eukaryotic host cells (such as COS or CHO cells). Once the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high-level expression of the nucleotide sequence and collection and purification of plasminogen.
  • Suitable expression vectors are usually replicated in the host organism as an episome or as an integrated part of the host chromosomal DNA.
  • the expression vector contains a selection marker (for example, ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance, or neomycin resistance) to facilitate the transformation of the desired DNA sequence for exogenous use Those cells are tested.
  • Escherichia coli is an example of a prokaryotic host cell that can be used to clone a polynucleotide encoding a subject antibody.
  • Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis and other enterobacteriaceae, such as Salmonella, Serratia, and various pseudomonas. Genus (Pseudomonas) species.
  • expression vectors can also be produced, which usually contain expression control sequences compatible with the host cell (for example, an origin of replication).
  • promoters such as the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system, or the promoter system from bacteriophage lambda. Promoters usually control expression, optionally in the case of manipulating gene sequences, and have ribosome binding site sequences, etc., to initiate and complete transcription and translation.
  • yeast can also be used for expression.
  • Yeast such as S. cerevisiae
  • Pichia Pichia
  • suitable yeast host cells in which suitable vectors have expression control sequences (such as promoters), origins of replication, termination sequences, etc., as required.
  • suitable promoters include 3-phosphoglycerate kinase and other glycolytic enzymes.
  • Inducible yeasts are initiated by specifically including promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for the utilization of maltose and galactose.
  • mammalian cells e.g., mammalian cells cultured in an in vitro cell culture
  • the anti-Tau antibodies of the present invention e.g., polynucleotides encoding the subject anti-Tau antibodies.
  • Suitable mammalian host cells include CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, and transformed B cells or hybridomas.
  • Expression vectors used in these cells may contain expression control sequences such as an origin of replication, promoters and enhancers (Queen et al., Immunol. Rev.
  • ribosome binding Site RNA splice site
  • polyadenylation site RNA splice site
  • transcription terminator sequence RNA splice site
  • suitable expression control sequences are promoters derived from white immunoglobulin gene, SV40, adenovirus, bovine papilloma virus, cytomegalovirus and the like. See Co et al., J. Immunol. 148:1149 (1992).
  • the plasminogen is substantially pure, for example at least about 80% to 85% pure, at least about 85% to 90% pure, at least about 90% to 95% pure, or 98% to 99% pure Or purer, for example, free of contaminants, such as cell debris, macromolecules other than the target product, and so on.
  • plasminogen activation pathway with the required purity or related compounds (such as plasminogen) with optional pharmaceutical carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences, 16 Edition, Osol, A. ed. (1980)) mixed to form a lyophilized preparation or an aqueous solution to prepare a therapeutic formulation.
  • pharmaceutical carriers such as plasminogen
  • excipients such as plasminogen
  • stabilizers Remington's Pharmaceutical Sciences, 16 Edition, Osol, A. ed. (1980)
  • Acceptable carriers, excipients, and stabilizers are non-toxic to recipients at the dose and concentration used, and include buffers such as phosphate, citrate and other organic acids; antioxidants include ascorbic acid and methionine; preservatives (such as Octadecyl dimethyl benzyl ammonium chloride; hexane diamine chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl p-hydroxybenzoic acid Esters such as methyl or propyl parabens; catechol; resorcinol; cyclohexanol; 3-pentanol; m-cresol); low molecular weight polypeptides (less than about 10 residues) ; Proteins such as serum albumin, gelatin or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine,
  • formulations of the present invention may also contain more than one active compound required for the specific condition to be treated, preferably those with complementary activities and no side effects between each other.
  • the plasminogen of the present invention can be encapsulated in microcapsules prepared by techniques such as coacervation or interfacial polymerization, for example, can be placed in a colloidal drug delivery system (e.g., liposomes, albumin microspheres, microemulsions, Nanoparticles and nanocapsules) or placed in hydroxymethyl cellulose or gel-microcapsules and poly-(methyl methacrylate) microcapsules in a coarse emulsion.
  • colloidal drug delivery system e.g., liposomes, albumin microspheres, microemulsions, Nanoparticles and nanocapsules
  • hydroxymethyl cellulose or gel-microcapsules and poly-(methyl methacrylate) microcapsules in a coarse emulsion.
  • the components of the plasminogen activation pathway of the present invention or related compounds (such as plasminogen) for in vivo administration must be sterile. This can be easily achieved by filtration through a sterile filter before or after freeze-drying and reformulation.
  • sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers having a certain shape and containing glycoproteins, such as films or microcapsules.
  • sustained-release matrices include polyesters, hydrogels such as poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater.
  • Polymers such as ethylene- Vinyl acetate and lactic-glycolic acid can continue to release molecules for more than 100 days, but some hydrogels release proteins for a short time.
  • a reasonable strategy for stabilizing the protein can be designed according to the relevant mechanism. For example, if the mechanism of aggregation is found to be The formation of intermolecular SS bonds through the exchange of thiodisulfide bonds can be stabilized by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling humidity, using appropriate additives, and developing specific polymer matrix compositions .
  • nasal inhalation nebulization
  • nasal drops or eye drops intravenous, intraperitoneal, subcutaneous, intracranial, intrathecal, intraarterial (for example, via the carotid artery), intramuscular, and rectal Drugs to achieve the administration of the pharmaceutical composition of the present invention.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, and so on. Preservatives and other additives may also be present, such as, for example, antimicrobial agents, antioxidants, chelating agents, and inert gases, among others.
  • the dosage range of the pharmaceutical composition containing plasminogen of the present invention can be, for example, about 0.0001 to 2000 mg/kg, or about 0.001 to 500 mg/kg (e.g., 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg per day). mg/kg, 10 mg/kg, 50 mg/kg, etc.) subject weight.
  • the dosage may be 1 mg/kg body weight or 50 mg/kg body weight or in the range of 1-50 mg/kg, or at least 1 mg/kg. Doses above or below this exemplified range are also covered, especially taking into account the factors mentioned above.
  • the intermediate dose in the above range is also included in the scope of the present invention.
  • the subject can administer such doses every day, every other day, every week, or according to any other schedule determined through empirical analysis.
  • An exemplary dosage schedule includes 0.01-100 mg/kg for consecutive days. During the administration of the drug of the present invention, it is necessary to evaluate the therapeutic effect and safety in real time.
  • One embodiment of the present invention relates to a product or a kit comprising a component of the plasminogen activation pathway or a related compound (for example, plasminogen).
  • the article preferably includes a container, label or package insert. Suitable containers are bottles, vials, syringes, etc.
  • the container can be made of various materials such as glass or plastic.
  • the container contains a composition that can effectively treat the disease or condition of the present invention and has a sterile access (for example, the container may be an intravenous solution pack or a vial, which contains a stopper that can be penetrated by a hypodermic injection needle of).
  • At least one active agent in the composition is a component of the plasminogen activation pathway or a related compound (for example, plasminogen).
  • the label on or attached to the container indicates that the composition is used to treat the condition of the present invention.
  • the preparation may further comprise a second container containing a pharmaceutically acceptable buffer, such as phosphate buffered saline, Ringer's solution, and dextrose solution. It may further contain other substances required from a commercial and user point of view, including other buffers, diluents, filters, needles and syringes.
  • the product includes a package insert with instructions for use, including, for example, a composition that instructs the user of the composition to include a component of the plasminogen activation pathway or a related compound (such as plasminogen) As well as other medications for the treatment of concomitant diseases.
  • a composition that instructs the user of the composition to include a component of the plasminogen activation pathway or a related compound (such as plasminogen) As well as other medications for the treatment of concomitant diseases.
  • the human plasminogen used in all the following examples is derived from donor plasma, based on the method described in the following literature: Kenneth C Robbins, Louis Summaria, David Elwyn et al. Further Studies on the Purification and Characterization of Human Plasminogen and Plasmin. Journal of Biological Chemistry,1965,240(1):541-550; Summaria L, Spitz F, Arzadon L et al. Isolation and characterization of the affinity chromatography forms of human Glu-and Lys-plasminogens and unplasmins. J Biol Chem. 1976 25; 251(12): 3693-9; HAGAN JJ, ABLONDI FB, DE RENZO EC. Purification and biochemical properties of human plasminogen. J Biol Chem. 1960 Apr; 235: 1005-10, and process optimization, donated from people Purified from the plasma. The purity of plasminogen monomer is >98%.
  • mice Twenty-eight male C57BL/6J mice aged 10-12 weeks were selected. One day before modeling, all mice were weighed and randomly divided into 2 groups according to their body weights, 8 in the blank control group and 20 in the model group. The mice in the blank control group were intraperitoneally injected with 200 ⁇ l of physiological saline solution, and the mice in the model group were injected intraperitoneally with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (1-methyl- 4-phenyl-1,2,3,6-tetrahydropyridine, MPTP) solution was injected continuously for 5 days to establish a Parkinson's model [1] .
  • 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
  • MPTP 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
  • MPTP solution preparation Take 45 mg of MPTP (Sigma, M0896) and dissolve it in 9 ml of physiological saline solution to prepare a final concentration of 5 mg/ml. After the completion of the modeling, all mice were subjected to body weight measurement and open field behavior testing on the 6th day of modeling. The mice in the model group were randomly divided into 2 groups according to body weight and open field results, 10 mice in the administration group and 10 mice in the vehicle group, and the administration was started, which was recorded as the first day.
  • the vehicle group was injected with 100 ⁇ l/only vehicle solution (10mM citric acid-sodium citrate solution, pH7.4), the administration was continued for 14 days, and the open field experiment was performed on the 15th day of administration .
  • MPTP is a specific strong substantia nigra toxin. Its metabolite MPP+ (1-methyl-4-phenylpyridinium) is an inhibitor of mitochondrial complex I, which can penetrate the blood-brain barrier and block the mitochondrial oxidative respiratory chain to damage the substantia nigra. Dopamine neurons in the body and striatum mimic human Parkinson's symptoms [2] .
  • mice were placed in the center of the bottom surface of the open field (40 ⁇ 40 ⁇ 40cm), and the camera and timekeeping were performed at the same time. The observation was continued for 5 minutes, and each mouse was subjected to 3 experiments. Recording parameters include total moving distance, border resting time rate, border zone movement distance, border zone movement distance percentage, border zone slow movement time percentage, border zone time percentage, number of times to enter the border zone, central zone movement distance, central zone movement The percentage of distance, the maximum speed of the central area, the percentage of time in the central area, the number of times to enter the central area, and the movement trajectory. After each experiment, 70% alcohol was used to wipe the box to prevent the preference of smell.
  • the design principle of the open field experiment is based on the avoidance of mice, which means that mice are afraid of open, unknown, and potentially dangerous places, so they have the nature of "sticking to the wall” activity.
  • the total distance and average speed are regarded as the main data reflecting the spontaneous activities of the mice, and the avoidance is evaluated by the activities of the mice in the surrounding areas (four corners and four sides) of the wilderness. Looking at the activity time in the surrounding area, which reflects the avoidance, the time is reduced, indicating that the mice are more "adventurous".
  • the significant increase in activity time in the central area indicates that avoidance and anxiety (depression) levels are low.
  • the total movement distance refers to the length of the movement track within the specified test time.
  • the boundary area is the surrounding area of the wilderness (four corners and four sides).
  • the resting time rate in the border zone refers to the ratio of the resting time in the border zone to the total resting time (including the resting time in the border zone and the resting time in the central zone).
  • the movement distance of the boundary zone refers to the length of the movement trajectory of the boundary zone within the specified test time.
  • the percentage of movement distance in the border zone refers to the ratio of the movement distance in the border area to the total movement distance (including the movement distance in the border area and the movement distance in the central area).
  • mice in the blank control group had a certain percentage of boundary movement distance; the percentage of movement distance in the boundary zone of the plasminogen group was significantly higher than that of the vehicle control group, and the statistical difference was extremely significant (** means P ⁇ 0.01); And the percentage of the boundary movement distance of mice in the plasminogen group was close to that of the blank control group ( Figure 4). It shows that plasminogen can enhance the avoidance behavior of Parkinson's model mice and relieve their anxiety.
  • the percentage of slow motion time in the boundary zone refers to the ratio of the time in the boundary zone slow motion during the test time to the total test time.
  • mice in the blank control group had a certain percentage of slow-moving time in the boundary zone; the percentage of slow-moving time in the boundary of the mice in the plasminogen group was significantly lower than that in the vehicle control group, and the statistical difference was extremely significant (** means P ⁇ 0.01 ); And the percentage of time for the border slow exercise in the plasminogen group was close to that of the blank control group ( Figure 5). It shows that plasminogen can enhance the avoidance of Parkinson's model mice and relieve their anxiety.
  • the percentage of time in the boundary zone refers to the ratio of the time in the boundary zone to the total test time.
  • the movement distance of the central area refers to the length of the movement trajectory of the central area during the test time.
  • the percentage of the central area's movement distance refers to the ratio of the length of the central area's movement trajectory to the total movement trajectory length during the test period.
  • the maximum movement speed of the central area refers to the fastest movement speed of the central area during the test time.
  • mice in the blank control group had a certain maximum movement speed in the central area; the maximum movement speed in the central area of the mice in the plasminogen administration group was significantly lower than that in the vehicle control group, and the statistical difference was extremely significant (** means P ⁇ 0.01). And the maximum movement speed of the central area of the plasminogen group was close to that of the blank control group ( Figure 10). It shows that plasminogen can enhance the avoidance of Parkinson's model mice and relieve their anxiety.
  • the percentage of time in the central area refers to the ratio of the exercise time in the central area to the total test time.
  • mice in the blank control group rarely entered the central area and showed normal avoidance; the mice in the plasminogen administration group entered the central area significantly less than the vehicle control group, and the statistical difference was significant (* means P ⁇ 0.05 ); And the number of times the mice in the plasminogen group entered the central area was close to that of the blank control group ( Figure 12). It shows that plasminogen can enhance the avoidance of Parkinson's model mice and relieve their anxiety.
  • mice in the blank control group had a certain total distance of movement and almost no activity in the central area; compared with the vehicle control group, the mice in the plasminogen group showed total distance of movement and activity in the central area. All have a significant decreasing trend, and are close to the blank control mice ( Figure 13). It shows that plasminogen can enhance the avoidance of Parkinson's model mice and relieve their anxiety.
  • Plasminogen can promote DTA expression in the substantia nigra of Parkinsonian model mice
  • mice Forty male C57BL/6J mice aged 10-12 weeks were selected. One day before modeling, all mice were weighed and randomly divided into 2 groups according to their body weights, 8 in the blank control group and 32 in the model group. The mice in the blank control group were injected intraperitoneally with 200 ⁇ l of solvent solution, and the mice in the model group were injected intraperitoneally with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (1-methyl-4 -phenyl-1,2,3,6-tetrahydropyridine, MPTP) solution was injected continuously for 5 days to establish a Parkinson's model [1] .
  • 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
  • MPTP 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
  • MPTP solution preparation Take 45 mg of MPTP (Sigma, M0896) and dissolve it in 9 ml of physiological saline solution to prepare a final concentration of 5 mg/ml.
  • the mice in the model group are randomly divided into two groups according to their body weight, the vehicle group and the administration group, each with 16 mice, and the administration starts, which is recorded as the first day, the administration group Mice were injected with 1 mg/100 ⁇ l/mouse of plasminogen solution via tail vein, and the vehicle group was injected with 100 ⁇ l/moist of vehicle solution (10mM citric acid-sodium citrate solution, pH7.4) for 14 days, on the 15th day of dosing They were sacrificed on the same day, and the substantia nigra of mice was collected and fixed in 10% neutral formaldehyde solution for 24-48 hours.
  • vehicle solution 10mM citric acid-sodium citrate solution, pH7.4
  • the fixed substantia nigra tissue was dehydrated by alcohol gradient and transparent with xylene before embedding in paraffin.
  • the slice thickness is 3 ⁇ m, and the slice is washed once after deparaffinization and rehydration.
  • Circle the tissue with PAP pen incubate with 3% hydrogen peroxide for 15 minutes, wash twice with 0.01MPBS, 5 minutes each time.
  • DAT rabbit anti-mouse dopamine transporter
  • Goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was incubated for 1 hour at room temperature, washed twice with 0.01M PBS, 5 minutes each time. The color was developed according to the DAB kit (Vector laboratories, Inc, USA), and the hematoxylin was counterstained for 30 seconds after washing 3 times, and rinsed with running water for 5 minutes. Gradient alcohol dehydration, transparent xylene and sealing with neutral gum, slices were observed under a 400x optical microscope.
  • Parkinson disease is a neurodegenerative disease. Its pathological features are the progressive death of dopaminergic neurons in the substantia nigra striatum and the formation of intracytoplasmic Lewy bodies in the remaining dopaminergic neurons. The lack of dopamine in the nodal region, which in turn leads to the classic dyskinesia symptoms of PD.
  • Dopamine transporter is located in the presynaptic membrane of dopamine neurons and can re-uptake the dopamine transmitter released into the synaptic cleft, reflecting the presynaptic function of dopamine neurons. The reduction of DAT is closely related to the development of PD [3] .
  • Plasminogen can promote the recovery of Nissl body in the striatum of Parkinson's model mice
  • mice Forty male C57BL/6J mice aged 10-12 weeks were selected. One day before modeling, all mice were weighed and randomly divided into 2 groups according to their body weights, 8 in the blank control group and 32 in the model group. The mice in the blank control group were injected intraperitoneally with 200 ⁇ l of solvent solution, and the mice in the model group were injected intraperitoneally with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (1-methyl-4 -phenyl-1,2,3,6-tetrahydropyridine, MPTP) solution was injected continuously for 5 days to establish a Parkinson's model [1] .
  • 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
  • MPTP 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
  • MPTP solution preparation Take 45 mg of MPTP (Sigma, M0896) and dissolve it in 9 ml of physiological saline solution to prepare a final concentration of 5 mg/ml.
  • the mice in the model group are randomly divided into two groups according to their body weight, the vehicle group and the administration group, each with 16 mice, and the administration starts, which is recorded as the first day, the administration group Mice were injected with 1 mg/100 ⁇ l/mouse of plasminogen solution via tail vein, and the vehicle group was injected with 100 ⁇ l/moist of vehicle solution (10mM citric acid-sodium citrate solution, pH7.4) for 14 days, on the 15th day of dosing They were sacrificed on the same day, and the mouse striatum was taken and fixed in 10% neutral formaldehyde solution for 24-48 hours.
  • vehicle solution 10mM citric acid-sodium citrate solution, pH7.4
  • the fixed striatum tissue was dehydrated by alcohol gradient and transparent with xylene before embedding in paraffin.
  • Nissl body also known as chromatin, is a unique structure of nerve cells. It is composed of many parallel rough endoplasmic reticulum and free ribosomes during distribution. It has the function of synthesizing protein. The functional status is closely related and is regarded as a sign of nerve cell survival [4] .
  • Example 4 Plasminogen can promote the recovery of the number of Nissl bodies in Parkinson's model mice
  • mice in the model group are randomly divided into two groups according to their body weight, the vehicle group and the administration group, each with 10 mice, and the administration starts, which is recorded as the first day, the administration group Mice were injected with 1 mg/100 ⁇ l/mouse of plasminogen solution via tail vein, and the vehicle group was injected with 100 ⁇ l/moist of vehicle solution (10mM citric acid-sodium citrate solution, pH7.4) for 14 days, on the 15th day of dosing They were sacrificed on the same day, and the substantia nigra of mice was collected and fixed in 10% neutral formaldehyde solution for 24-48 hours.
  • vehicle solution 10mM citric acid-sodium citrate solution, pH7.4
  • the fixed substantia nigra tissue was dehydrated by alcohol gradient and transparent with xylene before embedding in paraffin.
  • Plasminogen can promote the expression of GLP-1R in the substantia nigra of Parkinsonian model mice
  • mice Twelve 9-week-old C57 male mice were weighed one day before modeling. The mice were intraperitoneally injected with 5 mg/ml MPTP solution at 30 mg/kg body weight per day for 5 consecutive days to establish a Parkinson's model [6-7] .
  • MPTP solution preparation draw 10ml deionized water with a syringe, add it to 100mg MPTP powder (sigma, M0896) to prepare a 10mg/ml mother liquor, then draw 1ml mother liquor into an ampoule, then add 1ml deionized water to the final concentration It is 5mg/ml. After the model was completed, the mice were randomly divided into two groups.
