WO2024259975A1 - miR-15a-5p在治疗眼底疾病中的应用 - Google Patents

miR-15a-5p在治疗眼底疾病中的应用 Download PDF

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WO2024259975A1
WO2024259975A1 PCT/CN2024/073634 CN2024073634W WO2024259975A1 WO 2024259975 A1 WO2024259975 A1 WO 2024259975A1 CN 2024073634 W CN2024073634 W CN 2024073634W WO 2024259975 A1 WO2024259975 A1 WO 2024259975A1
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mirna
nucleotide sequence
seq
mir
retinal
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French (fr)
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李筱荣
张晓敏
张慧
于欣悦
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TIANJIN MEDICAL UNIVERSITY EYE HOSPITAL
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TIANJIN MEDICAL UNIVERSITY EYE HOSPITAL
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C12N2310/141—MicroRNAs, miRNAs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the current treatment method is intravitreal injection of monoclonal antibodies against vascular endothelial growth factor (VEGF), but its target is single and cannot improve retinal neurodegeneration and reduce the level of retinal inflammation; and because VEGF is an important neurotrophic factor, the use of anti-VEGF treatment can lead to further damage to retinal nerve cells and decreased retinal function.
  • VEGF vascular endothelial growth factor
  • Diabetic retinopathy (DR) which has a high incidence rate in the working age group, is also a microvascular disease characterized by neovascularization and neurodegeneration.
  • the causative factor is the persistent increase in blood sugar, but the subsequent pathological mechanism is more complicated.
  • MicroRNA is a type of non-coding single-stranded RNA molecule with a length of about 22 nucleotides encoded by endogenous genes. They participate in post-transcriptional gene expression regulation in animals and plants. The main characteristics are natural existence in the human body, multi-target regulation, and rich biological functions. Among them, miR-15a-5p is closely related to the occurrence and development of many diseases.
  • Patent document: CN109414459B discloses that exosomes containing miRNA such as miR-15a-5p can promote wound healing.
  • the nucleotide sequence of the miRNA includes ACGACGAU (SEQ ID NO: 10).
  • the nucleotide sequence of the miRNA includes SEQ ID NO: 10, and has 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 95% or more identity with the nucleotide sequence of SEQ ID NO: 1.
  • the fundus disease is selected from one or more of retinopathy of prematurity, retinal neovascularization disease, choroidal neovascularization disease or diabetic retinopathy.
  • the miRNA or its mimics comprises a sense strand and an antisense strand.
  • the sense strand comprises or is SEQ ID NO: 1 or a nucleotide sequence having 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 95% or 99% homology with the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence comprising substitution, deletion or insertion of one or more nucleotides, and the antisense strand comprises or is CACAA ACCAUUAUGUGCUGCUA (SEQ ID NO: 6) or a nucleotide sequence that has 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 95% or 99% or more homology to
  • the product comprises miRNA and/or modified miRNA and/or miRNA mimics, or a vector comprising miRNA and/or modified miRNA and/or miRNA mimics (the vector may be any vector that encapsulates, transcribes or expresses miRNA).
  • the vector is a viral vector or a non-viral vector.
  • the sequence of the miRNA contains modifications, such as modifications made at the bases.
  • Modified miRNAs include modifications made at the bases.
  • the product contains a sense strand and an antisense strand.
  • the dangling base is located at the 3' end of the sense strand and/or the antisense strand.
  • the dangling base is a deoxynucleoside.
  • the dangling base is dTdT, dTdC or dUdU.
  • the sense strand of the miRNA or its mimic may be UAGCAGCACAUAAUGGUUUGUGdTdT (SEQ ID NO: 7), and the antisense strand may be CACAAACCAUUAUGUGCUGCUAdTdT (SEQ ID NO: 8).
  • the sense strand comprises or is SEQ ID NO: 1 or has 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 95% or more homology with the nucleotide sequence of SEQ ID NO: 1 or comprises a substitution, deletion or other substitution of one or more nucleotides.