  • mice in the vehicle PBS control group and the plasminogen group were given 6 mice each, and the administration was started, which was recorded as day 1.
  • the mice in the plasminogen group were given the 1mg/0.1ml/head/day plasminogen solution was injected into the tail vein, and the vehicle PBS control group was given the same volume of PBS in the tail vein for 14 days.
  • the mice were sacrificed on the 15th day of administration, and the brains were quickly collected and fixed in 4% paraformaldehyde for 24-48 hours.
  • the fixed brain tissue was dehydrated by alcohol gradient and transparent with xylene before embedding in paraffin.
  • rabbit anti-mouse GLP-1R antibody rabbit anti-mouse GLP-1R antibody
  • Goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was incubated for 1 hour at room temperature, washed twice with 0.01M PBS, 5 minutes each time. Color was developed according to DAB kit (Vector laboratories, Inc., USA). After washing 3 times, it was counterstained with hematoxylin for 30 seconds, and washed with running water for 5 minutes. Gradient alcohol dehydration, transparent xylene and sealing with neutral gum, slices were observed under a 200X optical microscope.
  • Glucagon-like peptide 1 receptor is a member of the glucagon receptor family and is a G protein-coupled receptor that can regulate blood sugar by promoting the secretion of insulin Level [8-9] .
  • Parkinson’s disease is characterized by the absence of dopaminergic signals in neurons of the substantia nigra striatum, and the substantia nigra striatum also expresses GLP-1R [10] .
  • Plasminogen can promote the expression of TH in the substantia nigra of Parkinsonian model mice
  • mice Twelve 9-week-old C57 male mice were weighed one day before modeling. The mice were intraperitoneally injected with 5 mg/ml MPTP solution at 30 mg/kg body weight per day for 5 consecutive days to establish a Parkinson's model [6-7] .
  • MPTP solution preparation draw 10ml deionized water with a syringe, add it to 100mg MPTP powder (sigma, M0896) to prepare a 10mg/ml mother liquor, then draw 1ml mother liquor into an ampoule, then add 1ml deionized water to the final concentration It is 5mg/ml. After the model was completed, the mice were randomly divided into two groups.
  • mice in the vehicle PBS control group and the plasminogen group were given 6 mice each, and the administration was started, which was recorded as day 1.
  • the mice in the plasminogen group were given the 1mg/0.1ml/head/day plasminogen solution was injected into the tail vein, and the vehicle PBS control group was given the same volume of PBS in the tail vein for 14 days.
  • the mice were sacrificed on the 15th day of administration, and the substantia nigra of the mice was taken and fixed in 4% paraformaldehyde for 24-48 hours.
  • the fixed brain tissue was dehydrated by alcohol gradient and transparent with xylene before embedding in paraffin.
  • rabbit anti-mouse TH antibody Proteintech, 25859-1-AP
  • Goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was incubated for 1 hour at room temperature, washed twice with 0.01M PBS, 5 minutes each time. Color was developed according to DAB kit (Vector laboratories, Inc., USA). After washing 3 times, it was counterstained with hematoxylin for 30 seconds, and washed with running water for 5 minutes. Gradient alcohol dehydration, transparent xylene and sealing with neutral gum, slices were observed under a 400x optical microscope.
  • Tyrosine hydroxylase is the rate-limiting enzyme for the synthesis of levodopa (L-dopa) from tyrosine. It is only expressed in the cytoplasm and is abundant in dopamine neurons. Most of the TH-positive neurons in the substantia nigra are dopaminergic, so TH can be used as a marker of dopaminergic neurons in the substantia nigra, and the expression of TH in the substantia nigra becomes an indicator of PD detection [11] .
  • Example 7 Plasminogen affects the number of substantia nigra microglia in Parkinson's model mice
  • mice Twelve 9-week-old C57 male mice were weighed one day before modeling. The mice were injected with 5 mg/ml MPTP solution intraperitoneally at 30 mg/kg body weight every day for 5 consecutive days to establish a Parkinson's model [6-7] .
  • MPTP solution preparation draw 10ml deionized water with a syringe, add it to 100mg MPTP powder (sigma, M0896) to prepare a 10mg/ml mother liquor, then draw 1ml mother liquor into an ampoule, then add 1ml deionized water to the final concentration It is 5mg/ml. After the model was completed, the mice were randomly divided into two groups.
  • mice in the vehicle PBS control group and the plasminogen group were given 6 mice each, and the administration was started, which was recorded as the first day. 1mg/0.1ml/head/day plasminogen solution was injected into the tail vein, and the vehicle PBS control group was given the same volume of PBS in the tail vein for 14 days. The mice were sacrificed on the 15th day of administration, and the substantia nigra of the mice was fixed in 4% paraformaldehyde for 24-48 hours. The fixed substantia nigra was dehydrated by alcohol gradient and transparent with xylene before embedding in paraffin.
  • Goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was incubated for 1 hour at room temperature, washed twice with 0.01M PBS, 5 minutes each time. Color was developed according to DAB kit (Vector laboratories, Inc., USA). After washing 3 times, it was counterstained with hematoxylin for 30 seconds, and washed with running water for 5 minutes. Gradient alcohol dehydration, transparent xylene and sealing with neutral gum, slices were observed under a 400x optical microscope.
  • Microglia are innate immune cells of the central nervous system, which are activated when brain disease or injury occurs. Activated microglia migrate to the injured site and perform a variety of functions, such as phagocytosis of dead cells, increase of pro-inflammatory cytokines, etc., and participate in various central nervous system diseases [12-14] .
  • Iba-1 is a calcium binding protein of about 17kDa, which is specifically expressed in central nervous system microglia and has been widely used as a microglia marker [15-17] .
  • Plasminogen can promote the recovery of myelin sheath in the striatum of Parkinson's model mice
  • mice Twelve 9-week-old C57 male mice were weighed one day before modeling. The mice were intraperitoneally injected with 5 mg/ml MPTP solution at 30 mg/kg body weight per day for 5 consecutive days to establish a Parkinson's model [6-7] .
  • MPTP solution preparation draw 10ml deionized water with a syringe, add it to 100mg MPTP powder (sigma, M0896) to prepare a 10mg/ml mother liquor, then draw 1ml mother liquor into an ampoule, then add 1ml deionized water to the final concentration It is 5mg/ml.
  • the mice were randomly divided into two groups. The mice were given 6 mice each in the vehicle PBS control group and the plasminogen group.
  • the plasminogen solution was injected into the tail vein of 0.1 ml/head/day, and the same volume of PBS was given to the tail vein of the vehicle PBS control group for 14 days.
  • the mice were sacrificed on the 15th day of administration, and the mouse striatum was taken and fixed in 10% neutral formaldehyde solution for 24-48 hours.
  • the fixed striatum tissue was dehydrated by alcohol gradient and transparent with xylene before embedding in paraffin. The thickness of the tissue section is 3 ⁇ m. After dewaxing to water, it is dyed and sealed with 0.1% LFB dye solution for 8-16h. Gradient alcohol dehydration, transparent xylene, and sealing with neutral gum. The slices were observed and photographed under a 400x optical microscope.
  • Neurodegenerative diseases refer to diseases caused by the loss of neurons in the brain and spinal cord or their myelin sheaths.
  • LFB Longt al.
  • myelin sheath is a specific staining method for myelin sheath, which can reflect the damage of myelin sheath [18-19] .
  • Plasminogen can reduce the expression of substantia nigra ⁇ -synuclein in Parkinson's model mice
  • mice in the model group are randomly divided into two groups according to their body weight, the vehicle group and the administration group, 10 mice in each group, and the administration is started, which is recorded as the first day, the administration group Mice were injected with 1mg/100 ⁇ L/only plasminogen solution in the tail vein, and the vehicle group was injected with 100 ⁇ L/only vehicle solution (10mM citric acid-sodium citrate solution, pH7.4) for 14 days, on the 15th day of administration They were sacrificed on the same day, and the substantia nigra of mice was collected and fixed in 4% paraformaldehyde for 24-48 hours.
  • the fixed brain tissue was dehydrated by alcohol gradient and transparent with xylene before embedding in paraffin. Locate the substantia nigra of the section, the thickness of the section is 3 ⁇ m, and wash the section once with water after deparaffinization and rehydration. Circle the tissue with PAP pen, incubate with 3% hydrogen peroxide for 15 minutes, wash twice with 0.01M PBS, 5 minutes each time. Blocked with 5% normal goat serum (Vector laboratories, Inc., USA) for 30 minutes; when time is up, discard the goat serum and add rabbit anti-mouse ⁇ -synuclein antibody (Proteintech, 10842-1-AP) 4 Incubate overnight at °C, wash twice with 0.01M PBS, 5 minutes each time.
  • Goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was incubated for 1 hour at room temperature, washed twice with 0.01M PBS, 5 minutes each time. Color was developed according to DAB kit (Vector laboratories, Inc., USA). After washing 3 times, it was counterstained with hematoxylin for 30 seconds, and washed with running water for 5 minutes. Gradient alcohol dehydration, transparent xylene and sealing with neutral gum, slices were observed under a 400x optical microscope.
  • Parkinson's disease is caused by the absence of dopaminergic neurons in the substantia nigra of the midbrain and the appearance of Lewy bodies.
  • ⁇ -synuclein a neuronal protein composed of 140 amino acid residues, which can cause neuron damage and participate in the process of neurodegeneration in the central nervous system.
  • nerve cells Lewy bodies and nerves ⁇ -synuclein aggregated in synapses is a hallmark of brain lesions in Parkinson's disease [20] .
  • Example 10 Plasminogen can improve the axonal injury of the striatum of Parkinson's model mice
  • mice in the model group are randomly divided into two groups according to their body weight, the vehicle group and the administration group, 10 mice in each group, and the administration is started, which is recorded as the first day, the administration group Mice were injected with 1mg/100 ⁇ L/only plasminogen solution in the tail vein, and the vehicle group was injected with 100 ⁇ L/only vehicle solution (10mM citric acid-sodium citrate solution, pH7.4) for 14 days, on the 15th day of administration They were sacrificed on the same day, and the substantia nigra of mice was collected and fixed in 4% paraformaldehyde for 24-48 hours.
  • the fixed brain tissue was dehydrated by alcohol gradient and transparent with xylene before embedding in paraffin. Locate the substantia nigra of the section, the thickness of the section is 3 ⁇ m, and wash the section once with water after deparaffinization and rehydration. Circle the tissue with PAP pen, incubate with 3% hydrogen peroxide for 15 minutes, wash twice with 0.01M PBS, 5 minutes each time. Blocked with 5% normal goat serum (Vector laboratories, Inc., USA) for 30 minutes; when the time is up, discard the goat serum, drop rabbit anti-mouse NF antibody (Abcam, ab207176) and incubate overnight at 4°C, 0.01M Wash 2 times with PBS, 5 minutes each time.
  • Goat anti-rabbit IgG (HRP) antibody (Abcam, ab6721) secondary antibody was incubated for 1 hour at room temperature, washed twice with 0.01M PBS, 5 minutes each time. Color was developed according to DAB kit (Vector laboratories, Inc., USA). After washing 3 times, it was counterstained with hematoxylin for 30 seconds, and washed with running water for 5 minutes. Gradient alcohol dehydration, transparent xylene and sealing with neutral gum, slices were observed under a 400x optical microscope.
  • Neurofilament Neurofilament
  • NF Neurofilament
  • mice striatum of the blank control group had a certain amount of NF (arrow mark), and the amount of NF in the mouse striatum of the vehicle group (Figure 22B) was lower than that of the blank control group.
  • Figure 22C The amount of NF in the mouse striatum was significantly higher than that in the vehicle group, and the statistical difference was extremely significant (** means P ⁇ 0.01) ( Figure 22D). It shows that plasminogen can promote the recovery of NF in the striatum of Parkinson's model mice and repair the injury of Parkinson's axon.
  • Plasminogen can reduce the expression of GFAP in the striatum of Parkinson's model mice
  • mice in the model group are randomly divided into two groups according to their body weight, the vehicle group and the administration group, each with 10 mice, and the administration starts, which is recorded as the first day, the administration group Mice were injected with 1mg/100 ⁇ L/only plasminogen solution in the tail vein, and the vehicle group was injected with 100 ⁇ L/only vehicle solution (10mM citric acid-sodium citrate solution, pH7.4), continued administration for 14 days, on the 15th day of administration They were sacrificed on the same day, and the substantia nigra of mice was collected and fixed in 4% paraformaldehyde for 24-48 hours.
  • the fixed brain tissue was dehydrated by alcohol gradient and transparent with xylene before embedding in paraffin. Position the substantia nigra of the section, the thickness of the section is 3 ⁇ m, the section is washed once with water after deparaffinization and rehydration. Circle the tissue with PAP pen, incubate with 3% hydrogen peroxide for 15 minutes, wash twice with 0.01M PBS, 5 minutes each time. Blocked with 5% normal goat serum (Vector laboratories, Inc., USA) for 30 minutes; when the time is up, discard the goat serum, drop rabbit anti-mouse GFAP antibody (Abcam, ab7260) and incubate overnight at 4°C, 0.01M Wash 2 times with PBS, 5 minutes each time.
  • Goat anti-rabbit IgG (HRP) antibody (Abcam, ab6721) secondary antibody was incubated for 1 hour at room temperature, washed twice with 0.01M PBS, 5 minutes each time. The color was developed according to DAB kit (Vector laboratories, Inc., USA), and after washing 3 times, it was counterstained with hematoxylin for 30 seconds, and rinsed with running water for 5 minutes. Gradient alcohol dehydration, transparent xylene and sealing with neutral gum, slices were observed under a 400x optical microscope.
  • GliaI fibrillary acidic protein is an important component of astrocyte soma collagen, and it only exists in the intermediate filaments of astrocyte glial fibrils, which is astrocyte. A characteristic marker of plasma cell activation [22] , it has inflammatory damage to neurons and degenerates neurons [23] , which leads to the occurrence of Parkinson's.
  • mice striatum of the blank control group ( Figure 23A) expressed a small amount of GFAP (arrow mark), and the expression of GFAP in the mouse striatum of the vehicle group ( Figure 23B) was significantly higher than that of the blank control group.
  • the expression level of GFAP in the striatum of the original group of mice ( Figure 23C) was lower than that of the vehicle group. It shows that plasminogen can reduce the expression of GFAP in the striatum of Parkinson's model mice and reduce the damage of striatal neurons.
  • Example 12 Plasminogen promotes the degradation of ⁇ -synuclein in the brain homogenate of Parkinson's model mice
  • mice were subjected to an open-field experiment, and the modeling was successful. After all mice were sacrificed, the entire brain was taken and weighed, and 1 ⁇ PBS (Thermo Fisher, pH 7.4; 10010-031) was added at 150 mg tissue/mL PBS, homogenized at 4°C (1 min, 3-4 times), and homogenized. After the slurry was centrifuged at 4°C (12000 rpm, 20 min), the supernatant was taken and placed in a new EP tube.
  • 1 ⁇ PBS Thermo Fisher, pH 7.4; 10010-031
  • Eppendorf (EP) tubes Take Eppendorf (EP) tubes as 1 blank control group, 2 vehicle control group, 3 plasminogen group, and set 5 parallel in each group.
  • the blank control group was added with 21.5 ⁇ L of normal saline, 4.6 ⁇ L of plasminogen solution (2mg/mL), and 23.9 ⁇ L of mouse brain homogenate;
  • the vehicle control group was added with 21.5 ⁇ L of ⁇ -synuclein solution (Shanghai Qiangyao Biotechnology Co., Ltd.
  • a 12% gel was prepared according to the instructions of the Tris-Tricine-SDS-PAGE gel preparation kit (Solarbio, P1320).
  • the samples of each group were mixed with 4 ⁇ loading buffer (TaKaRa, e2139) at a volume ratio of 3:1, heated at 100°C for 5 minutes, cooled and centrifuged for 2 minutes, and then 20 ⁇ L was loaded.
  • the electrophoresis conditions were 30V for 1.5h, and then 100V electrophoresis to the bottom of the gel.
  • the gel was stripped and placed in 1 ⁇ Coomassie Brilliant Blue staining solution (1g Coomassie Brilliant Blue R250 dissolved in 1000ml ethanol: glacial acetic acid: purified water with a volume ratio of 5:2:13) and stained for 30 minutes, and then used
  • the gel was photographed under the biomolecular imager and quantitatively scanned and analyzed.
  • Example 13 Plasminogen promotes the degradation of ⁇ -synuclein in the brain homogenate of Parkinson's model mice
  • mice were subjected to an open-field experiment, and the modeling was successful. After all mice were sacrificed, the entire brain was taken and weighed, and 1 ⁇ PBS (Thermo Fisher, pH 7.4; 10010-031) was added at 150 mg tissue/mL PBS, homogenized at 4°C (1 min, 3-4 times), and homogenized. After the slurry was centrifuged at 4°C (12000 rpm, 20 min), the supernatant was taken and placed in a new EP tube.
  • 1 ⁇ PBS Thermo Fisher, pH 7.4; 10010-031
  • Eppendorf (EP) tubes Take Eppendorf (EP) tubes as 1 blank group, 2 blank control group, 3 vehicle control group, 4 plasminogen group, and set 5 parallel groups in each group.
  • the blank group was added with 21.5 ⁇ L of physiological saline, 4.6 ⁇ L of solvent solution (10mM sodium citrate, 2% arginine hydrochloride, 3% mannitol, pH7.4), 23.9 ⁇ L of mouse brain homogenate;
  • the blank control group was added with 21.5 ⁇ L of physiological Saline, 4.6 ⁇ L plasminogen solution (2mg/mL), 23.9 ⁇ L mouse brain homogenate;
  • the vehicle control group was added with 21.5 ⁇ L ⁇ -synuclein solution (Shanghai Qiangyao Biotechnology Co., Ltd., customized expression of human ⁇ - Nuclein, UniProtKB-P37840, 1.0mg/mL), 4.6 ⁇ L solvent solution, 23.9 ⁇ L mouse brain homogenate; 21.5mL ⁇ -sy
  • a 12% gel was prepared according to the instructions of the Tris-Tricine-SDS-PAGE gel preparation kit (Solarbio, P1320).
  • the samples of each group were mixed with 4 ⁇ loading buffer (TaKaRa, e2139) at a volume ratio of 3:1, heated at 100°C for 5 minutes, cooled and centrifuged for 2 minutes, and then 20 ⁇ L was loaded.
  • the electrophoresis conditions were 30V for 1.5h, and then 100V electrophoresis to the bottom of the gel. After the electrophoresis, the gel was stripped and transferred to a PVDF membrane (GE, A29433753).
  • the electrophoresis condition was 15V, 2h.
  • the transferred PVDF membrane is immersed in a blocking solution (5% degreasing emulsion) and sealed overnight in a refrigerator at 4°C. After washing 4 times with TBST (0.01M Tris-NaCl, pH7.6 buffer), add rabbit anti-human ⁇ -transition Nuclein antibody (Proteintech, 10842-1-AP) was incubated at room temperature for 3 hours, TBST was washed 4 times, and goat anti-rabbit IgG (HRP) antibody (Abcam, ab6721) was added to incubate at room temperature for 1 hour.
  • TBST 0.01M Tris-NaCl, pH7.6 buffer
  • HRP goat anti-rabbit IgG
  • Example 14 Plasminogen promotes the lysis of Pro-BDNF in the brain homogenate of Parkinson's model mice
  • mice After all mice were sacrificed, the entire brain was taken and weighed, and 1 ⁇ PBS (Thermo Fisher, pH 7.4; 10010-031) was added at 150 mg tissue/mL PBS, homogenized at 4°C (1 min, 3-4 times), and homogenized. After the slurry was centrifuged at 4°C (12000 rpm, 20 min), the supernatant, namely the brain homogenate, was placed in a new EP tube.
  • PBS Thermo Fisher, pH 7.4; 10010-031
  • Eppendorf (EP) tubes Take Eppendorf (EP) tubes as 1 blank group, 2 blank control group, 3 vehicle control group, 4 plasminogen group, and set 5 parallel groups in each group.
  • the blank group was added with 21.5 ⁇ L of physiological saline, 4.6 ⁇ L of solvent solution (10mM sodium citrate, 2% arginine hydrochloride, 3% mannitol, pH7.4), 23.9 ⁇ L of mouse brain homogenate;
  • the blank control group was added with 21.5 ⁇ L of physiological Saline, 4.6 ⁇ L plasminogen solution (2mg/mL), 23.9 ⁇ L mouse brain homogenate; 21.5 ⁇ L Pro-BDNF was added to the vehicle control group (Nanjing GenScript, custom-expressed human Pro-BDNF, UniProtKB-P23560, 1.0 mg/mL), 4.6 ⁇ L solvent solution, 23.9 ⁇ L mouse brain homogenate; add 21 ⁇ L Pro-BDNF (1.0mg/mL), 4.6 ⁇ L plasminogen solution (2mg/m
  • Brain-derived neurotrophic factor is a type of basic protein with a molecular weight of 12.3kD. It consists of 119 amino acid residues and contains 3 pairs of disulfide bonds. It is a dimer in the body. It exists in the form and is synthesized in the form of BDNF precursor.
  • BDNF precursor Pro-BDNF
  • Pro-BDNF can be cleaved by enzymatic hydrolysis to form mature BDNF. It is reported in the literature that Pro-BDNF has the opposite effect to the mature BDNF formed by cleavage. Pro-BDNF promotes nerve cell apoptosis and reduces nerve synaptic plasticity. Mature BDNF and its receptors are widely distributed in the central nervous system.
  • central nervous system During the development of the central nervous system, they play an important role in the survival, differentiation, growth and development of neurons, and can prevent the death of neurons from damage and improve the pathology of neurons. It promotes the regeneration and differentiation of injured neurons and other biological effects, and it is also necessary for the mature neurons of the central and peripheral nervous system to maintain survival and normal physiological functions [24] .
  • Example 15 Plasminogen promotes the cleavage of Pro-BDNF in the brain homogenate of Parkinson's model mice to form mature BDNF
  • mice were subjected to an open-field experiment, and the modeling was successful. After all mice were sacrificed, the entire brain was taken and weighed, and 1 ⁇ PBS (Thermo Fisher, pH 7.4; 10010-031) was added at 150 mg tissue/mL PBS, homogenized at 4°C (1 min, 3-4 times), and homogenized. After the slurry was centrifuged at 4°C (12000 rpm, 20 min), the supernatant, namely the brain homogenate, was placed in a new EP tube.
  • 1 ⁇ PBS Thermo Fisher, pH 7.4; 10010-031
  • Eppendorf (EP) tubes Take Eppendorf (EP) tubes as 1 blank group, 2 blank control group, 3 vehicle control group, 4 plasminogen group, and set 5 parallel groups in each group.
  • the blank control group was added with 21.5 ⁇ L of normal saline, 4.6 ⁇ L of plasminogen solution (2mg/mL), and 23.9 ⁇ L of mouse brain homogenate;
  • the vehicle control group was added with 21.5 ⁇ L of Pro-BDNF (Nanjing GenScript, customized expressing human Pro- BDNF, UniProtKB-P23560, 1.0mg/mL), 4.6 ⁇ L solvent solution (citric acid-sodium citrate solution), 23.9 ⁇ L mouse brain homogenate;
  • plasminogen group added 21. ⁇ L Pro-BDNF (1.0mg/ mL), 4.6 ⁇ L plasminogen solution (2mg/mL), 23.9 ⁇ L mouse brain homogenate. After the samples of each group were added, they were incubated at 37°C for 6 hours, and then 50
  • GLP-1 receptor stimulation preserves primary cortical and dopaminergic neurons in cellular and rodent models of stroke and Parkinsonism.Proc.Natl.Acad.Sci.USA. 106, 1285-1290.

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Abstract

提供一种预防和治疗帕金森病的方法，包括给药受试者治疗有效量的纤维蛋白溶酶原激活途径组分；涉及用于治疗上述病症的包含纤维蛋白溶酶原激活途径组分的药物、药物组合物、制品、试剂盒。

Description

一种治疗帕金森病的方法和药物 技术领域
本发明涉及一种治疗帕金森病的方法，包括给药受试者有效量的纤维蛋白溶酶原激活途径的组分或其相关化合物，例如纤溶酶原，以改善临床症状和体征。
背景技术
帕金森病(Parkinson’s disease，PD)是一种常见的神经系统变性疾病，老年人多见，平均发病年龄为60岁左右，40岁以下起病的青年帕金森病较少见。大部分帕金森病患者为散发病例，仅有不到10％的患者有家族史。帕金森病最主要的病理改变是中脑黑质多巴胺(dopamine,DA)能神经元的变性死亡，由此而引起纹状体DA含量显著性减少而致病。导致这一病理改变的确切病因目前仍不清楚，遗传因素、环境因素、年龄老化、氧化应激等均可能参与PD多巴胺能神经元的变性死亡过程。
帕金森病突出的病理改变是中脑黑质多巴胺(dopamine,DA)能神经元的变性死亡、纹状体DA含量显著性减少以及黑质残存神经元胞质内出现嗜酸性包涵体，即路易小体(Lewy body)。除多巴胺能系统外，帕金森病患者的非多巴胺能系统也有明显的受损。如Meynert基底核的胆碱能神经元，蓝斑的去甲肾上腺素能神经元，脑干中缝核的5-羟色胺能神经元，以及大脑皮质、脑干、脊髓、以及外周自主神经系统的神经元。纹状体多巴胺含量显著下降与帕金森病运动症状的出现密切相关。中脑-边缘系统和中脑-皮质系统多巴胺浓度的显著降低与帕金森病患者出现智能减退、情感障碍等密切相关。
帕金森病起病隐袭，进展缓慢。首发症状通常是一侧肢体的震颤或活动笨拙，进而累及对侧肢体。临床上主要表现为静止性震颤、运动迟缓、肌强直和姿势步态障碍。近年来人们越来越多的注意到抑郁、便秘和睡眠 障碍等非运动症状也是帕金森病患者常见的主诉，它们对患者生活质量的影响甚至超过运动症状。
药物治疗是帕金森病最主要的治疗手段，能在一定程度上改善症状，但不能阻止病情的进展，需要寻找其他的治疗方法和药物。
发明概述
本发明研究发现纤溶酶原能够促进帕金森病受试者记忆功能恢复、改善认知能力、促进黑质DTA表达、促进纹状体尼氏体恢复、促进黑质GLP-1R表达、增加黑质TH阳性细胞数量、促进纹状体髓鞘修复、促进脑组织中α-突触核蛋白降解、促使纹状体NF表达、促进轴索损伤修复、减少纹状体GFAP表达、减轻纹状体神经元损伤、促进脑组织中Pro-BDNF裂解形成BDNF、改善抑郁或焦虑症状，从而具有开发成为治疗帕金森病药物的潜力。
具体地，本发明涉及如下各项：
1.一方面，本申请涉及一种预防和治疗帕金森病的方法，包括给药帕金森病受试者治疗有效量的选自如下的一种或多种化合物：纤维蛋白溶酶原激活途径的组分、能够直接激活纤维蛋白溶酶原或通过激活纤维蛋白溶酶原激活途径上游组分而间接激活纤维蛋白溶酶原的化合物、模拟纤维蛋白溶酶原或纤维蛋白溶酶之活性的化合物、能够上调纤维蛋白溶酶原或纤维蛋白溶酶原激活剂表达的化合物、纤维蛋白溶酶原类似物、纤维蛋白溶酶类似物、tPA或uPA类似物和纤溶抑制剂的拮抗剂。
一方面，本申请涉及选自如下的一种或多种化合物在制备治疗帕金森病的药物中的用途，所述一种或多种化合物选自：纤维蛋白溶酶原激活途径的组分、能够直接激活纤维蛋白溶酶原或通过激活纤维蛋白溶酶原激活途径上游组分而间接激活纤维蛋白溶酶原的化合物、模拟纤维蛋白溶酶原或纤维蛋白溶酶之活性的化合物、能够上调纤维蛋白溶酶原或纤维蛋白溶酶原激活剂表达的化合物、纤维蛋白溶酶原类似物、纤维蛋白溶酶类似物、tPA或uPA类似物和纤溶抑制剂的拮抗剂。
一方面，本申请涉及用于治疗帕金森病的包含选自如下的一种或多种化合物的药物或药物组合物，所述一种或多种化合物选自：纤维蛋白溶酶 原激活途径的组分、能够直接激活纤维蛋白溶酶原或通过激活纤维蛋白溶酶原激活途径上游组分而间接激活纤维蛋白溶酶原的化合物、模拟纤维蛋白溶酶原或纤维蛋白溶酶之活性的化合物、能够上调纤维蛋白溶酶原或纤维蛋白溶酶原激活剂表达的化合物、纤维蛋白溶酶原类似物、纤维蛋白溶酶类似物、tPA或uPA类似物和纤溶抑制剂的拮抗剂。
2.项1所述的方法、用途、药物或药物组合物，其中所述纤维蛋白溶酶原激活途径的组分选自纤维蛋白溶酶原、重组人纤维蛋白溶酶、Lys-纤维蛋白溶酶原、Glu-纤维蛋白溶酶原、纤维蛋白溶酶、含有纤维蛋白溶酶原和纤维蛋白溶酶的一个或多个kringle结构域和蛋白酶结构域的纤维蛋白溶酶原和纤维蛋白溶酶变体及类似物、小纤维蛋白溶酶原(mini-plasminogen)、小纤维蛋白溶酶(mini-plasmin)、微纤溶酶原(micro-plasminogen)、微纤溶酶(micro-plasmin)、delta-纤溶酶原、delta-纤溶酶(delta-plasmin)、纤维蛋白溶酶原激活剂、tPA和uPA。
3.项1的方法、用途、药物或药物组合物，所述纤溶抑制剂的拮抗剂为PAI-1、补体C1抑制物、α2抗纤溶酶或α2巨球蛋白的抑制剂，例如抗体。
4.项1-3任一项的方法、用途、药物或药物组合物，其中所述化合物对帕金森病受试者具有如下一项或多项作用：促进记忆功能恢复、改善认知能力、促进黑质DTA表达、促进纹状体尼氏体恢复、促进黑质GLP-1R表达、增加黑质TH阳性细胞数量、促进纹状体髓鞘修复、促进脑组织中α-突触核蛋白降解、促使纹状体NF表达、促进轴索损伤修复、减少纹状体GFAP表达、减轻纹状体神经元损伤、促进脑组织中Pro-BDNF裂解形成BDNF、缓解抑郁或焦虑症状。
5.项1-4任一项的方法、用途、药物或药物组合物，其中所述化合物为纤溶酶原。
6.项1-5任一项的方法、用途、药物或药物组合物，其中所述纤溶酶原为人全长纤溶酶原或其保守取代变体。
7.项1-5任一项的方法、用途、药物或药物组合物，其中所述纤溶酶原与序列2具有至少75％、80％、85％、90％、95％、96％、97％、98％或 99％的序列同一性，并且仍然具有纤溶酶原的赖氨酸结合活性或蛋白水解活性。
8.项1-5任一项的方法、用途、药物或药物组合物，所述纤溶酶原包含与序列14具有至少80％、90％、95％、96％、97％、98％、99％氨基酸序列同一性的氨基酸序列、并且仍然具有纤溶酶原的蛋白水解活性的蛋白质。
9.项1-5任一项的方法、用途、药物或药物组合物，所述纤溶酶原选自Glu-纤溶酶原、Lys-纤溶酶原、小纤溶酶原、微纤溶酶原、delta-纤溶酶原或它们的保留纤溶酶原的蛋白水解活性的变体。
10.项1-5任一项的方法、用途、药物或药物组合物，所述纤溶酶原包含序列2、6、8、10、12所示的氨基酸序列或包含序列2、6、8、10、12所示氨基酸序列的保守取代变体。
11.项1-10任一项的方法、用途、药物或药物组合物，其中所述化合物与一种或多种其他治疗方法或药物联合使用。
12.项11的方法、用途、药物或药物组合物，其中所述其他治疗方法包括细胞治疗(包括干细胞治疗)和物理治疗。
13.项11的方法、用途、药物或药物组合物，其中所述其他药物为治疗帕金森病的其它药物。
14.项1-13任一项的方法、用途、药物或药物组合物，其中所述化合物通过鼻腔吸入、雾化吸入、滴鼻液、滴眼液、滴耳液、静脉内、腹膜内、皮下、颅内、鞘内、动脉内(例如经由颈动脉)或肌肉内给药。在本发明的上述任一实施方案中，所述纤溶酶原可与序列2、6、8、10或12具有至少75％、80％、85％、90％、95％、96％、97％、98％或99％的序列同一性，并且仍然具有纤溶酶原活性，例如赖氨酸结合活性或蛋白水解活性。在一些实施方案中，所述纤溶酶原是在序列2、6、8、10或12的基础上，添加、删除和/或取代1-100、1-90、1-80、1-70、1-60、1-50、1-45、1-40、1-35、1-30、1-25、1-20、1-15、1-10、1-5、1-4、1-3、1-2、1个氨基酸，并且仍然具有纤溶酶原活性，例如赖氨酸结合活性或蛋白水解活性的蛋白质。
在一些实施方案中，所述纤溶酶原是包含纤溶酶原活性片段、并且仍然具有纤溶酶原活性，例如赖氨酸结合活性或蛋白水解活性的蛋白质。在一些实施方案中，所述纤溶酶原选自Glu-纤溶酶原、Lys-纤溶酶原、小纤 溶酶原、微纤溶酶原、delta-纤溶酶原或它们的保留纤溶酶原活性，例如蛋白水解活性的变体。在一些实施方案中，所述纤溶酶原为天然或合成的人纤溶酶原、或其仍然保留纤溶酶原活性，例如赖氨酸结合活性或蛋白水解活性的变体或片段。在一些实施方案中，所述纤溶酶原为来自灵长类动物或啮齿类动物的人纤溶酶原直向同系物或其仍然保留纤溶酶原活性，例如赖氨酸结合活性或蛋白水解活性的变体或片段。在一些实施方案中，所述纤溶酶原的氨基酸如序列2、6、8、10或12所示。在一些实施方案中，所述纤溶酶原是人天然纤溶酶原。
在一些实施方案中，所述受试者是人。在一些实施方案中，所述受试者缺乏或缺失纤溶酶原。在一些实施方案中，所述缺乏或缺失是先天的、继发的和/或局部的。
在一些实施方案中，所述药物组合物包含药学上可接受的载剂和用于前述方法的纤溶酶原。在一些实施方案中，所述试剂盒可以是预防性或治疗性试剂盒，其包含：(i)用于前述方法的纤溶酶原和(ii)用于递送所述纤溶酶原至所述受试者的构件(means)。在一些实施方案中，所述构件为注射器或小瓶。在一些实施方案中，所述试剂盒还包含标签或使用说明书，该标签或使用说明书指示将所述纤溶酶原投予所述受试者以实施前述任一方法。
在一些实施方案中，所述制品包含：含有标签的容器；和包含(i)用于前述方法的纤溶酶原或包含纤溶酶原的药物组合物，其中所述标签指示将所述纤溶酶原或组合物投予所述受试者以实施前述任一方法。
在一些实施方案中，所述试剂盒或制品还包含另外的一个或多个构件或容器，该构件或容器中含有其他药物。
在前述方法的一些实施方案中，所述纤溶酶原通过全身或局部给药，优选通过以下途径施用：静脉内、肌内、皮下给予纤溶酶原来进行治疗。在前述方法的一些实施方案中，所述纤溶酶原与适当的多肽载体或稳定剂组合施用。在前述方法的一些实施方案中，所述纤溶酶原以每天0.0001-2000mg/kg、0.001-800mg/kg、0.01-600mg/kg、0.1-400mg/kg、1-200mg/kg、1-100mg/kg、10-100mg/kg(以每公斤体重计算)或0.0001-2000mg/cm2、0.001-800mg/cm2、0.01-600mg/cm2、0.1-400mg/cm2、1- 200mg/cm2、1-100mg/cm2、10-100mg/cm2(以每平方厘米体表面积计算)的剂量施用，优选至少重复一次，优选至少每天施用。
本发明明确涵盖了属于本发明实施方案之间的技术特征的所有组合，并且这些组合后的技术方案在本申请中已经明确公开，就像上述技术方案已经单独且明确公开一样。另外，本发明还明确涵盖各个实施方案及其要素的之间的组合，该组合后的技术方案在本文中明确公开。
附图简述