  • the antisense strand comprises SEQ ID NO: 6 modified with 2 thio backbones at the 5' end, 4 thio backbones at the 3' end, cholesterol at the 3' end and methoxy modification on the whole chain, or a nucleotide sequence having 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 95% or more homology with the nucleotide sequence of SEQ ID NO: 6, or comprising substitution, deletion or insertion of one or more nucleotides.
  • the treatment and/or prevention of fundus diseases is selected from one or more of inhibiting the activity of retinal vascular endothelial cells (HRMECs), inhibiting the proliferation of HRMECs (especially reducing the proliferation of HRMECs induced by VEGF), inhibiting the phosphorylation of Smad2, inhibiting the expression of total Smad2, inhibiting fibrosis, inhibiting the expression of VEGF, inhibiting retinal inflammation, resisting neovascularization or promoting the recovery of non-perfused areas, improving retinal thinning, restoring visual function or promoting nerve damage repair.
  • HRMECs retinal vascular endothelial cells
  • VEGF retinal vascular endothelial cells
  • the anti-angiogenesis comprises inhibiting retinal neovascularization and/or choroidal neovascularization.
  • the treatment and/or prevention of fundus diseases comprises administering miRNA and/or modified miRNA or miRNA mimics and/or administering a vector containing miRNA and/or modified miRNA or miRNA mimics (eg, a vector containing a nucleic acid transcribed into miRNA) to a subject in need thereof.
  • a vector containing miRNA and/or modified miRNA or miRNA mimics eg, a vector containing a nucleic acid transcribed into miRNA
  • the present invention provides a vector comprising a nucleic acid transcribed into miRNA or a mimic thereof.
  • the nucleotide sequence of the miRNA includes SEQ ID NO: 10.
  • the nucleotide sequence of the miRNA includes SEQ ID NO: 10, and has 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 95% or more identity with the nucleotide sequence of SEQ ID NO: 1.
  • the nucleotide sequence of the miRNA includes SEQ ID NO: 10, and has a nucleotide sequence containing one or more nucleotide substitutions, deletions or insertions compared with the nucleotide sequence of SEQ ID NO: 1, preferably a nucleotide sequence containing no more than ten, nine, eight, seven, six, five, four, three, two or one nucleotide substitutions, deletions or insertions.
  • the miRNA is miR-15a-5p.
  • nucleotide sequence of the miRNA is SEQ ID NO: 1.
  • the miRNA sequence contains modifications, such as modifications on the bases.
  • the base modifications are located in the sense strand and/or the antisense strand, preferably including one or more of cholesterol modification at the 3' end, two sulfide backbone modifications at the 5' end, four sulfide backbone modifications at the 3' end, or full-chain methoxy modification.
  • the nucleic acid transcribed into miRNA contains TAGCAGCA (SEQ ID NO: 11).
  • the nucleic acid transcribed into miRNA comprises SEQ ID NO: 11, and has 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 95% or more identity with the nucleotide sequence of SEQ ID NO: 9.
  • the nucleic acid transcribed into miRNA comprises SEQ ID NO: 11, and has a nucleotide sequence containing one or more nucleotide substitutions, deletions or insertions compared with the nucleotide sequence of SEQ ID NO: 9, preferably a nucleotide sequence containing no more than ten, nine, eight, seven, six, five, four, three, two or one nucleotide substitutions, deletions or insertions.
  • the nucleic acid transcribed into miRNA is SEQ ID NO: 9.
  • the miRNA or its mimic comprises a sense strand and an antisense strand.
  • the sense strand comprises SEQ ID NO: 1 or has 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 95% or more homology with the nucleotide sequence of SEQ ID NO: 1 or comprises one or more nucleotides
  • the antisense strand comprises SEQ ID NO: 6 or a nucleotide sequence having 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 95% or 99% or more homology with the nucleotide sequence of SEQ ID NO: 6, or a nucleotide sequence comprising substitution, deletion or insertion of one or more nucleotides.