图1给予纤溶酶原14天后帕金森模型小鼠旷场实验总运动路程统计结果。结果显示，空白对照组小鼠在实验期间会运动一定路程；给药纤溶酶原组小鼠总运动路程明显短于溶媒对照组，统计差异显著(*表示P<0.05)，且给纤溶酶原组小鼠总运动路程接近于空白对照组。说明纤溶酶原可增强帕金森模型小鼠趋避性，缓解其焦虑。
图2给予纤溶酶原14天后帕金森模型小鼠旷场实验边界区静息时间率统计结果。结果显示，空白对照组小鼠具有一定的边界区静息时间率；给纤溶酶原组小鼠边界区静息时间率明显大于溶媒对照组，统计差异极为显著(**表示P<0.01)，且给纤溶酶原组边界区静息时间率接近于空白对照组。说明纤溶酶原可增强帕金森模型小鼠趋避性，缓解其焦虑。
图3给予纤溶酶原14天后帕金森模型小鼠旷场实验边界区运动路程统计结果。结果显示，空白对照组小鼠具有一定的边界区运动路程；给纤溶酶原组小鼠边界区运动路程明显小于溶媒对照组，统计差异接近显著(P＝0.05)，且给药纤溶酶原组边界区运动路程接近于空白对照组。说明纤溶酶原可增强帕金森模型小鼠趋避性，缓解其焦虑。
图4给予纤溶酶原14天后帕金森模型小鼠旷场实验边界区运动路程百分率统计结果。结果显示，空白对照组具有一定的边界区运动路程百分率；给纤溶酶原组小鼠边界区运动路程百分率明显大于溶媒对照组，统计极为差异显著(**表示P<0.01)，且给纤溶酶原组边界区运动路程百分率接近于空白对照组。说明纤溶酶原可增强帕金森模型小鼠趋避性，缓解其焦虑。
图5给予纤溶酶原14天后帕金森模型小鼠旷场实验边界区慢速运动时间百分率计算结果。结果显示，空白对照组小鼠具有一定的边界区慢速运动时间百分率；给药纤溶酶原组小鼠边界慢速运动时间百分率明显低于溶媒对照组，统计差异极为显著(**表示P<0.01)；且给纤溶酶原组边界慢速运动时间率接近于空白对照组。说明纤溶酶原可增强帕金森模型小鼠趋避性，缓解其焦虑。
图6给予纤溶酶原14天后帕金森模型小鼠旷场实验边界区时间百分率统计结果。结果显示，空白对照组小鼠具有一定的边界区时间百分率；给纤溶酶原组小鼠边界区时间百分率高于溶媒对照组，统计差异接近显著(P＝0.06)，且给药纤溶酶原组边界区时间百分率接近空白对照组。说明纤溶酶原可提高帕金森模型小鼠趋避性行为水平，缓解其焦虑。
图7给予纤溶酶原14天后帕金森模型小鼠旷场实验进入边界区次数统计结果。结果显示，空白对照组小鼠具有一定进入边界区次数；给药纤溶酶原组小鼠进入边界区次数明显少于溶媒组，统计差异显著(*表示P<0.05)，且给药纤溶酶原组小鼠进入边界次数接近空白对照组。说明纤溶酶原可增强帕金森模型小鼠趋避性，缓解其焦虑。
图8给予纤溶酶原14天后帕金森模型小鼠旷场实验中心区运动路程统计结果。结果显示，空白对照组小鼠具有一定的中心区运动路程；给药纤溶酶原组小鼠中心区运动路程明显低于溶媒对照组小鼠，统计差异显著(*表示P<0.05)；且给药纤溶酶原组小鼠中心区运动路程接近空白对照组。说明纤溶酶原可增强帕金森模型小鼠趋避性，缓解其焦虑。
图9给予纤溶酶原14天后帕金森模型小鼠旷场实验中心区运动路程百分率统计结果。结果显示，空白对照组小鼠具有一定的中心区运动路程百分率；给药纤溶酶原组小鼠中心区运动路程百分率明显低于溶媒对照组小鼠，统计差异显著(*表示P<0.05)；且给药纤溶酶原组小鼠中心区运动路程百分率接近空白对照组。说明纤溶酶原可增强帕金森模型小鼠趋避性，缓解其焦虑。
图10给予纤溶酶原14天后帕金森模型小鼠旷场实验中心区最大运动速度统计结果。结果显示，空白对照组小鼠具有一定的中心区最大运动速度；给药纤溶酶原组小鼠中心区最大运动速度明显小于溶媒对照组，统计 差异极为显著(**表示P<0.01)，且给纤溶酶原组中心区最大运动速度接近于空白对照组。说明纤溶酶原可增强帕金森模型小鼠趋避性，缓解其焦虑。
图11给予纤溶酶原14天后帕金森模型小鼠旷场实验中心区时间百分率统计结果。结果显示，空白对照组小鼠具有一定地进入中心区时间百分率；给药纤溶酶原组小鼠中心区时间百分率明显小于溶媒对照组，统计差异接近显著(P＝0.06)；且给纤溶酶原组小鼠进入中心区时间百分率接近于空白对照组。说明纤溶酶原可增强帕金森模型小鼠趋避性，缓解其焦虑。
图12给予纤溶酶原14天后帕金森模型小鼠旷场实验进入中心区次数统计结果。结果显示，空白对照组小鼠具有一定的进入中心区次数；给药纤溶酶原组小鼠进入中心区次数明显少于溶媒对照组，统计差异明显(*表示P<0.05)；且给药纤溶酶原组小鼠进入中心区次数接近空白对照组。说明纤溶酶原可增强帕金森模型小鼠趋避性，缓解其焦虑。
图13给予纤溶酶原14天后帕金森模型小鼠旷场实验运动轨迹代表性图片。结果显示，空白对照组小鼠几乎没有中心区活动；给药纤溶酶原组小鼠表现出中心区活动明显减少的趋势相比于溶媒对照组，且接近于空白对照组小鼠。说明纤溶酶原可增强帕金森模型小鼠趋避性，缓解其焦虑。
图14 A-C给予纤溶酶原14天后帕金森模型小鼠黑质DTA免疫组化结果。A为空白对照组，B为溶媒对照组，C为给纤溶酶原组。结果显示，空白对照组小鼠黑质多巴胺神经元表达一定量的DTA(箭头标识)，溶媒组小鼠黑质DTA的表达量低于空白对照组，而给药纤溶酶原组小鼠黑质DTA的表达量高于溶媒组，且接近空白对照组。该结果表明纤溶酶原可促进帕金森模型小鼠黑质DTA的表达。
图15 A-D给予纤溶酶原14天后帕金森模型小鼠纹状体焦油紫染色结果。A为空白对照组，B为溶媒对照组，C为给纤溶酶原组，D为纹状体尼氏体数量分析结果。结果显示，空白对照组小鼠纹状体神经元存在一定数量的尼氏体(箭头标识)；溶媒对照组小鼠纹状体神经元尼氏体数量明显多于空白对照组，且统计差异接近显著(P＝0.085)；给纤溶酶原组小鼠纹状体神经元尼氏体数量与空白对照组无明显差异，但明显低于溶媒对照 组，且统计差异显著(*表示P<0.05)。该结果表明纤溶酶原能够影响帕金森模型小鼠纹状体尼氏体数量。
图16 A-C给予纤溶酶原14天后帕金森模型小鼠黑质焦油紫染色结果。A为空白对照组，B为溶媒对照组，C为给纤溶酶原组。结果显示，空白对照组小鼠黑质中有一定量的尼氏体(箭头标识)；溶媒对照组小鼠黑质中尼氏体数少于空白对照组；给纤溶酶原组小鼠黑质尼氏体数高于溶媒组。该结果表明纤溶酶原可促进帕金森模型小鼠黑质尼氏体恢复。
图17 A-C给予纤溶酶原14天后帕金森模型小鼠黑质GLP-1R免疫组化结果。A为溶媒PBS组，B为给纤溶酶原组，C为平均光密度定量分析结果。结果显示，给纤溶酶原组小鼠黑质GLP-1R的表达量(箭头标识)明显多于溶媒PBS对照组，且统计差异显著(*表示P<0.05)。该结果表明，纤溶酶原能够促进帕金森模型小鼠黑质GLP-1R的表达。
图18 A-D给予纤溶酶原14天后帕金森模型小鼠黑质TH免疫组化结果。A为空白对照组，B为溶媒PBS组，C为给纤溶酶原组，D为定量分析结果。结果显示，空白对照组小鼠黑质中存在一定量的TH阳性细胞(箭头标识)，溶媒组小鼠黑质中TH阳性细胞数减少，给纤溶酶原组小鼠黑质中TH阳性细胞数明显高于溶媒组。该结果表明纤溶酶原可恢复帕金森模型小鼠黑质中TH阳性细胞。
图19 A-C给予纤溶酶原14天后帕金森模型小鼠黑质Iba-1免疫组化结果。A为空白对照组，B为溶媒PBS组，C为给纤溶酶原组。结果显示，空白对照组小鼠黑质有一定量的小胶质细胞(箭头标识)，溶媒组小鼠黑质中小胶质细胞数高于空白对照组，给纤溶酶原组小鼠黑质中小胶质细胞数明显低于溶媒对照组，且接近于空白对照组。该结果显示纤溶酶原可促使帕金森模型小鼠黑质小胶质细胞恢复。
图20 A-C给予纤溶酶原14天后帕金森模型小鼠纹状体LFB免疫组化结果。A为空白对照组，B为溶媒PBS组，C为给纤溶酶原组。结果显示，空白对照组小鼠纹状体存在一定量的髓鞘，溶媒组小鼠纹状体髓鞘数少于空白对照组，给纤溶酶原组小鼠纹状体髓鞘数明显高于溶媒组。该结果显示纤溶酶原可使帕金森模型小鼠纹状体髓鞘恢复。
图21 A-D给予纤溶酶原14天后帕金森模型小鼠黑质α-突触核蛋白(α-synuclein)免疫组化结果。A为空白对照组，B为溶媒组，C为给纤溶酶原组，D为平均光密度定量分析结果。结果显示，空白对照组小鼠黑质仅有少量的α-突触核蛋白；溶媒组小鼠黑质α-突触核蛋白的量明显高于空白对照组(*表示P<0.05)；给纤溶酶原组小鼠黑质α-突触核蛋白的量明显低于溶媒组，且统计差异显著(*表示P<0.05)；给纤溶酶原组小鼠黑质α-突触核蛋白的量趋近于空白对照组的。说明纤溶酶原可减少帕金森模型小鼠黑质α-突触核蛋白的表达，改善神经损伤变性。
图22 A-D给予纤溶酶原14天后帕金森模型小鼠纹状体NF免疫组化结果。A为空白对照组，B为溶媒组，C为给纤溶酶原组，D为平均光密度定量分析结果。结果显示，空白对照组小鼠纹状体有一定量的NF(箭头标识)；溶媒组小鼠纹状体NF的量低于空白对照组；给纤溶酶原组小鼠纹状体中NF的量明显高于溶媒组，且统计差异极显著(**表示P<0.01)。说明纤溶酶原可促使帕金森模型小鼠纹状体NF表达的恢复，改善帕金森纹状体轴索损伤。
图23 A-C给予纤溶酶原14天后帕金森模型小鼠纹状体GFAP免疫组化结果。A为空白对照组，B为溶媒组，C为给纤溶酶原组。结果显示，空白对照组小鼠纹状体有少量的GFAP表达(箭头标识)，溶媒组小鼠纹状体GFAP的表达明显高于空白对照组；给纤溶酶原组小鼠纹状体GFAP的表达低于溶媒组。说明纤溶酶原可减少帕金森模型小鼠纹状体GFAP表达，减轻纹状体损伤。
图24 A-D在小鼠脑匀浆中纤溶酶原对α-突触核蛋白的作用结果。A为Tricine-page，B、C、D分别为α-突触核蛋白、聚合物a、聚合物b条带扫描定量分析结果。结果显示，在帕金森模型小鼠脑匀浆中，纤溶酶原组α-突触核蛋白的量明显低于溶媒对照组(***代表P＜0.001)，且其聚合物a、b的量均明显低于溶媒对照组(**代表P＜0.01，***代表P＜0.001)；在正常小鼠脑匀浆中，纤溶酶原组α-突触核蛋白的量明显低于溶媒对照组(***代表P＜0.001)，且其聚合物a、b的量均明显低于溶媒对照组(*代表P＜0.05，**代表P＜0.01)。表明在帕金森模型小鼠和正常小鼠脑匀浆中，纤溶酶原可有效地促进人α-突触核蛋白及其聚合物降解。
图25 A-C在小鼠脑匀浆中纤溶酶原对重组人α-突触核蛋白的作用结果。A为Western-blotting图，B、C分别为α-突触核蛋白、聚合物条带扫描定量分析结果。结果显示，在帕金森模型小鼠脑匀浆中，纤溶酶原组α-突触核蛋白的量明显低于溶媒对照组(**代表P＜0.01)，且其聚合物的量均明显低于溶媒对照组(***代表P＜0.001)；在正常小鼠脑匀浆中，纤溶酶原组α-突触核蛋白的量明显低于溶媒对照组(**代表P＜0.01)，且其聚合物的量均明显低于溶媒对照组(***代表P＜0.001)。表明在帕金森模型小鼠和正常小鼠脑匀浆中，纤溶酶原可有效地促进重组人α-突触核蛋白及其聚合物降解。
图26 A-B在帕金森模型小鼠脑匀浆液中，纤溶酶原对重组人Pro-BDNF的作用，A为SDS-PAGE成像图片，B为SDS-PAGE条带定量分析结果。结果显示，在帕金森模型小鼠脑匀浆液中，给药纤溶酶原组Pro-BDNF的量明显低于溶媒对照组，差异极显著(***代表P＜0.001)。提示纤溶酶原能够促进帕金森模型小鼠脑匀浆中重组人Pro-BDNF裂解。
图27 A-C在帕金森模型小鼠脑匀浆液中，纤溶酶原对重组人Pro-BDNF的作用。A为Western blot成像图片，B为Western blot中Pro-BDNF条带光密度(OD)值分析结果，C为Western blot中BDNF条带光密度(OD)值分析结果。结果显示，在帕金森模型小鼠脑匀浆液中，给药纤溶酶原组Pro-BDNF的量明显低于溶媒对照组，差异显著(*代表P＜0.05，***表示P＜0.001)；给药纤溶酶原组BDNF的量明显高于溶媒对照组，差异极为显著。提示纤溶酶原能够促进帕金森模型小鼠脑匀浆液重组人Pro-BDNF裂解和成熟BDNF形成。
发明详述
纤维蛋白溶解系统(Fibrinolytic system)也称纤溶系统，为参与纤维蛋白溶解(纤溶)过程的一系列化学物质组成的系统，主要包括纤维蛋白溶解酶原(纤溶酶原)、纤溶酶、纤溶酶原激活物、纤溶抑制剂。纤溶酶原激活物包括组织型纤溶酶原激活物(t-PA)和尿激酶型纤溶酶原激活物(u-PA)。t-PA是一种丝氨酸蛋白酶，由血管内皮细胞合成。t-PA激活纤溶酶原，此过程主要在纤维蛋白上进行；尿激酶型纤溶酶原激活物(u- PA)由肾小管上皮细胞和血管内皮细胞产生，可以直接激活纤溶酶原而不需要纤维蛋白作为辅因子。纤溶酶原(PLG)由肝脏合成，当血液凝固时，PLG大量吸附在纤维蛋白网上，在t-PA或u-PA的作用下，被激活为纤溶酶，促使纤维蛋白溶解。纤溶酶(PL)是一种丝氨酸蛋白酶，作用如下：降解纤维蛋白和纤维蛋白原；水解多种凝血因子Ⅴ、Ⅷ、Ⅹ、Ⅶ、Ⅺ、Ⅱ等；使纤溶酶原转变为纤溶酶；水解补体等。纤溶抑制物：包括纤溶酶原激活物抑制剂(PAI)和α2抗纤溶酶(α2-AP)。PAI主要有PAI-1和PAI-2两种形式，能特异性与t-PA以1：1比例结合，从而使其失活，同时激活PLG。α2-AP由肝脏合成，与PL以1：1比例结合形成复合物，抑制PL活性；FⅩⅢ使α2-AP以共价键与纤维蛋白结合，减弱了纤维蛋白对PL作用的敏感性。体内抑制纤溶系统活性的物质：PAI-1，补体C1抑制物；α2抗纤溶酶；α2巨球蛋白。
本发明的术语“纤维蛋白溶酶原激活途径的组分”涵盖：
1.纤维蛋白溶酶原、Lys-纤维蛋白溶酶原、Glu-纤维蛋白溶酶原、微纤溶酶原(micro-plasminogen)、delta-纤溶酶原；它们的变体或类似物；
2.纤维蛋白溶酶以及它们的变体或类似物；和
3.纤维蛋白溶酶原激活剂，例如tPA和uPA以及包含一个或多个tPA或uPA的结构域(如一个或多个kringle结构域和蛋白水解结构域)的tPA或uPA变体和类似物。
上述纤维蛋白溶酶原、纤维蛋白溶酶、tPA和uPA的“变体”包括所有天然存在的人类遗传变体以及这些蛋白质的其他哺乳动物形式，以及通过添加、删除和/或取代例如1-100、1-90、1-80、1-70、1-60、1-50、1-45、1-40、1-35、1-30、1-25、1-20、1-15、1-10、1-5、1-4、1-3、1-2、1个氨基酸、仍然具有纤维蛋白溶酶原、纤维蛋白溶酶、tPA或uPA活性的蛋白质。例如，纤维蛋白溶酶原、纤维蛋白溶酶、tPA和uPA的“变体”包括通过例如1-100、1-90、1-80、1-70、1-60、1-50、1-45、1-40、1-35、1-30、1-25、1-20、1-15、1-10、1-5、1-4、1-3、1-2、1个保守性氨基酸取代获得的这些蛋白质的突变变体。
本发明的“纤溶酶原变体”涵盖与序列2、6、8、10或12具有至少75％、80％、85％、90％、95％、96％、97％、98％或99％的序列同一性，并 且仍然具有纤溶酶原活性，例如赖氨酸结合活性或蛋白水解活性的蛋白质。例如本发明的“纤溶酶原变体”可以是在序列2、6、8、10或12的基础上，添加、删除和/或取代1-100、1-90、1-80、1-70、1-60、1-50、1-45、1-40、1-35、1-30、1-25、1-20、1-15、1-10、1-5、1-4、1-3、1-2、1个氨基酸，并且仍然具有纤溶酶原活性，例如赖氨酸结合活性或蛋白水解活性的蛋白质。具体地，本发明纤溶酶原变体包括所有天然存在的人类遗传变体以及这些蛋白质的其他哺乳动物形式，以及通过保守性氨基酸取代例如1-100、1-90、1-80、1-70、1-60、1-50、1-45、1-40、1-35、1-30、1-25、1-20、1-15、1-10、1-5、1-4、1-3、1-2、1个氨基酸获得的这些蛋白质的突变变体。
本发明的纤溶酶原可以为来自灵长类动物或啮齿类动物的人纤溶酶原直向同系物或其仍然保留纤溶酶原活性，例如赖氨酸结合活性或蛋白水解活性的变体，例如序列2、6、8、10或12所示的纤溶酶原，例如序列2所示的人天然纤溶酶原。
上述纤维蛋白溶酶原、纤维蛋白溶酶、tPA和uPA的“类似物”包括分别提供与纤维蛋白溶酶原、纤维蛋白溶酶、tPA或uPA基本相似的作用的化合物。
上述纤维蛋白溶酶原、纤维蛋白溶酶、tPA和uPA的“变体”和“类似物”涵盖包含一个或多个结构域(例如一个或多个kringle结构域和蛋白水解结构域)的纤维蛋白溶酶原、纤维蛋白溶酶、tPA和uPA的“变体”和“类似物”。例如，纤维蛋白溶酶原的“变体”和“类似物”涵盖包含一个或多个纤溶酶原结构域(例如一个或多个kringle结构域和蛋白水解结构域)的纤维蛋白溶酶原变体和类似物，例如小纤维蛋白溶酶原(mini-plasminogen)。纤维蛋白溶酶的“变体”和“类似物”涵盖包含一个或多个纤维蛋白溶酶结构域(例如一个或多个kringle结构域和蛋白水解结构域)的纤维蛋白溶酶“变体”和“类似物”，例如小纤维蛋白溶酶(mini-plasmin)和δ-纤维蛋白溶酶(delta-plasmin)。
上述纤维蛋白溶酶原、纤维蛋白溶酶、tPA或uPA的“变体”或“类似物”是否分别具有纤维蛋白溶酶原、纤维蛋白溶酶、tPA或uPA的活性，或者是否分别提供与纤维蛋白溶酶原、纤维蛋白溶酶、tPA或uPA基本相似的作用可以通过本领域已知方法进行检测，例如，通过基于酶谱法 (enzymography)、ELISA(酶联免疫吸附测定)和FACS(荧光激活细胞分选方法)通过激活的纤维蛋白溶酶活性水平来衡量，例如可以参照选自如下文献中记载的方法测量：Ny，A.，Leonardsson，G.，Hagglund，A.C，Hagglof，P.，Ploplis，V.A.，Carmeliet，P.and Ny，T.(1999).Ovulation inplasminogen-deficient mice.Endocrinology 140，5030-5035；Silverstein RL,Leung LL,Harpel PC,Nachman RL(November 1984)."Complex formation of platelet thrombospondin with plasminogen.Modulation of activation by tissue activator".J.Clin.Invest.74(5):1625–33；Gravanis I,Tsirka SE(February2008)."Tissue-type plasminogen activator as a therapeutic target in stroke".Expert Opinion on Therapeutic Targets.12(2):159–70；Geiger M,Huber K,Wojta J,Stingl L,Espana F,Griffin JH,Binder BR(Aug 1989)."Complex formation between urokinase and plasma protein C inhibitor in vitro and in vivo".Blood.74(2):722–8.
在本发明的一些实施方案中，本发明的“纤维蛋白溶酶原激活途径的组分”为纤溶酶原。在一些实施方案中，所述纤溶酶原为人全长纤溶酶原或其保留纤溶酶原活性(例如其赖氨酸结合活性和蛋白水解活性)的保守取代变体。在一些实施方案中，所述纤溶酶原选自Glu-纤溶酶原、Lys-纤溶酶原、小纤溶酶原、微纤溶酶原、delta-纤溶酶原或它们的保留纤溶酶原活性(例如其赖氨酸结合活性或蛋白水解活性)的变体。在一些实施方案中，所述纤溶酶原为天然或合成的人纤溶酶原、或其仍然保留纤溶酶原活性(例如其赖氨酸结合活性或蛋白水解活性)的保守取代变体或其片段。在一些实施方案中，所述纤溶酶原为来自灵长类动物或啮齿类动物的人纤溶酶原直向同系物或其仍然保留纤溶酶原活性的保守取代变体或其片段。在一些实施方案中，所述纤溶酶原包含如序列2、6、8、10或12所示氨基酸序列。在一些实施方案中，所述纤溶酶原包含序列2、6、8、10或12所示的氨基酸序列的保守取代序列。在一些实施方案中，所述纤溶酶原的氨基酸如序列2、6、8、10或12所示。在一些实施方案中，所述纤溶酶原为序列2、6、8、10或12所示的纤溶酶原的保守取代变体。在一些实施方案中，所述纤溶酶原是人天然纤溶酶原或其保守突变体。在一些实施方案中，所述纤溶酶原是如序列2所示的人天然纤溶酶原或其保守取代变体。
“能够直接激活纤维蛋白溶酶原或通过激活纤维蛋白溶酶原激活途径上游组分而间接激活纤维蛋白溶酶原的化合物”指能够直接激活纤维蛋白溶酶原或通过激活纤维蛋白溶酶原激活途径上游组分而间接激活纤维蛋白溶酶原的任何化合物，例如tPA、uPA、链激酶、沙芦普酶、阿替普酶、瑞替普酶、替奈普酶、阿尼普酶、孟替普酶、拉诺替普酶、帕米普酶、葡激酶。
本发明“纤溶抑制剂的拮抗剂”为拮抗、减弱、封闭、阻止纤溶抑制剂作用的化合物。所述纤溶抑制剂例如PAI-1、补体C1抑制物、α2抗纤溶酶和α2巨球蛋白。所述拮抗剂例如PAI-1、补体C1抑制物、α2抗纤溶酶或α2巨球蛋白的抗体，或阻断或下调例如PAI-1、补体C1抑制物、α2抗纤溶酶或α2巨球蛋白表达的反义RNA或小RNA，或占据PAI-1、补体C1抑制物、α2抗纤溶酶或α2巨球蛋白的结合位点但无PAI-1、补体C1抑制物、α2抗纤溶酶或α2巨球蛋白功能的化合物”，或封闭PAI-1、补体C1抑制物、α2抗纤溶酶或α2巨球蛋白的结合结构域和/或活性结构域的化合物。
纤溶酶是纤溶酶原激活系统(PA系统)的关键组分。它是一种广谱的蛋白酶，能够水解细胞外基质(ECM)的几个组分，包括纤维蛋白、明胶、纤连蛋白、层粘连蛋白和蛋白聚糖。此外，纤溶酶能将一些金属蛋白酶前体(pro-MMPs)激活形成具有活性的金属蛋白酶(MMPs)。因此纤溶酶被认为是胞外蛋白水解作用的一个重要的上游调节物。纤溶酶是由纤溶酶原通过两种生理性的PAs：组织型纤溶酶原激活剂(tPA)或尿激酶型纤溶酶原激活剂(uPA)蛋白水解形成的。由于纤溶酶原在血浆和其他体液中相对水平较高，传统上认为PA系统的调节主要通过PAs的合成和活性水平实现。PA系统组分的合成受不同因素严格调节，如激素、生长因子和细胞因子。此外，还存在纤溶酶和PAs的特定生理抑制剂。纤溶酶的主要抑制剂是α2-抗纤溶酶(α2-antiplasmin)。PAs的活性同时被uPA和tPA的纤溶酶原激活剂抑制剂-1(PAI-1)抑制以及主要抑制uPA的溶酶原激活剂抑制剂-2(PAI-2)调节。某些细胞表面具有直接水解活性的uPA特异性细胞表面受体(uPAR)。
纤溶酶原是一个单链糖蛋白，由791个氨基酸组成，分子量约为92kDa。纤溶酶原主要在肝脏合成，大量存在于胞外液中。血浆中纤溶酶原含量约为2μM。因此纤溶酶原是组织和体液中蛋白质水解活性的一个巨大的 潜在来源。纤溶酶原存在两种分子形式：谷氨酸-纤溶酶原(Glu-plasminogen)和赖氨酸-纤溶酶原(Lys-plasminogen)。天然分泌和未裂解形式的纤溶酶原具有一个氨基末端(N-末端)谷氨酸，因此被称为谷氨酸-纤溶酶原。然而，在纤溶酶存在时，谷氨酸-纤溶酶原在Lys76-Lys77处水解成为赖氨酸-纤溶酶原。与谷氨酸-纤溶酶原相比，赖氨酸-纤溶酶原与纤维蛋白具有更高的亲和力，并可以更高的速率被PAs激活。这两种形式的纤溶酶原的Arg560-Val561肽键可被uPA或tPA切割，导致二硫键连接的双链蛋白酶纤溶酶的形成。纤溶酶原的氨基末端部分包含五个同源三环，即所谓的kringles，羧基末端部分包含蛋白酶结构域。一些kringles含有介导纤溶酶原与纤维蛋白及其抑制剂α2-AP特异性相互作用的赖氨酸结合位点。最新发现一个纤溶酶原为38kDa的片段，其中包括kringles1-4，是血管生成的有效抑制剂。这个片段被命名为血管抑素，可通过几个蛋白酶水解纤溶酶原产生。
纤溶酶的主要底物是纤维蛋白，纤维蛋白的溶解是预防病理性血栓形成的关键。纤溶酶还具有对ECM几个组分的底物特异性，包括层粘连蛋白、纤连蛋白、蛋白聚糖和明胶，表明纤溶酶在ECM重建中也起着重要作用。间接地，纤溶酶还可以通过转化某些蛋白酶前体为活性蛋白酶来降解ECM的其他组分，包括MMP-1，MMP-2，MMP-3和MMP-9。因此，有人提出，纤溶酶可能是细胞外蛋白水解的一个重要的上游调节器。此外，纤溶酶具有激活某些潜在形式的生长因子的能力。在体外，纤溶酶还能水解补体系统的组分并释放趋化补体片段。
“纤溶酶”是存在于血液中的一种非常重要的酶，能将纤维蛋白凝块水解为纤维蛋白降解产物和D-二聚体。
“纤溶酶原”是纤溶酶的酶原形式，根据swiss prot中的序列，按含有信号肽的天然人源纤溶酶原氨基酸序列(序列4)计算由810个氨基酸组成，分子量约为90kD，主要在肝脏中合成并能够在血液中循环的糖蛋白，编码该氨基酸序列的cDNA序列如序列3所示。全长的纤溶酶原包含七个结构域：位于C末端的丝氨酸蛋白酶结构域、N末端的Pan Apple(PAp)结构域以及5个Kringle结构域(Kringle1-5)。参照swiss prot中的序列，其信号肽包括残基Met1-Gly19，PAp包括残基Glu20-Val98，Kringle1包括残基 Cys103-Cys181，Kringle2包括残基Glu184-Cys262，Kringle3包括残基Cys275-Cys352，Kringle4包括残基Cys377-Cys454，Kringle5包括残基Cys481-Cys560。根据NCBI数据，丝氨酸蛋白酶域包括残基Val581-Arg804。
Glu-纤溶酶原是人天然全长的纤溶酶原，由791个氨基酸组成(不含有19个氨基酸的信号肽)，编码该序列的cDNA序列如序列1所示，其氨基酸序列如序列2所示。在体内，还存在一种是从Glu-纤溶酶原的第76-77位氨基酸处水解从而形成的Lys-纤溶酶原，如序列6所示，编码该氨基酸序列的cDNA序列如序列5所示。Delta-纤溶酶原(δ-plasminogen)是全长纤溶酶原缺失了Kringle2-Kringle5结构的片段，仅含有Kringle1和丝氨酸蛋白酶域(也称蛋白酶结构域(protease domain，PD))，有文献报道了delta-纤溶酶原的氨基酸序列(序列8)，编码该氨基酸序列的cDNA序列如序列7所示。小纤溶酶原(Mini-plasminogen)由Kringle5和丝氨酸蛋白酶域组成，有文献报道其包括残基Val443-Asn791(以不含有信号肽的Glu-纤溶酶原序列的Glu残基为起始氨基酸)，其氨基酸序列如序列10所示，编码该氨基酸序列的cDNA序列如序列9所示。而微纤溶酶原(Micro-plasminogen)仅含有丝氨酸蛋白酶结构域，有文献报道其氨基酸序列包括残基Ala543-Asn791(以不含有信号肽的Glu-纤溶酶原序列的Glu残基为起始氨基酸)，也有专利文献CN102154253A报道其序列包括残基Lys531-Asn791(以不含有信号肽的Glu-纤溶酶原序列的Glu残基为起始氨基酸)，在本专利申请中微纤溶酶原序列参考专利文献CN102154253A，其氨基酸序列如序列12所示，编码该氨基酸序列的cDNA序列如序列11所示。
全长纤溶酶原的结构也描述在Aisina等(Aisina R B,Mukhametova L I.Structure and function of plasminogen/plasmin system[J].Russian Journal of Bioorganic Chemistry,2014,40(6):590-605)的文章中。在该文章中，Aisina等描述纤溶酶原包括Kringle1、2、3、4、5结构域和丝氨酸蛋白酶结构域(也称蛋白酶结构域(protease domain，PD))，其中，Kringles负责纤溶酶原与低分子量和高分子量的配体结合(即赖氨酸结合活性)，导致纤溶酶原转变成一个更加开放的构型，从而更容易被活化；蛋白酶结构域 (PD)为残基Val562-Asn791，tPA和UPA特异性切割纤溶酶原的Arg561-Val562位活化键，从而使纤溶酶原形成纤溶酶，因此，蛋白酶结构域(PD)是赋予纤溶酶原蛋白水解活性的区域。本发明的“纤溶酶”与“纤维蛋白溶酶”、“纤维蛋白溶解酶”可互换使用，含义相同；“纤溶酶原”与“纤维蛋白溶酶原”、“纤维蛋白溶解酶原”可互换使用，含义相同。
在本申请中，所述纤溶酶原“缺乏”的含义或活性为受试者体内纤溶酶原的含量比正常人低，低至足以影响所述受试者的正常生理功能；所述纤溶酶原“缺失”的含义或活性为受试者体内纤溶酶原的含量显著低于正常人，甚至活性或表达极微，只有通过外源提供才能维持正常生理功能。
本领域技术人员可以理解，本发明纤溶酶原的所有技术方案适用于纤溶酶，因此，本发明描述的技术方案涵盖了纤溶酶原和纤溶酶。在循环过程中，纤溶酶原采用封闭的非活性构象，但当结合至血栓或细胞表面时，在纤溶酶原激活剂(plasminogen activator，PA)的介导下，其转变为呈开放性构象的活性纤溶酶。具有活性的纤溶酶可进一步将纤维蛋白凝块水解为纤维蛋白降解产物和D-二聚体，进而溶解血栓。其中纤溶酶原的PAp结构域包含维持纤溶酶原处于非活性封闭构象的重要决定簇，而KR结构域则能够与存在于受体和底物上的赖氨酸残基结合。已知多种能够作为纤溶酶原激活剂的酶，包括：组织纤溶酶原激活剂(tPA)、尿激酶纤溶酶原激活剂(uPA)、激肽释放酶和凝血因子XII(哈格曼因子)等。
“纤溶酶原活性片段”是指具有与底物靶序列中赖氨酸结合的活性(赖氨酸结合活性)、或发挥蛋白水解功能的活性(蛋白水解活性)、或蛋白水解活性和赖氨酸结合活性的片段。本发明涉及纤溶酶原的技术方案涵盖了用纤溶酶原活性片段代替纤溶酶原的技术方案。在一些实施方案中，本发明所述的纤溶酶原活性片段包含纤溶酶原的丝氨酸蛋白酶结构域或由纤溶酶原的丝氨酸蛋白酶结构域组成。在一些实施方案中，本发明所述的纤溶酶原活性片段包含序列14、或包含与序列14具有至少80％、90％、95％、96％、97％、98％、99％同一性的氨基酸序列，或由序列14组成、或由与序列14具有至少80％、90％、95％、96％、97％、98％、99％同一性的氨基酸序列组成。在一些实施方案中，本发明所述的纤溶酶原活性片段包含选自Kringle 1、Kringle 2、Kringle 3、Kringle 4、Kringle 5中一个或多个 的区域或其保守取代变体，或由选自Kringle 1、Kringle 2、Kringle 3、Kringle 4、Kringle 5中一个或多个的区域或其保守取代变体组成。在一些实施方案中，本发明所述的纤溶酶原包括含有上述纤溶酶原活性片段的蛋白质。目前，对于血液中纤溶酶原及其活性测定方法包括：对组织纤溶酶原激活剂活性的检测(t-PAA)、血浆组织纤溶酶原激活剂抗原的检测(t-PAAg)、对血浆组织纤溶酶原活性的检测(plgA)、血浆组织纤溶酶原抗原的检测(plgAg)、血浆组织纤溶酶原激活剂抑制物活性的检测、血浆组织纤溶酶原激活剂抑制物抗原的检测、血浆纤维蛋白溶酶-抗纤维蛋白溶酶复合物检测(PAP)。其中最常用的检测方法为发色底物法：向受检血浆中加链激酶(SK)和发色底物，受检血浆中的PLG在SK的作用下，转变成PLM，后者作用于发色底物，随后用分光光度计测定，吸光度增加与纤溶酶原活性成正比。此外也可采用免疫化学法、凝胶电泳、免疫比浊法、放射免疫扩散法等对血液中的纤溶酶原活性进行测定。
“直系同源物或直系同系物(ortholog)”指不同物种之间的同源物，既包括蛋白同源物也包括DNA同源物，也称为直向同源物、垂直同源物。其具体指不同物种中由同一祖先基因进化而来的蛋白或基因。本发明的纤溶酶原包括人的天然纤溶酶原，还包括来源于不同物种的、具有纤溶酶原活性的纤溶酶原直系同源物或直系同系物。
“保守取代变体”是指其中一个给定的氨基酸残基改变但不改变蛋白质或酶的整体构象和功能，这包括但不限于以相似特性(如酸性，碱性，疏水性，等)的氨基酸取代亲本蛋白质中氨基酸序列中的氨基酸。具有类似性质的氨基酸是众所周知的。例如，精氨酸、组氨酸和赖氨酸是亲水性的碱性氨基酸并可以互换。同样，异亮氨酸是疏水氨基酸，则可被亮氨酸，蛋氨酸或缬氨酸替换。因此，相似功能的两个蛋白或氨基酸序列的相似性可能会不同。例如，基于MEGALIGN算法的70％至99％的相似度(同一性)。“保守取代变体”还包括通过BLAST或FASTA算法确定具有60％以上的氨基酸同一性的多肽或酶，若能达75％以上更好，最好能达85％以上，甚至达90％以上为最佳，并且与天然或亲本蛋白质或酶相比具有相同或基本相似的性质或功能。
“分离的”纤溶酶原是指从其天然环境分离和/或回收的纤溶酶原蛋白。在一些实施方案中，所述纤溶酶原会纯化(1)至大于90％、大于95％、或大于98％的纯度(按重量计)，如通过Lowry法所确定的，例如超过99％(按重量计)，(2)至足以通过使用旋转杯序列分析仪获得N端或内部氨基酸序列的至少15个残基的程度，或(3)至同质性，该同质性是通过使用考马斯蓝或银染在还原性或非还原性条件下的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)确定的。分离的纤溶酶原也包括通过生物工程技术从重组细胞制备，并通过至少一个纯化步骤分离的纤溶酶原。
术语“多肽”、“肽”和“蛋白质”在本文中可互换使用，指任何长度的氨基酸的聚合形式，其可以包括遗传编码的和非遗传编码的氨基酸，化学或生物化学修饰的或衍生化的氨基酸，和具有经修饰的肽主链的多肽。该术语包括融合蛋白，包括但不限于具有异源氨基酸序列的融合蛋白，具有异源和同源前导序列(具有或没有N端甲硫氨酸残基)的融合物；等等。
关于参照多肽序列的“氨基酸序列同一性百分数(％)”定义为在必要时引入缺口以实现最大百分比序列同一性后，且不将任何保守替代视为序列同一性的一部分时，候选序列中与参照多肽序列中的氨基酸残基相同的氨基酸残基的百分率。为测定百分比氨基酸序列同一性目的的对比可以以本领域技术范围内的多种方式实现，例如使用公众可得到的计算机软件，诸如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员能决定用于比对序列的适宜参数，包括对所比较序列全长实现最大对比需要的任何算法。然而，为了本发明的目的，氨基酸序列同一性百分数值是使用序列比较计算机程序ALIGN-2产生的。
在采用ALIGN-2来比较氨基酸序列的情况中，给定氨基酸序列A相对于给定氨基酸序列B的％氨基酸序列同一性(或者可表述为具有或包含相对于、与、或针对给定氨基酸序列B的某一％氨基酸序列同一性的给定氨基酸序列A)如下计算：
分数X/Y乘100
其中X是由序列比对程序ALIGN-2在该程序的A和B比对中评分为相同匹配的氨基酸残基的数目，且其中Y是B中的氨基酸残基的总数。应当领会，在氨基酸序列A的长度与氨基酸序列B的长度不相等的情况下， A相对于B的％氨基酸序列同一性会不等于B相对于A的％氨基酸序列同一性。除非另有明确说明，本文中使用的所有％氨基酸序列同一性值都是依照上一段所述，使用ALIGN-2计算机程序获得的。
如本文中使用的，术语“治疗”指获得期望的药理和/或生理效果。所述效果可以是完全或部分预防疾病或其症状的发生、发作，部分或完全减轻疾病和/或其症状，和/或部分或完全治愈疾病和/或其症状，包括：(a)预防疾病在受试者体内发生或发作，所述受试者可以具有疾病的素因，但是尚未诊断为具有疾病；(b)抑制疾病，即阻滞其形成；和(c)减轻疾病和/或其症状，即引起疾病和/或其症状消退或消失。
术语“个体”、“受试者”和“患者”在本文中可互换使用，指哺乳动物，包括但不限于鼠(大鼠、小鼠)、非人灵长类、人、犬、猫、有蹄动物(例如马、牛、绵羊、猪、山羊)等。
“治疗有效量”或“有效量”指在对哺乳动物或其它受试者施用以治疗疾病时足以实现对疾病的所述预防和/或治疗的纤维蛋白溶酶原激活途径的组分或其相关化合物(例如纤溶酶原)的量。“治疗有效量”会根据所使用的纤维蛋白溶酶原激活途径的组分或其相关化合物(例如纤溶酶原)、要治疗的受试者的疾病和/或其症状的严重程度以及年龄、体重等而变化。
本发明纤溶酶原的制备
纤溶酶原可以从自然界分离并纯化用于进一步的治疗用途，也可以通过标准的化学肽合成技术来合成。当通过化学合成多肽时，可以经液相或固相进行合成。固相多肽合成(SPPS)(其中将序列的C末端氨基酸附接于不溶性支持物，接着序贯添加序列中剩余的氨基酸)是适合纤溶酶原化学合成的方法。各种形式的SPPS，诸如Fmoc和Boc可用于合成纤溶酶原。用于固相合成的技术描述于Barany和Solid-Phase Peptide Synthesis；第3-284页于The Peptides:Analysis,Synthesis,Biology.第2卷：Special Methods in Peptide Synthesis,Part A.,Merrifield,等J.Am.Chem.Soc.,85:2149-2156(1963)；Stewart等,Solid Phase Peptide Synthesis,2nd ed.Pierce Chem.Co.,Rockford,Ill.(1984)；和Ganesan A.2006 Mini Rev.Med Chem.6:3-10和Camarero JA等2005 Protein Pept Lett.12:723-8中。简言之，用其上构建有肽链的功能性单元处理小的不溶性多孔珠。在偶联/去保护的重复循环后， 将附接的固相游离N末端胺与单个受N保护的氨基酸单元偶联。然后，将此单元去保护，露出可以与别的氨基酸附接的新的N末端胺。肽保持固定在固相上，之后将其切掉。
可以使用标准重组方法来生产本发明的纤溶酶原。例如，将编码纤溶酶原的核酸插入表达载体中，使其与表达载体中的调控序列可操作连接。表达调控序列包括但不限于启动子(例如天然关联的或异源的启动子)、信号序列、增强子元件、和转录终止序列。表达调控可以是载体中的真核启动子系统，所述载体能够转化或转染真核宿主细胞(例如COS或CHO细胞)。一旦将载体掺入合适的宿主中，在适合于核苷酸序列的高水平表达及纤溶酶原的收集和纯化的条件下维持宿主。
合适的表达载体通常在宿主生物体中作为附加体或作为宿主染色体DNA的整合部分复制。通常，表达载体含有选择标志物(例如氨苄青霉素抗性、潮霉素抗性、四环素抗性、卡那霉素抗性或新霉素抗性)以有助于对外源用期望的DNA序列转化的那些细胞进行检测。