  • the sense strand and/or antisense strand contain hanging bases.
  • the dangling base is located at the 3' end of the sense strand and/or the antisense strand.
  • the hanging bases are deoxynucleosides.
  • the hanging base is dTdT, dTdC or dUdU.
  • the vector is a viral vector or a non-viral vector.
  • the viral vector includes one or more of a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, a poxvirus vector or a herpesvirus vector.
  • the non-viral vector comprises one or more of liposomes, lipid nanoparticles, polymers, polypeptides, antibodies, aptamers or N-acetylgalactosamine.
  • the third aspect of the present invention provides a modified miR-15a-5p or miR-15a-5p mimic.
  • the modified miR-15a-5p includes modifications made on the bases.
  • the modification of the base is located in the antisense strand, preferably including one or more of cholesterol modification at the 3' end, two thio-skeleton modifications at the 5' end, four thio-skeleton modifications at the 3' end, or methoxy modification of the entire strand.
  • the sense strand and/or antisense strand contained in the miR-15a-5p or its mimics is subjected to one or more of full-chain methoxy modification, 3'-end cholesterol modification, 5'-end thio skeleton modification or 3'-end thio skeleton modification.
  • the antisense strand of miR-15a-5p or its mimics is modified with full-chain methoxy, 3'-end cholesterol, 5'-end two thio backbones and 3'-end four thio backbones.
  • the sense strand comprises SEQ ID NO: 1 or a nucleotide sequence having 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 95% or more homology with the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence comprising substitution, deletion or insertion of one or more nucleotides, and the antisense strand comprises 2 SEQ ID NO: 6 with thiolate backbone modification, 4 thiolate backbone modifications at the 3' end, cholesterol modification at the 3' end and full-chain methoxy modification, or a nucleotide sequence with 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 95% or more
  • the fourth aspect of the present invention provides a medicine or a pharmaceutical composition, which comprises the above-mentioned miRNA and/or modified miRNA and/or miRNA mimics and/or the above-mentioned vector containing nucleic acid transcribed into miRNA or miRNA mimics, and pharmaceutically acceptable excipients.
  • the nucleotide sequence of the miRNA includes ACGACGAU (SEQ ID NO: 10).
  • the nucleotide sequence of the miRNA includes SEQ ID NO: 10, and the nucleotide sequence of SEQ ID NO:
  • the amino acid sequences have 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 95% or more identity.
  • the nucleotide sequence of the miRNA includes SEQ ID NO: 10, and has a nucleotide sequence containing one or more nucleotide substitutions, deletions or insertions compared with the nucleotide sequence of SEQ ID NO: 1, preferably a nucleotide sequence containing no more than ten, nine, eight, seven, six, five, four, three, two or one nucleotide substitutions, deletions or insertions.
  • the miRNA is miR-15a-5p.
  • nucleotide sequence of the miRNA is SEQ ID NO: 1.
  • Modified miRNAs include modifications made at the bases.
  • the modification performed on the base includes one or more of cholesterol modification at the 3' end, two sulfide backbone modifications at the 5' end, four sulfide backbone modifications at the 3' end, or full-chain methoxy modification.
  • the medicine or pharmaceutical composition can treat fundus diseases.
  • the pharmaceutical composition may also contain other nucleic acids, polypeptides, proteins, compounds, etc. for treating or preventing fundus diseases or nucleic acids, polypeptides, proteins, compounds, etc. for reducing side effects.
  • the drug or pharmaceutical composition can be administered by any suitable route, such as enteral administration (e.g., oral administration) or parenteral administration (e.g., intravenous, intramuscular, subcutaneous, intradermal, intraorgan, intranasal, intraocular, instillation, intracerebral, intrathecal, transdermal, intrarectal, etc.).