大肠杆菌(Escherichia coli)是可以用于克隆主题抗体编码多核苷酸的原核宿主细胞的例子。适合于使用的其它微生物宿主包括杆菌，诸如枯草芽孢杆菌(Bacillus subtilis)和其他肠杆菌科(enterobacteriaceae)，诸如沙门氏菌属(Salmonella)、沙雷氏菌属(Serratia)、和各种假单胞菌属(Pseudomonas)物种。在这些原核宿主中，也可以生成表达载体，其通常会含有与宿主细胞相容的表达控制序列(例如复制起点)。另外，会存在许多公知的启动子，诸如乳糖启动子系统，色氨酸(trp)启动子系统，beta-内酰胺酶启动子系统，或来自噬菌体λ的启动子系统。启动子通常会控制表达，任选在操纵基因序列的情况中，并且具有核糖体结合位点序列等，以启动并完成转录和翻译。
其他微生物，诸如酵母也可用于表达。酵母(例如酿酒酵母(S.cerevisiae))和毕赤酵母(Pichia)是合适的酵母宿主细胞的例子，其中合适的载体根据需要具有表达控制序列(例如启动子)、复制起点、终止序列等。典型的启动子包含3-磷酸甘油酸激酶和其它糖分解酶。诱导型酵母启动于特别包括来自醇脱氢酶、异细胞色素C、和负责麦芽糖和半乳糖利用的酶的启动子。
在微生物外，哺乳动物细胞(例如在体外细胞培养物中培养的哺乳动物细胞)也可以用于表达并生成本发明的抗-Tau抗体(例如编码主题抗-Tau抗体的多核苷酸)。参见Winnacker,From Genes to Clones,VCH Publishers,N.Y.,N.Y.(1987)。合适的哺乳动物宿主细胞包括CHO细胞系、各种Cos细胞系、HeLa细胞、骨髓瘤细胞系、和经转化的B细胞或杂交瘤。用于这些细胞的表达载体可以包含表达控制序列，如复制起点，启动子和增强子(Queen等,Immunol.Rev.89:49(1986))，以及必需的加工信息位点，诸如核糖体结合位点，RNA剪接位点，多聚腺苷酸化位点，和转录终止子序列。合适的表达控制序列的例子是白免疫球蛋白基因、SV40、腺病毒、牛乳头瘤病毒、巨细胞病毒等衍生的启动子。参见Co等,J.Immunol.148:1149(1992)。
一旦合成(化学或重组方式)，可以依照本领域的标准规程，包括硫酸铵沉淀，亲和柱，柱层析，高效液相层析(HPLC)，凝胶电泳等来纯化本发明所述的纤溶酶原。该纤溶酶原是基本上纯的，例如至少约80％至85％纯的，至少约85％至90％纯的，至少约90％至95％纯的，或98％至99％纯的或更纯的，例如不含污染物，所述污染物如细胞碎片，除目标产物以外的大分子，等等。
药物配制剂
可以通过将具有所需纯度的纤维蛋白溶酶原激活途径的组分或其相关化合物(例如纤溶酶原)与可选的药用载体，赋形剂，或稳定剂(Remington's Pharmaceutical Sciences,16版，Osol,A.ed.(1980))混合形成冻干制剂或水溶液制备治疗配制剂。可接受的载体、赋形剂、稳定剂在所用剂量及浓度下对受者无毒性，并包括缓冲剂例如磷酸盐，柠檬酸盐及其它有机酸；抗氧化剂包括抗坏血酸和蛋氨酸；防腐剂(例如十八烷基二甲基苄基氯化铵；氯化己烷双胺；氯化苄烷铵(benzalkonium chloride)，苯索氯铵；酚、丁醇或苯甲醇；烷基对羟基苯甲酸酯如甲基或丙基对羟基苯甲酸酯；邻苯二酚；间苯二酚；环己醇；3-戊醇；间甲酚)；低分子量多肽(少于约10个残基)；蛋白质如血清白蛋白，明胶或免疫球蛋白；亲水聚合物如聚乙烯吡咯烷酮；氨基酸如甘氨酸，谷氨酰胺、天冬酰胺、组氨酸、精氨酸或赖氨酸；单糖，二糖及其它碳水化合物包括葡萄糖、甘露糖、或糊精； 螯合剂如EDTA；糖类如蔗糖、甘露醇、岩藻糖或山梨醇；成盐反离子如钠；金属复合物(例如锌-蛋白复合物)；和/或非离子表面活性剂，例如TWEENTM，PLURONICSTM或聚乙二醇(PEG)。优选冻干的抗-VEGF抗体配制剂在WO 97/04801中描述，其包含在本文中作为参考。
本发明的配制剂也可含有需治疗的具体病症所需的一种以上的活性化合物，优选活性互补并且相互之间没有副作用的那些。
本发明的纤溶酶原可包裹在通过诸如凝聚技术或界面聚合而制备的微胶囊中，例如，可置入在胶质药物传送系统(例如，脂质体，白蛋白微球，微乳剂，纳米颗粒和纳米胶囊)中或置入粗滴乳状液中的羟甲基纤维素或凝胶-微胶囊和聚-(甲基丙烯酸甲酯)微胶囊中。这些技术公开于Remington's Pharmaceutical Sciences 16th edition,Osol,A.Ed.(1980)。
用于体内给药的本发明的纤维蛋白溶酶原激活途径的组分或其相关化合物(例如纤溶酶原)必需是无菌的。这可以通过在冷冻干燥和重新配制之前或之后通过除菌滤膜过滤而轻易实现。
本发明的纤维蛋白溶酶原激活途径的组分或其相关化合物(例如纤溶酶原)可制备缓释制剂。缓释制剂的适当实例包括具有一定形状且含有糖蛋白的固体疏水聚合物半通透基质，例如膜或微胶囊。缓释基质实例包括聚酯、水凝胶(如聚(2-羟基乙基-异丁烯酸酯)(Langer等，J.Biomed.Mater.Res.,15:167-277(1981)；Langer,Chem.Tech.,12:98-105(1982))或聚(乙烯醇)，聚交酯(美国专利3773919,EP 58,481)，L-谷氨酸与γ乙基-L-谷氨酸的共聚物(Sidman，等，Biopolymers 22:547(1983))，不可降解的乙烯－乙烯乙酸酯(ethylene-vinyl acetate)(Langer，等，出处同上)，或可降解的乳酸-羟基乙酸共聚物如Lupron DepotTM(由乳酸-羟基乙酸共聚物和亮氨酰脯氨酸(leuprolide)乙酸酯组成的可注射的微球体)，以及聚D-(-)-3-羟丁酸。聚合物如乙烯-乙酸乙烯酯和乳酸-羟基乙酸能持续释放分子100天以上，而一些水凝胶释放蛋白的时间却较短。可以根据相关机理来设计使蛋白稳定的合理策略。例如，如果发现凝聚的机理是通过硫代二硫键互换而形成分子间S-S键，则可通过修饰巯基残基、从酸性溶液中冻干、控制湿度、采用合适的添加剂、和开发特定的聚合物基质组合物来实现稳定。
给药和剂量
可以通过不同方式，例如通过鼻腔吸入、雾化吸入、滴鼻液或滴眼液，静脉内，腹膜内、皮下、颅内、鞘内、动脉内(例如经由颈动脉)、肌内、直肠给药来实现本发明药物组合物的施用。
用于胃肠外施用的制备物包括无菌水性或非水性溶液、悬浮液和乳剂。非水性溶剂的例子是丙二醇、聚乙二醇、植物油如橄榄油，和可注射有机酯，如油酸乙酯。水性载体包括水、醇性/水性溶液、乳剂或悬浮液，包括盐水和缓冲介质。胃肠外媒介物包含氯化钠溶液、林格氏右旋糖、右旋糖和氯化钠、或固定油。静脉内媒介物包含液体和营养补充物、电解质补充物，等等。也可以存在防腐剂和其他添加剂，诸如例如，抗微生物剂、抗氧化剂、螯合剂、和惰性气体，等等。
医务人员会基于各种临床因素确定剂量方案。如医学领域中公知的，任一患者的剂量取决于多种因素，包括患者的体型、体表面积、年龄、要施用的具体化合物、性别、施用次数和路径、总体健康、和同时施用的其它药物。本发明包含纤溶酶原的药物组合物的剂量范围可以例如为每天约0.0001至2000mg/kg，或约0.001至500mg/kg(例如0.02mg/kg，0.25mg/kg，0.5mg/kg，0.75mg/kg，10mg/kg，50mg/kg等等)受试者体重。例如，剂量可以是1mg/kg体重或50mg/kg体重或在1-50mg/kg的范围，或至少1mg/kg。高于或低于此例示性范围的剂量也涵盖在内，特别是考虑到上述的因素。上述范围中的中间剂量也包含在本发明的范围内。受试者可以每天、隔天、每周或根据通过经验分析确定的任何其它日程表施用此类剂量。例示性的剂量日程表包括连续几天0.01-100mg/kg。在本发明的药物施用过程中需要实时评估治疗效果和安全性。
制品或药盒
本发明的一个实施方案涉及一种制品或药盒，其包含纤维蛋白溶酶原激活途径的组分或其相关化合物(例如纤溶酶原)。所述制品优选包括一个容器，标签或包装插页。适当的容器有瓶子，小瓶，注射器等。容器可由各种材料如玻璃或塑料制成。所述容器含有组合物，所述组合物可有效治疗本发明的疾病或病症并具有无菌入口(例如所述容器可为静脉内溶液包或小瓶，其含有可被皮下注射针穿透的塞子的)。所述组合物中至少一种活性剂为纤维蛋白溶酶原激活途径的组分或其相关化合物(例如纤溶酶 原)。所述容器上或所附的标签说明所述组合物用于治疗本发明所述病症。所述制品可进一步包含含有可药用缓冲液的第二容器，诸如磷酸盐缓冲的盐水，林格氏溶液以及葡萄糖溶液。其可进一步包含从商业和使用者角度来看所需的其它物质，包括其它缓冲液，稀释剂，过滤物，针和注射器。此外，所述制品包含带有使用说明的包装插页，包括例如指示所述组合物的使用者将包含纤维蛋白溶酶原激活途径的组分或其相关化合物(例如纤溶酶原)的组合物以及治疗伴随的疾病的其它药物给药患者。
实施例
以下所有实施例中使用的人纤溶酶原来自捐赠者血浆，基于如下文献描述的方法：KennethC Robbins,Louis Summaria,David Elwyn et al.Further Studies on the Purification and Characterization of Human Plasminogen and Plasmin.Journal of Biological Chemistry,1965,240(1):541-550；Summaria L,Spitz F,Arzadon L et al.Isolation and characterization of the affinity chromatography forms of human Glu-and Lys-plasminogens and plasmins.J Biol Chem.1976 Jun 25；251(12):3693-9；HAGAN JJ,ABLONDI FB,DE RENZO EC.Purification and biochemical properties of human plasminogen.J Biol Chem.1960 Apr；235:1005-10，并进行工艺优化，从人捐赠者血浆中纯化所得。纤溶酶原单体的纯度>98％。
实施例1纤溶酶原改善帕金森模型小鼠自发活动及趋避性行为
取10-12周龄雄性C57BL/6J小鼠28只，造模前1天所有小鼠称重并根据体重随机分为2组，空白对照组8只，模型组20只。空白对照组小鼠腹腔注射生理盐水溶液200μl，模型组小鼠按照35mg/kg/只腹腔注射1-甲基-4-苯基-1,2,3,6-四氢吡啶(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine，MPTP)溶液，连续注射5天，建立帕金森模型 [1]。MPTP溶液配制：取45mg MPTP(Sigma，M0896)溶解在9ml生理盐水溶液中，配制最终浓度为5mg/ml。造模完成后即造模第6天所有小鼠进行体重测量及旷场行为学检测。模型组小鼠按体重及旷场结果随机分为2组，给药组10只小鼠和溶媒组10只小鼠，并开始给药，记为第1天，给药组小鼠尾静脉注射1mg/100μl/只纤溶酶原溶液，溶媒组注射100μl/只溶媒溶液(10mM柠檬酸-柠檬酸钠溶液，pH7.4)，持续给药14天，于给药第15天进行旷场实验。
MPTP是一种特定的强黑质毒素，其代谢产物MPP+(1-methyl-4-phenylpyridinium)是线粒体复合体Ⅰ抑制剂，可透过血脑屏障，阻断线粒体氧化呼吸链等途径损伤黑质体和纹状体中多巴胺神经元，模拟人类帕金森症状 [2]。
旷场实验
实验时，小鼠放入旷场(40×40×40cm)底面中心，同时进行摄像和计时，持续观察5分钟，每只小鼠进行3次实验。记录参数包括总移动距离、边界静息时间率、边界区运动路程、边界区运动路程百分率、边界区慢速运动时间百分率、边界区时间百分率、进入边界区次数、中心区运动路程、中心区运动路程百分率、中心区最大运动速度、中心区时间百分率、进入中心区次数、运动轨迹。每次实验后采用70％酒精擦拭箱体防止嗅觉产生的偏好。
旷场实验的设计原理是基于小鼠的趋避性，指的是小鼠畏惧开阔、未知、可能存在潜在危险的场所，因而其有“贴墙”活动的天性。总路程和平均速度被视为反映小鼠自发活动的主要数据，趋避性是以小鼠在旷野周边区(四个角和四个边)的活动来评价的。从反映趋避性的周边区活动时间看，时间减少，说明小鼠更富有“冒险”倾向。在中央区活动时间显著增多，说明趋避性和焦虑(抑郁)水平较低。
总体活动情况
总运动路程
总运动路程是指规定测试时间内运动轨迹的长度。
结果显示，空白对照组在实验期间会运动一定路程；给纤溶酶原组小鼠总运动路程明显短于溶媒对照组，统计差异显著(*表示P<0.05)，且给纤溶酶原组总运动路程接近于空白对照组(图1)。说明纤溶酶原可使帕金森模型小鼠自发活动行为恢复。
边界区活动情况
边界区静息时间率
边界区为旷野周边区(四个角和四个边)。边界区静息时间率是指：边界区静息时间与总静息时间(包括边界区静息时间和中心区静息时间)的比值。结果显示，空白对照组小鼠具有一定的边界区静息时间率；给纤 溶酶原组小鼠边界区静息时间率明显大于溶媒对照组，统计差异极为显著(**表示P<0.01)，且给纤溶酶原组边界静息时间率接近于空白对照组(图2)。说明纤溶酶原可增强帕金森模型小鼠趋避性行为，缓解其焦虑。
边界区运动路程
边界区运动路程指规定测试时间内边界区运动轨迹的长度。结果显示，空白对照组小鼠具有一定的边界运动路程；溶媒对照组小鼠边界运动路程长于给纤溶酶原组，统计差异接近显著(P＝0.05)；且给纤溶酶原组边界区运动路程接近空白对照组(图3)。说明纤溶酶原可增强帕金森模型小鼠的趋避性，缓解其焦虑。
边界区运动路程百分率
边界区运动路程百分率指边界区运动路程与总运动路程(包括边界区运动路程和中心区运动路程)的比值。
结果显示，空白对照组小鼠具有一定的边界运动路程百分率；给药纤溶酶原组小鼠边界区运动路程百分率明显高于溶媒对照组，统计差异极为显著(**表示P<0.01)；且给药纤溶酶原组小鼠边界运动路程百分率接近空白对照组(图4)。说明纤溶酶原可增强帕金森模型小鼠趋避性行为，缓解其焦虑。
边界区慢速运动时间百分率
边界区慢速运动时间百分率指测试时间内边界区慢速运动的时间与总测试时间的比值。
结果显示，空白对照组小鼠具有一定的边界区慢速运动时间百分率；给纤溶酶原组小鼠边界慢速运动时间百分率明显小于溶媒对照组，统计差异极为显著(**表示P<0.01)；且给纤溶酶原组边界慢速运动时间百分率接近于空白对照组(图5)。说明纤溶酶原可增强帕金森模型小鼠趋避性，缓解其焦虑。
边界区时间百分率
边界区时间百分率指边界区时间与总的测试时间的比值。
结果显示，空白对照组小鼠具有一定的边界区时间百分率；给纤溶酶原组小鼠边界区时间百分率明显高于溶媒对照组小鼠，统计差异接近显著 (P＝0.06)，且给药纤溶酶原组边界区时间百分率接近空白对照组(图6)。说明纤溶酶原可提高帕金森模型小鼠趋避性行为水平，缓解其焦虑。
进入边界区次数
结果显示，空白对照组小鼠具有一定进入边界区次数；给纤溶酶原组小鼠进入边界区次数明显低于溶媒对照组小鼠，统计差异显著(*表示P<0.05)；且给药纤溶酶原组小鼠进入边界区的次数接近空白对照组(图7)。说明纤溶酶原可增强帕金森模型小鼠趋避性，缓解其焦虑。
中心区活动情况
中心区运动路程
中心区运动路程指测试时间内中心区运动轨迹的长度。
结果显示，空白对照组小鼠具有一定的中心区运动路程；给药纤溶酶原组小鼠中心区运动路程明显低于溶媒对照组小鼠，统计差异显著(*表示P<0.05)；且给药纤溶酶原组小鼠中心区运动路程接近空白对照组(图8)。说明纤溶酶原可增强帕金森模型小鼠的趋避性，缓解其焦虑。
中心区运动路程百分率
中心区运动路程百分率指测试时间内中心区运动轨迹的长度与总的运动轨迹长度的比值。
结果显示，空白对照组小鼠具有一定的中心区运动路程百分率；给药纤溶酶原组小鼠中心区运动路程百分率明显低于溶媒对照组小鼠，统计差异显著(*表示P<0.05)；且给药纤溶酶原组小鼠中心区运动路程百分率接近空白对照组(图9)。说明纤溶酶原可增强帕金森模型小鼠趋避性，缓解其焦虑。
中心区最大运动速度
中心区最大运动速度指测试时间内中心区的最快运动速度。
结果显示，空白对照组小鼠具有一定的中心区最大运动速度；给药纤溶酶原组小鼠中心区最大运动速度明显小于溶媒对照组，统计差异极为明显(**表示P<0.01)，且给纤溶酶原组中心区最大运动速度接近于空白对照组(图10)。说明纤溶酶原可增强帕金森模型小鼠趋避性，缓解其焦虑。
中心区时间百分率
中心区时间百分率指中心区的运动时间与总测试时间的比值。
结果显示，空白对照组小鼠具有一定地进入中心区时间百分率；给药纤溶酶原组小鼠中心区时间百分率明显小于溶媒对照组，统计差异接近显著(P＝0.06)；且给纤溶酶原组中心区时间百分率接近于空白对照组(图11)。说明纤溶酶原可增强帕金森模型小鼠趋避性，缓解其焦虑。
进入中心区次数
结果显示，空白对照组小鼠很少进入中心区，表现出正常的趋避天性；给药纤溶酶原组小鼠进入中心区次数明显少于溶媒对照组，统计差异明显(*表示P<0.05)；且给药纤溶酶原组小鼠进入中心区次数接近空白对照组(图12)。说明纤溶酶原可增强帕金森模型小鼠趋避性，缓解其焦虑。
运动轨迹
结果显示，旷场实验期间空白对照组小鼠具有一定的总运动路程并且几乎没有中心区活动；相比于溶媒对照组，给药纤溶酶原组小鼠表现出总运动路程和中心区活动均有明显减少的趋势，且接近于空白对照组小鼠(图13)。说明纤溶酶原可增强帕金森模型小鼠趋避性，缓解其焦虑。
实施例2纤溶酶原可促进帕金森模型小鼠黑质DTA表达
取10-12周龄雄性C57BL/6J小鼠40只，造模前1天所有小鼠称重并根据体重随机分为2组，空白对照组8只，模型组32只。空白对照组小鼠腹腔注射溶媒溶液200μl，模型组小鼠按照40mg/kg/只腹腔注射1-甲基-4-苯基-1,2,3,6-四氢吡啶(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine，MPTP)溶液，连续注射5天，建立帕金森模型 [1]。MPTP溶液配制：取45mg MPTP(Sigma，M0896)溶解在9ml生理盐水溶液中，配制最终浓度为5mg/ml。造模完成后即造模第6天，模型组小鼠按体重随机分为两组，溶媒组和给药组，每组各16只，并开始给药，记为第1天，给药组小鼠尾静脉注射1mg/100μl/只纤溶酶原溶液，溶媒组注射100μl/只溶媒溶液(10mM柠檬酸-柠檬酸钠溶液，pH7.4)，持续给药14天，于给药第15天处死，取材小鼠黑质于10％中性甲醛溶液固定24-48小时。固定后的黑质组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。切片厚度为3μm，切片脱蜡复水后水洗1次。PAP笔圈出组织，以3％双氧水孵育15分钟， 0.01MPBS洗2次，每次5分钟。5％的正常羊血清液(Vector laboratories,Inc.,USA)封闭30分钟；时间到后，弃除羊血清液，滴加兔抗鼠多巴胺转运蛋白(dopamine transporter，DAT)抗体(ab7260,Abcam)4℃孵育过夜，0.01M PBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，0.01M PBS洗2次，每次5分钟。按DAB试剂盒(Vector laboratories,Inc,USA)显色，水洗3次后苏木素复染30秒，流水冲洗5分钟。梯度酒精脱水，二甲苯透明并中性树胶封片，切片在400倍光学显微镜下观察。
帕金森病(Parkinson disease，PD)是一种神经退行性疾病，其病理特征为黑质纹状体多巴胺能神经元进行性死亡，以及残存的多巴胺能神经元胞浆内路易小体形成，基底节区多巴胺缺乏，进而导致发生PD经典运动障碍症状。多巴胺转运蛋白(dopamine transporter，DAT)位于多巴胺神经元突触前膜，能重新摄取释放至突触间隙的多巴胺递质，反映多巴胺神经元突触前功能。DAT减少与PD发展关系密切 [3]。
结果显示，空白对照组(图14A)小鼠黑质多巴胺神经元表达一定量的DTA(箭头标识)，溶媒组(图14B)小鼠黑质DTA的表达量低于空白对照组，给药纤溶酶原组(图14C)小鼠黑质DTA的表达量高于溶媒组。该结果表明纤溶酶原可增强帕金森模型小鼠黑质DTA的表达。
实施例3纤溶酶原可促使帕金森模型小鼠纹状体尼氏体恢复
取10-12周龄雄性C57BL/6J小鼠40只，造模前1天所有小鼠称重并根据体重随机分为2组，空白对照组8只，模型组32只。空白对照组小鼠腹腔注射溶媒溶液200μl，模型组小鼠按照40mg/kg/只腹腔注射1-甲基-4-苯基-1,2,3,6-四氢吡啶(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine，MPTP)溶液，连续注射5天，建立帕金森模型 [1]。MPTP溶液配制：取45mg MPTP(Sigma，M0896)溶解在9ml生理盐水溶液中，配制最终浓度为5mg/ml。造模完成后即造模第6天，模型组小鼠按体重随机分为两组，溶媒组和给药组，每组各16只，并开始给药，记为第1天，给药组小鼠尾静脉注射1mg/100μl/只纤溶酶原溶液，溶媒组注射100μl/只溶媒溶液(10mM柠檬酸-柠檬酸钠溶液，pH7.4)，持续给药14天，于给药第15天处死，取材小鼠纹状体于10％中性甲醛溶液固定24-48小时。固定后的纹 状体组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，脱蜡至水后，用0.4％焦油紫染液(pH＝3)进行染色。梯度酒精脱水，二甲苯透明，中性树胶封片。切片在400倍光学显微镜下观察拍照。
PD主要的病理变化是黑质纹状体多巴胺能神经元进行性变性坏死，导致纹状体多巴胺递质水平下降，从而引起整个基底神经节环路功能紊乱 [3]。尼氏体又称嗜染质，是神经细胞所特有的结构，由许多平行排列的粗面内质网和分布期间的游离核糖体组成，具有合成蛋白的功能，其数量和分布与神经元的功能状态密切相关，被视为神经细胞存活的标志 [4]。结果显示，空白对照组(图15A)小鼠纹状体神经元存在一定数量的尼氏体(箭头标识)；溶媒对照组(图15B)小鼠纹状体神经元尼氏体数量明显多于空白对照组，且统计差异接近显著(P＝0.085)；给纤溶酶原组(图15C)小鼠纹状体神经元尼氏体数量与空白对照组无明显差异，但明显低于溶媒对照组，且统计差异显著(*表示P<0.05)(图15D)。该结果表明纤溶酶原能够促进帕金森模型小鼠纹状体尼氏体恢复。
实施例4纤溶酶原可促使帕金森模型小鼠黑质尼氏体数量恢复
取10～12周龄C57BL/6J雄性小鼠28只，造模前1天称重，根据体重随机分为2组，空白对照组8只，模型组20只。造模时间定在每日早上9点，空白对照组腹腔注射200μl生理盐水；模型组腹腔注射35mg/kg/只5mg/ml MPTP溶液，连续注射5天，建立帕金森模型 [5]。MPTP溶液配制：取45mg MPTP(sigma，M0896)溶解在9ml生理盐水溶液中，配制最终浓度为5mg/ml。造模完成后即造模第6天，模型组小鼠按体重随机分为两组，溶媒组和给药组，每组各10只，并开始给药，记为第1天，给药组小鼠尾静脉注射1mg/100μl/只纤溶酶原溶液，溶媒组注射100μl/只溶媒溶液(10mM柠檬酸-柠檬酸钠溶液，pH7.4)，持续给药14天，于给药第15天处死，取材小鼠黑质于10％中性甲醛溶液固定24-48小时。固定后的黑质组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，脱蜡至水后，用0.4％焦油紫染液(pH＝3)进行染色。梯度酒精脱水，二甲苯透明，中性树胶封片。切片在400倍光学显微镜下观察拍照。
结果显示，空白对照组(图16A)小鼠黑质中有一定量的尼氏体(箭头标识)，溶媒组(图16B)小鼠黑质中尼氏体数减少，给纤溶酶原组 (图16C)小鼠黑质尼氏体数高于溶媒组。该结果表明纤溶酶原可促进帕金森模型小鼠黑质尼氏体数量恢复。
实施例5纤溶酶原可促进帕金森模型小鼠黑质GLP-1R的表达
取9周龄C57雄性小鼠12只，造模前1天称重，小鼠按照30mg/kg体重每天腹腔注射5mg/ml MPTP溶液，连续注射5天，建立帕金森模型 [6-7]。MPTP溶液配制：用注射器吸取10ml去离子水，加入到100mg MPTP粉末(sigma，M0896)中，配制成10mg/ml的母液，然后吸取1ml母液于安瓶中，再加入1ml去离子水，最终浓度为5mg/ml。造模完成后小鼠随机分为两组，给溶媒PBS对照组和给纤溶酶原组各6只小鼠，并开始给药，记为第1天，给纤溶酶原组小鼠按照1mg/0.1ml/只/天尾静脉注射纤溶酶原溶液，给溶媒PBS对照组尾静脉给予相同体积PBS，持续给药14天。于给药第15天处死小鼠，快速取材大脑于4％多聚甲醛固定24-48小时。固定后的脑组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。定位切片黑质，切片厚度为4μm，切片脱蜡复水后水洗1次。PAP笔圈出组织，以3％双氧水孵育15分钟，0.01M PBS洗2次，每次5分钟。5％的正常羊血清液(Vector laboratories,Inc.,USA)封闭30分钟；时间到后，弃除羊血清液，滴加兔抗小鼠GLP-1R抗体(NOVUS,NBP1-97308)4℃孵育过夜，0.01M PBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，0.01M PBS洗2次，每次5分钟。按DAB试剂盒(Vector laboratories,Inc.,USA)显色，水洗3次后苏木素复染30秒，流水冲洗5分钟。梯度酒精脱水，二甲苯透明并中性树胶封片，切片在200倍光学显微镜下观察。
胰高血糖素样肽1受体(glucagon-like peptide 1receptor，GLP-1R)是胰高血糖素受体家族成员之一，是一种G蛋白偶联受体，能通过促进胰岛素的分泌调节血糖水平 [8-9]。帕金森氏病的特点是黑质纹状体神经元多巴胺能信号缺失，而黑质纹状体也表达GLP-1R [10]。
结果显示，给纤溶酶原组(图17B)小鼠黑质GLP-1R的表达量(箭头标识)明显多于溶媒PBS对照组(图17A)，且统计差异显著(*表示P<0.05)(图17C)。该结果表明，纤溶酶原能够增强帕金森模型小鼠黑质GLP-1R的表达。
实施例6纤溶酶原可促进帕金森模型小鼠黑质TH的表达
取9周龄C57雄性小鼠12只，造模前1天称重，小鼠按照30mg/kg体重每天腹腔注射5mg/ml MPTP溶液，连续注射5天，建立帕金森模型 [6-7]。MPTP溶液配制：用注射器吸取10ml去离子水，加入到100mg MPTP粉末(sigma，M0896)中，配制成10mg/ml的母液，然后吸取1ml母液于安瓶中，再加入1ml去离子水，最终浓度为5mg/ml。造模完成后小鼠随机分为两组，给溶媒PBS对照组和给纤溶酶原组各6只小鼠，并开始给药，记为第1天，给纤溶酶原组小鼠按照1mg/0.1ml/只/天尾静脉注射纤溶酶原溶液，给溶媒PBS对照组尾静脉给予相同体积PBS，持续给药14天。于给药第15天处死小鼠，取材小鼠黑质于4％多聚甲醛固定24-48小时。固定后的脑组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。定位切片黑质，切片厚度为3μm，切片脱蜡复水后水洗1次。PAP笔圈出组织，以3％双氧水孵育15分钟，0.01M PBS洗2次，每次5分钟。5％的正常羊血清液(Vector laboratories,Inc.,USA)封闭30分钟；时间到后，弃除羊血清液，滴加兔抗小鼠TH抗体(Proteintech,25859-1-AP)4℃孵育过夜，0.01M PBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，0.01M PBS洗2次，每次5分钟。按DAB试剂盒(Vector laboratories,Inc.,USA)显色，水洗3次后苏木素复染30秒，流水冲洗5分钟。梯度酒精脱水，二甲苯透明并中性树胶封片，切片在400倍光学显微镜下观察。
酪氨酸羟化酶(tyrosine hydroxylase,TH)是酪氨酸合成左旋多巴(L-dopa)的限速酶，仅在胞浆中表达，多巴胺神经元中含量丰富。黑质中TH阳性神经元大多是多巴胺能的，因此TH可作为黑质中多巴胺能神经元的标志物，黑质中TH表达量成为检测PD的指标 [11]。
结果显示，空白对照组(图18A)小鼠黑质中有一定量的TH阳性细胞(箭头标识)，溶媒组(图18B)小鼠黑质中TH阳性细胞数减少，给纤溶酶原组(图18C)小鼠黑质中TH阳性细胞数明显高于溶媒组。该结果表明纤溶酶原可增加帕金森模型小鼠黑质中TH阳性细胞数量。
实施例7纤溶酶原影响帕金森模型小鼠黑质小胶质细胞数量
取9周龄C57雄性小鼠12只，造模前1天称重，小鼠按照30mg/kg体重每天腹腔注射5mg/ml MPTP溶液，连续注射5天，建立帕金森模型 [6-7]。MPTP溶液配制：用注射器吸取10ml去离子水，加入到100mg MPTP粉末(sigma，M0896)中，配制成10mg/ml的母液，然后吸取1ml母液于安瓶中，再加入1ml去离子水，最终浓度为5mg/ml。造模完成后小鼠随机分为两组，给溶媒PBS对照组和给纤溶酶原组各6只小鼠，并开始给药，记为第1天，给纤溶酶原组小鼠按照1mg/0.1ml/只/天尾静脉注射纤溶酶原溶液，给溶媒PBS对照组尾静脉给予相同体积PBS，持续给药14天。于给药第15天处死小鼠，取材小鼠中脑黑质于4％多聚甲醛固定24-48小时。固定后的黑质经酒精梯度脱水和二甲苯透明后进行石蜡包埋。定位切片黑质，切片厚度为3μm，切片脱蜡复水后水洗1次。PAP笔圈出组织，以3％双氧水孵育15分钟，0.01M PBS洗2次，每次5分钟。5％的正常羊血清液(Vector laboratories,Inc.,USA)封闭30分钟；时间到后，弃除羊血清液，滴加兔抗小鼠Iba-1抗体(Abcam,ab178847)4℃孵育过夜，0.01M PBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，0.01M PBS洗2次，每次5分钟。按DAB试剂盒(Vector laboratories,Inc.,USA)显色，水洗3次后苏木素复染30秒，流水冲洗5分钟。梯度酒精脱水，二甲苯透明并中性树胶封片，切片在400倍光学显微镜下观察。
小胶质细胞是中枢神经系统固有免疫细胞，在脑病变或损伤时被活化。活化的小胶质细胞迁移到损伤部位，发挥多种功能，例如吞噬死细胞、增加促炎性细胞因子等，参与各种中枢神经系统疾病 [12-14]。Iba-1(Ionized calcium binding adapter molecule 1)是一约17kDa的钙结合蛋白，在中枢神经系统小胶质细胞中特异性表达，已被广泛用作小胶质细胞标记物 [15-17]。
结果显示，空白对照组(图19A)小鼠黑质有一定数量的小胶质细胞(箭头标识)，溶媒对照组(图19B)小鼠黑质中小胶质细胞数高于空白对照组，给纤溶酶原组(图19C)小鼠黑质中小胶质细胞数明显低于溶媒对照组，且接近于空白对照组。该结果显示纤溶酶原影响帕金森模型小鼠黑质小胶质细胞数量。
实施例8纤溶酶原可促进帕金森模型小鼠纹状体髓鞘恢复
取9周龄C57雄性小鼠12只，造模前1天称重，小鼠按照30mg/kg体重每天腹腔注射5mg/ml MPTP溶液，连续注射5天，建立帕金森模型 [6-7]。MPTP溶液配制：用注射器吸取10ml去离子水，加入到100mg MPTP粉末(sigma，M0896)中，配制成10mg/ml的母液，然后吸取1ml母液于安瓶中，再加入1ml去离子水，最终浓度为5mg/ml。造模完成后小鼠随机分为两组，给溶媒PBS对照组和给纤溶酶原组各6只，并开始给药，记为第1天，给纤溶酶原组小鼠按照1mg/0.1ml/只/天尾静脉注射纤溶酶原溶液，给溶媒PBS对照组尾静脉给予相同体积PBS，持续给药14天。于给药第15天处死小鼠，取材小鼠纹状体于10％中性甲醛溶液固定24-48小时。固定后的纹状体组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。组织切片厚度为3μm，脱蜡至水后，用0.1％LFB染液进行染色密封浸染8-16h。梯度酒精脱水，二甲苯透明，中性树胶封片。切片在400倍光学显微镜下观察拍照。
神经退行性疾病是指由大脑和脊髓的神经元或其髓鞘的丧失所导致的疾病。LFB(Luxol fast blue)是一种髓鞘特异性染色方法，可反映髓鞘受损情况 [18-19]。
结果显示，空白对照组(图20A)小鼠纹状体存在一定量的髓鞘，溶媒组(图20B)小鼠纹状体髓鞘染色少于空白对照组，给纤溶酶原组(图20C)小鼠纹状体髓鞘染色明显多于溶媒组，且接近空白对照组。该结果显示纤溶酶原可使帕金森模型小鼠纹状体髓鞘一定程度地恢复。
实施例9纤溶酶原可减少帕金森模型小鼠黑质α-突触核蛋白表达
取10～12周龄C57BL/6J雄性小鼠28只，造模前1天称重，根据体重随机分为2组，空白对照组8只，模型组20只。造模时间定在每日早上9点，空白对照组腹腔注射200μL生理盐水；模型组腹腔注射35mg/kg/只5mg/mL MPTP溶液，连续注射5天，建立帕金森模型 [5]。MPTP溶液配制：取45mg MPTP(sigma，M0896)溶解在9mL生理盐水溶液中，配制最终浓度为5mg/mL。造模完成后即造模第6天，模型组小鼠按体重随机分为两组，溶媒组和给药组，每组各10只，并开始给药，记为第1天，给药组小鼠尾静脉注射1mg/100μL/只纤溶酶原溶液，溶媒组注射100μL/只溶媒 溶液(10mM柠檬酸-柠檬酸钠溶液，pH7.4)，持续给药14天，于给药第15天处死，取材小鼠黑质于4％多聚甲醛固定24-48小时。固定后的脑组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。定位切片黑质，切片厚度为3μm，切片脱蜡复水后水洗1次。PAP笔圈出组织，以3％双氧水孵育15分钟，0.01M PBS洗2次，每次5分钟。5％的正常羊血清液(Vector laboratories,Inc.,USA)封闭30分钟；时间到后，弃除羊血清液，滴加兔抗小鼠α-synuclein抗体(Proteintech,10842-1-AP)4℃孵育过夜，0.01M PBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam)二抗室温孵育1小时，0.01M PBS洗2次，每次5分钟。按DAB试剂盒(Vector laboratories,Inc.,USA)显色，水洗3次后苏木素复染30秒，流水冲洗5分钟。梯度酒精脱水，二甲苯透明并中性树胶封片，切片在400倍光学显微镜下观察。
目前认为帕金森病是由于中脑黑质多巴胺能神经元缺失及出现路易小体所导致。α-突触核蛋白(α-synuclein)是一个由140个氨基酸残基组成的神经元蛋白，可导致神经元损伤，而且参与中枢神经系统神经变性的过程，研究表明神经细胞路易小体和神经突触内聚合的α-synuclein是帕金森病中大脑病变的标志 [20]。
结果显示，空白对照组(图21A)小鼠黑质仅有少量的α-突触核蛋白(α-synuclein)，溶媒组(图21B)小鼠黑质α-突触核蛋白的量明显高于空白对照组(*表示P<0.05)，给纤溶酶原组(图21C)小鼠黑质α-突触核蛋白的量明显低于溶媒组，且趋近于空白对照组，统计差异显著(*表示P<0.05)(图21D)。说明纤溶酶原可减少帕金森模型小鼠黑质α-突触核蛋白表达，改善神经元损伤变性。
实施例10纤溶酶原可改善帕金森模型小鼠纹状体轴索损伤
取10～12周龄C57BL/6J雄性小鼠28只，造模前1天称重，根据体重随机分为2组，空白对照组8只，模型组20只。造模时间定在每日早上9点，空白对照组腹腔注射200μL生理盐水；模型组腹腔注射35mg/kg/只5mg/mL MPTP溶液，连续注射5天，建立帕金森模型 [5]。MPTP溶液配制：取45mg MPTP(sigma，M0896)溶解在9mL生理盐水溶液中，配制最终浓度为5mg/mL。造模完成后即造模第6天，模型组小鼠按体重随机分 为两组，溶媒组和给药组，每组各10只，并开始给药，记为第1天，给药组小鼠尾静脉注射1mg/100μL/只纤溶酶原溶液，溶媒组注射100μL/只溶媒溶液(10mM柠檬酸-柠檬酸钠溶液，pH7.4)，持续给药14天，于给药第15天处死，取材小鼠黑质于4％多聚甲醛固定24-48小时。固定后的脑组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。定位切片黑质，切片厚度为3μm，切片脱蜡复水后水洗1次。PAP笔圈出组织，以3％双氧水孵育15分钟，0.01M PBS洗2次，每次5分钟。5％的正常羊血清液(Vector laboratories,Inc.,USA)封闭30分钟；时间到后，弃除羊血清液，滴加兔抗小鼠NF抗体(Abcam,ab207176)4℃孵育过夜，0.01M PBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam，ab6721)二抗室温孵育1小时，0.01M PBS洗2次，每次5分钟。按DAB试剂盒(Vector laboratories,Inc.,USA)显色，水洗3次后苏木素复染30秒，流水冲洗5分钟。梯度酒精脱水，二甲苯透明并中性树胶封片，切片在400倍光学显微镜下观察。
帕金森患者疾病过程中存在轴索损伤，且轴索损伤严重程度可能随病程延长持续加重。神经丝蛋白(Neurofilaments，NF)是神经轴索的主要骨架蛋白，对维持神经元正常形态和轴索运输具有至关重要的作用，可作为轴索损伤的相关生物标记物 [21]。
结果显示，空白对照组(图22A)小鼠纹状体有一定量的NF(箭头标识)，溶媒组(图22B)小鼠纹状体NF的量低于空白对照组，给纤溶酶原组(图22C)小鼠纹状体中NF的量明显高于溶媒组，且统计差异极显著(**表示P<0.01)(图22D)。说明纤溶酶原可促使帕金森模型小鼠纹状体NF恢复，修复帕金森轴索损伤。
实施例11纤溶酶原可减少帕金森模型小鼠纹状体GFAP表达
取10～12周龄C57BL/6J雄性小鼠28只，造模前1天称重，根据体重随机分为2组，空白对照组8只，模型组20只。造模时间定在每日早上9点，空白对照组腹腔注射200μL生理盐水；模型组腹腔注射35mg/kg/只5mg/mL MPTP溶液，连续注射5天，建立帕金森模型[5]。MPTP溶液配制：取45mg MPTP(sigma，M0896)溶解在9mL生理盐水溶液中，配制最终浓度为5mg/mL。造模完成后即造模第6天，模型组小鼠按体重随机分为两组，溶媒组和给药组，每组各10只，并开始给药，记为第1天，给药 组小鼠尾静脉注射1mg/100μL/只纤溶酶原溶液，溶媒组注射100μL/只溶媒溶液(10mM柠檬酸-柠檬酸钠溶液，pH7.4)，持续给药14天，于给药第15天处死，取材小鼠黑质于4％多聚甲醛固定24-48小时。固定后的脑组织经酒精梯度脱水和二甲苯透明后进行石蜡包埋。定位切片黑质，切片厚度为3μm，切片脱蜡复水后水洗1次。PAP笔圈出组织，以3％双氧水孵育15分钟，0.01M PBS洗2次，每次5分钟。5％的正常羊血清液(Vector laboratories,Inc.,USA)封闭30分钟；时间到后，弃除羊血清液，滴加兔抗小鼠GFAP抗体(Abcam,ab7260)4℃孵育过夜，0.01M PBS洗2次，每次5分钟。山羊抗兔IgG(HRP)抗体(Abcam，ab6721)二抗室温孵育1小时，0.01M PBS洗2次，每次5分钟。按DAB试剂盒(Vector laboratories,Inc.,USA)显色，水洗3次后苏木素复染30秒，流水冲洗5分钟。梯度酒精脱水，二甲苯透明并中性树胶封片，切片在400倍光学显微镜下观察。
胶质细胞纤维酸性蛋白(gilaI fibrillary acidic protein,GFAP)是构成星形胶质细胞胞体胶原蛋白的重要成分，且其仅存在于星形胶质细胞胶质原纤维中间丝中，是星形胶质细胞活化的特征性标记物 [22]，对神经元具有炎性损坏作用，使神经元变性 [23]，从而引起帕金森的发生。
结果显示，空白对照组(图23A)小鼠纹状体表达少量的GFAP(箭头标识)，溶媒组(图23B)小鼠纹状体GFAP的表达量明显高于空白对照组，给纤溶酶原组小鼠(图23C)纹状体GFAP的表达量低于溶媒组。说明纤溶酶原可减少帕金森模型小鼠纹状体GFAP表达，减轻纹状体神经元损伤。
实施例12纤溶酶原促进α-突触核蛋白在帕金森模型小鼠脑匀浆中降解
取11～12周龄、18-25g的C57BL/6J雄性小鼠8只，造模前1天称重，根据体重随机分为2组，空白对照组4只，模型组4只。造模时间定在每日早上9点，空白对照组腹腔注射200μL生理盐水；模型组腹腔注射35mg/kg/只5mg/mL MPTP溶液，连续注射5天，建立帕金森模型 [5]。MPTP溶液配制：取45mg MPTP(sigma，M0896)溶解在9mL生理盐水溶液中，配制最终浓度为5mg/mL。造模完成后即造模第6天，所有小鼠进行旷场实验，鉴定造模成功。所有小鼠处死后取整个脑部并称重，按150mg组织/mLPBS分别加入1×PBS(Thermo Fisher，pH7.4；10010- 031，4℃下匀浆(1min，3-4次)，匀浆后于4℃离心(12000rpm，20min)，取上清液置于新的EP管中。
取Eppendorf(EP)管分别设为①空白对照组、②溶媒对照组、③纤溶酶原组，各组设置5个平行。空白对照组加入21.5μL生理盐水、4.6μL纤溶酶原溶液(2mg/mL)、23.9μL小鼠脑匀浆；溶媒对照组加入21.5μLα-突触核蛋白溶液(上海强耀生物科技有限公司，定制表达人α-突触核蛋白，UniProtKB-P37840，1.0mg/mL)、4.6μL溶媒溶液(10mM柠檬酸钠，2％盐酸精氨酸，3％甘露醇，PH7.4)、23.9μL小鼠脑匀浆；纤溶酶原组加入21.5mLα-突触核蛋白溶液(1.0mg/mL)、4.6μL纤溶酶原溶液(2mg/mL)、23.9μL小鼠脑匀浆。各组样品加好后，37℃温育6h后分别加入50μL 0.1％三氟乙酸溶液终止反应。
根据Tris-Tricine-SDS-PAGE凝胶制备试剂盒(Solarbio，P1320)配胶说明制备12％凝胶。各组样品分别与4×上样缓冲液(TaKaRa，e2139)以体积比为3:1混匀后，100℃加热5min，冷却后离心2min，然后取20μL上样。电泳条件为30V跑1.5h，然后100V电泳至胶底。电泳结束后剥凝胶置于1‰考马斯亮蓝染色液(1g考马斯亮蓝R250溶于1000ml乙醇：冰醋酸：纯化水体积比为5:2:13的混合液中)中染色30min，再用脱色液(纯化水：冰醋酸：无水乙醇＝17:2:1体积比混合)脱色至干净。凝胶在生物分子成像仪下拍照并定量扫描分析。
结果显示，在帕金森模型小鼠脑匀浆中，纤溶酶原组α-突触核蛋白的量明显低于溶媒对照组，差异极显著(***代表P＜0.001)，且其聚合物a、b的量均明显低于溶媒对照组，差异极显著(**代表P＜0.01，***代表P＜0.001)；在正常小鼠脑匀浆中，纤溶酶原组α-突触核蛋白的量明显低于溶媒对照组，差异极显著(***代表P＜0.001)，且其聚合物a、b的量均明显低于溶媒对照组，差异显著(*代表P＜0.05，**代表P＜0.01)(图24)。表明在帕金森模型小鼠和正常小鼠脑匀浆中，纤溶酶原可有效地降解人α-突触核蛋白及其聚合物。
实施例13纤溶酶原促进α-突触核蛋白在帕金森模型小鼠脑匀浆中降解
取11～12周龄、18-25g的C57BL/6J雄性小鼠8只，造模前1天称重，根据体重随机分为2组，空白对照组4只，模型组4只。造模时间定在每 日早上9点，空白对照组腹腔注射200μL生理盐水；模型组腹腔注射35mg/kg/只5mg/mL MPTP溶液，连续注射5天，建立帕金森模型 [5]。MPTP溶液配制：取45mg MPTP(sigma，M0896)溶解在9mL生理盐水溶液中，配制最终浓度为5mg/mL。造模完成后即造模第6天，所有小鼠进行旷场实验，鉴定造模成功。所有小鼠处死后取整个脑部并称重，按150mg组织/mLPBS分别加入1×PBS(Thermo Fisher，pH7.4；10010-031，4℃下匀浆(1min，3-4次)，匀浆后于4℃离心(12000rpm，20min)，取上清液置于新的EP管中。
取Eppendorf(EP)管分别设为①空白组、②空白对照组、③溶媒对照组、④纤溶酶原组，各组设置5个平行。空白组加入21.5μL生理盐水、4.6μL溶媒溶液(10mM柠檬酸钠，2％盐酸精氨酸，3％甘露醇，pH7.4)、23.9μL小鼠脑匀浆；空白对照组加入21.5μL生理盐水、4.6μL纤溶酶原溶液(2mg/mL)、23.9μL小鼠脑匀浆；溶媒对照组加入21.5μLα-突触核蛋白溶液(上海强耀生物科技有限公司，定制表达人α-突触核蛋白，UniProtKB-P37840，1.0mg/mL)、4.6μL溶媒溶液、23.9μL小鼠脑匀浆；纤溶酶原组加入21.5mLα-突触核蛋白溶液(1.0mg/mL)(1.0mg/mL)、4.6μL纤溶酶原溶液(2mg/mL)、23.9μL小鼠脑匀浆。各组样品加好后，37℃温育6h后分别加入50μL 0.1％三氟乙酸溶液终止反应。
根据Tris-Tricine-SDS-PAGE凝胶制备试剂盒(Solarbio，P1320)配胶说明制备12％凝胶。各组样品分别与4×上样缓冲液(TaKaRa，e2139)以体积比为3:1混匀后，100℃加热5min，冷却后离心2min，然后取20μL上样。电泳条件为30V跑1.5h，然后100V电泳至胶底。电泳结束后剥取凝胶转移到PVDF膜(GE，A29433753)上，电泳条件为15V，2h。转移后的PVDF膜浸泡在封闭液(5％脱脂乳液)中于4℃冰箱中封闭过夜，TBST(0.01M Tris-NaCl，pH7.6缓冲液)洗4次后，加入兔抗人α-突触核蛋白抗体(Proteintech,10842-1-AP)室温孵育3h，TBST洗4次后，加入山羊抗兔IgG(HRP)抗体(Abcam，ab6721)二抗室温孵育1h，TBST洗4次后，将PVDF膜放于干净成像板上，加入Immobilon Western HRP Substrate(MILLIPORE，WBKLS0100)显色，在生物分子成像仪下拍照并用Image J定量分析。
结果显示，在帕金森模型小鼠脑匀浆中，纤溶酶原组α-突触核蛋白的量明显低于溶媒对照组，差异极显著(**代表P＜0.01)，且其聚合物的量均明显低于溶媒对照组，差异极显著(***代表P＜0.001)；在正常小鼠脑匀浆中，纤溶酶原组α-突触核蛋白的量明显低于溶媒对照组，差异极显著(**代表P＜0.01)，且其聚合物的量均明显低于溶媒对照组，差异显著(***代表P＜0.001)(图25)。表明在帕金森模型小鼠和正常小鼠脑匀浆中，纤溶酶原可有效地降解人α-突触核蛋白及其聚合物。
实施例14纤溶酶原促进Pro-BDNF在帕金森模型小鼠脑匀浆中裂解
取11～12周龄、18-25g的C57BL/6J雄性小鼠4只。造模时间定在每日早上9点，空白对照组腹腔注射200μL生理盐水；模型组腹腔注射35mg/kg/只5mg/mL MPTP溶液，连续注射5天，建立帕金森模型 [5]。MPTP溶液配制：取45mg MPTP(sigma，M0896)溶解在9mL生理盐水溶液中，配制最终浓度为5mg/mL。造模完成后即造模第6天，所有小鼠进行旷场实验，鉴定造模成功。所有小鼠处死后取整个脑部并称重，按150mg组织/mLPBS分别加入1×PBS(Thermo Fisher，pH7.4；10010-031，4℃下匀浆(1min，3-4次)，匀浆后于4℃离心(12000rpm，20min)，取上清液即脑匀浆液置于新的EP管中。
取Eppendorf(EP)管分别设为①空白组、②空白对照组、③溶媒对照组、④纤溶酶原组，各组设置5个平行。空白组加入21.5μL生理盐水、4.6μL溶媒溶液(10mM柠檬酸钠，2％盐酸精氨酸，3％甘露醇，pH7.4)、23.9μL小鼠脑匀浆；空白对照组加入21.5μL生理盐水、4.6μL纤溶酶原溶液(2mg/mL)、23.9μL小鼠脑匀浆；溶媒对照组加入21.5μL Pro-BDNF(南京金斯瑞，定制表达人Pro-BDNF，UniProtKB-P23560，1.0mg/mL)、4.6μL溶媒溶液、23.9μL小鼠脑匀浆；纤溶酶原组加入21.μL Pro-BDNF(1.0mg/mL)、4.6μL纤溶酶原溶液(2mg/mL)、23.9μL小鼠脑匀浆。各组样品加好后，37℃温育6h后分别加入50μL 0.1％三氟乙酸溶液终止反应。
根据SDS-PAGE配胶说明制备12％凝胶。各组样品分别与4×上样缓冲液(TaKaRa，e2139)以体积比为3:1混匀后，100℃加热5min，冷却后离心2min，然后取20μL上样。电泳条件为30V跑45min，然后100V电泳 至胶底。电泳结束后剥凝胶于1‰考马斯亮蓝染色液(1g考马斯亮蓝R250溶于1000ml乙醇：冰醋酸：纯化水体积比为5:2:13的混合液中)染色30min，再用脱色液(纯化水：冰醋酸：无水乙醇＝17:2:1体积比混合)脱色至干净。凝胶在生物分子成像仪下拍照并定量扫描分析。
脑源性神经营养因子(brain-derived neurotrophic factor，BDNF)是一类分子量为12.3kD的碱性蛋白质，由119个氨基酸残基构成，并含有3对二硫键，在体内以二聚体的形式存在，以BDNF前体的形式合成，BDNF前体(Pro-BDNF)可经酶解作用裂解形成成熟的BDNF。文献报道Pro-BDNF与其裂解形成的成熟的BDNF具有相反的作用。Pro-BDNF促进神经细胞凋亡，降低神经突触可塑性。成熟的BDNF及其受体广泛分布于中枢神经系统内，在中枢神经系统发育过程中，对神经元存活、分化、生长发育起重要作用，并能防止神经元受损伤死亡，改善神经元的病理状态，促进受损伤神经元再生及分化等生物效应，而且也是成熟的中枢及周围神经系统的神经元维持生存及正常生理功能所必需的 [24]。
结果显示，在帕金森模型小鼠脑匀浆液中，纤溶酶原组Pro-BDNF的量明显低于溶媒对照组，差异极显著(***代表P＜0.001)(图26)。提示纤溶酶原能够促进帕金森模型小鼠脑匀浆中Pro-BDNF裂解。
实施例15纤溶酶原促进Pro-BDNF在帕金森模型小鼠脑匀浆中裂解形成成熟的BDNF
取11～12周龄、18-25g的C57BL/6J雄性小鼠8只，造模前1天称重，根据体重随机分为2组，空白对照组4只，模型组4只。造模时间定在每日早上9点，空白对照组腹腔注射200μL生理盐水；模型组腹腔注射35mg/kg/只5mg/mL MPTP溶液，连续注射5天，建立帕金森模型 [5]。MPTP溶液配制：取45mg MPTP(sigma，M0896)溶解在9mL生理盐水溶液中，配制最终浓度为5mg/mL。造模完成后即造模第6天，所有小鼠进行旷场实验，鉴定造模成功。所有小鼠处死后取整个脑部并称重，按150mg组织/mLPBS分别加入1×PBS(Thermo Fisher，pH7.4；10010-031，4℃下匀浆(1min，3-4次)，匀浆后于4℃离心(12000rpm，20min)，取上清液即脑匀浆液置于新的EP管中。
取Eppendorf(EP)管分别设为①空白组、②空白对照组、③溶媒对照组、④纤溶酶原组，各组设置5个平行。空白对照组加入21.5μL生理盐水、4.6μL纤溶酶原溶液(2mg/mL)、23.9μL小鼠脑匀浆；溶媒对照组加入21.5μL Pro-BDNF(南京金斯瑞，定制表达人Pro-BDNF，UniProtKB-P23560，1.0mg/mL)、4.6μL溶媒溶液(柠檬酸-柠檬酸钠溶液)、23.9μL小鼠脑匀浆；纤溶酶原组加入21.μL Pro-BDNF(1.0mg/mL)、4.6μL纤溶酶原溶液(2mg/mL)、23.9μL小鼠脑匀浆。各组样品加好后，37℃温育6h后分别加入50μL 0.1％三氟乙酸溶液终止反应。
根据SDS-PAGE配胶说明制备12％凝胶。各组样品分别与4×上样缓冲液(TaKaRa，e2139)以体积比为3:1混匀后，100℃加热5min，冷却后离心2min，然后取20μL上样。电泳条件为30V跑45min，然后100V电泳至胶底。电泳结束后剥取凝胶转移到活化的PVDF膜(GE，A29433753)上，电泳条件为15V，2.5h。转移后的PVDF膜浸泡在封闭液(5％脱脂乳液)中于4℃冰箱中封闭过夜，TBST(0.01M Tris-NaCl，pH7.6缓冲液)洗4次后，加入兔抗人BDNF抗体(Boster Biological Technology,PB9075)室温孵育3h，TBST洗4次后，加入山羊抗兔IgG(HRP)抗体(Abcam，ab6721)二抗室温孵育1h，TBST洗4次后，将PVDF膜放于干净成像板上，加入Immobilon Western HRP Substrate(MILLIPORE，WBKLS0100)显色，在生物分子成像仪下拍照并用Image J定量分析。
结果显示，在帕金森模型小鼠脑匀浆液中，纤溶酶原组Pro-BDNF的量明显低于溶媒对照组，差异显著(*代表P＜0.05，***表示P＜0.001)；纤溶酶原组BDNF的量明显高于溶媒对照组，差异极为显著(图27)。提示纤溶酶原能够促进帕金森模型小鼠脑匀浆液Pro-BDNF裂解和成熟BDNF形成。
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Figure PCTCN2021082715-appb-000001
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Claims (14)

  1. 一种预防和治疗帕金森病的方法，包括给药帕金森病受试者治疗有效量的选自如下的一种或多种化合物：纤维蛋白溶酶原激活途径的组分、能够直接激活纤维蛋白溶酶原或通过激活纤维蛋白溶酶原激活途径上游组分而间接激活纤维蛋白溶酶原的化合物、模拟纤维蛋白溶酶原或纤维蛋白溶酶之活性的化合物、能够上调纤维蛋白溶酶原或纤维蛋白溶酶原激活剂表达的化合物、纤维蛋白溶酶原类似物、纤维蛋白溶酶类似物、tPA或uPA类似物和纤溶抑制剂的拮抗剂。
  2. 权利要求1所述的方法，其中所述纤维蛋白溶酶原激活途径的组分选自纤维蛋白溶酶原、重组人纤维蛋白溶酶、Lys-纤维蛋白溶酶原、Glu-纤维蛋白溶酶原、纤维蛋白溶酶、含有纤维蛋白溶酶原和纤维蛋白溶酶的一个或多个kringle结构域和蛋白酶结构域的纤维蛋白溶酶原和纤维蛋白溶酶变体及类似物、小纤维蛋白溶酶原(mini-plasminogen)、小纤维蛋白溶酶(mini-plasmin)、微纤溶酶原(micro-plasminogen)、微纤溶酶(micro-plasmin)、delta-纤溶酶原、delta-纤溶酶(delta-plasmin)、纤维蛋白溶酶原激活剂、tPA和uPA。
  3. 权利要求1的方法，所述纤溶抑制剂的拮抗剂为PAI-1、补体C1抑制物、α2抗纤溶酶或α2巨球蛋白的抑制剂，例如抗体。
  4. 权利要求1-3任一项的方法，其中所述化合物对帕金森病受试者具有如下一项或多项作用：促进记忆功能恢复、改善认知能力、促进黑质DTA表达、促进纹状体尼氏体恢复、促进黑质GLP-1R表达、增加黑质TH阳性细胞数量、促进纹状体髓鞘修复、促进脑组织中α-突触核蛋白降解、促使纹状体NF表达、促进轴索损伤修复、减少纹状体GFAP表达、减轻纹状体神经元损伤、促进脑组织中Pro-BDNF裂解形成BDNF、缓解抑郁或焦虑症状。
  5. 权利要求1-4任一项的方法，其中所述化合物为纤溶酶原。
  6. 权利要求1-5任一项的方法，其中所述纤溶酶原为人全长纤溶酶原或其保守取代变体。
  7. 权利要求1-5任一项的方法，其中所述纤溶酶原与序列2具有至少75％、80％、85％、90％、95％、96％、97％、98％或99％的序列同一性，并且仍然具有纤溶酶原的赖氨酸结合活性或蛋白水解活性。
  8. 权利要求1-5任一项的方法，所述纤溶酶原包含与序列14具有至少80％、90％、95％、96％、97％、98％、99％氨基酸序列同一性的氨基酸序列、并且仍然具有纤溶酶原的蛋白水解活性的蛋白质。
  9. 权利要求1-5任一项的方法，所述纤溶酶原选自Glu-纤溶酶原、Lys-纤溶酶原、小纤溶酶原、微纤溶酶原、delta-纤溶酶原或它们的保留纤溶酶原的蛋白水解活性的变体。
  10. 权利要求1-5任一项的方法，所述纤溶酶原包含序列2、6、8、10、12所示的氨基酸序列或包含序列2、6、8、10、12所示氨基酸序列的保守取代变体。
  11. 权利要求1-10任一项的方法，其中所述化合物与一种或多种其他治疗方法或药物联合使用。
  12. 权利要求11的方法，其中所述其他治疗方法包括细胞治疗(包括干细胞治疗)和物理治疗。
  13. 权利要求11的方法，其中所述其他药物为治疗帕金森病的其它药物。
  14. 权利要求1-13任一项的方法，其中所述化合物通过鼻腔吸入、雾化吸入、滴鼻液、滴眼液、滴耳液、静脉内、腹膜内、皮下、颅内、鞘内、动脉内(例如经由颈动脉)或肌肉内给药。
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CN115697386A (zh) 2023-02-03
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