  • enteral administration e.g., oral administration
  • parenteral administration e.g., intravenous, intramuscular, subcutaneous, intradermal, intraorgan, intranasal, intraocular, instillation, intracerebral, intrathecal, transdermal, intrarectal, etc.
  • the drug or pharmaceutical composition can be in any suitable dosage form, such as a dosage form for gastrointestinal administration or a dosage form for parenteral administration, preferably including but not limited to tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, microemulsions, suspensions, injections, sprays, aerosols, powder sprays, lotions, ointments, plasters, pastes, patches, eye drops, nasal drops, sublingual tablets, suppositories, aerosols, effervescent tablets, pills, gels, etc.
  • a dosage form for gastrointestinal administration or a dosage form for parenteral administration preferably including but not limited to tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, microemulsions, suspensions, injections, sprays, aerosols, powder sprays, lotions, ointments, plasters, pastes, patches, eye drops, nasal drops, sub
  • Various dosage forms of the drug or pharmaceutical composition can be prepared according to conventional production methods in the pharmaceutical field.
  • the drug or pharmaceutical composition may contain the miRNA, modified miRNA, or a vector containing miRNA or modified miRNA in a weight ratio of 0.01-99.5% (specifically, 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%).
  • the medicine or pharmaceutical composition can be a human medicine or a veterinary medicine.
  • the fifth aspect of the present invention provides a method for treating and/or preventing fundus diseases, which comprises administering an effective amount of miRNA and/or modified miRNA and/or miRNA mimics and/or a vector and/or pharmaceutical composition comprising miRNA or modified miRNA or miRNA mimics to a subject in need.
  • the nucleotide sequence of the miRNA includes ACGACGAU (SEQ ID NO: 10).
  • the nucleotide sequence of the miRNA includes SEQ ID NO: 10, and has 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 95% or more identity with the nucleotide sequence of SEQ ID NO: 1.
  • the nucleotide sequence of the miRNA includes SEQ ID NO: 10, and has a nucleotide sequence containing one or more nucleotide substitutions, deletions or insertions compared to the nucleotide sequence of SEQ ID NO: 1, preferably a nucleotide sequence containing no more than ten, nine, eight, seven, six, five, four, three, two or one nucleotide substitution, deletion or insertion.
  • the miRNA is miR-15a-5p.
  • nucleotide sequence of the miRNA is SEQ ID NO: 1.
  • the fundus disease is a fundus disease that can be prevented and/or treated by inhibiting VEGF and/or TGF- ⁇ 1.
  • the fundus disease is selected from one or more of vitreous lesions, retinal lesions, optic neuropathy or choroidal lesions.
  • the miRNA or its mimics comprises a sense strand and an antisense strand, wherein the sense strand comprises SEQ ID NO: 1 or has 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 95% or more homology with the nucleotide sequence of SEQ ID NO: 1 or comprises a substitution of one or more nucleotides,
  • the invention relates to a nucleotide sequence comprising a deletion or insertion, wherein the antisense strand comprises SEQ ID NO: 6 or a nucleotide sequence having 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 95% or more homology with the nucleotide sequence of SEQ ID NO: 6 or a nu
  • the modified miRNA includes modifications made on the bases, including one or more of cholesterol modification at the 3' end, two thio-skeleton modifications at the 5' end, four thio-skeleton modifications at the 3' end, or full-chain methoxy modification.
  • the treatment and/or prevention of fundus diseases is selected from one or more of inhibiting HRMECs activity, inhibiting HRMECs proliferation (especially reducing VEGF-induced HRMECs proliferation), inhibiting Smad2 phosphorylation, inhibiting total Smad2 expression, inhibiting fibrosis, inhibiting VEGF expression, inhibiting retinal inflammation, resisting neovascularization or promoting recovery of non-perfused areas, improving retinal thinning, restoring visual function or promoting nerve damage repair.
  • the anti-angiogenesis comprises inhibiting retinal neovascularization and/or choroidal neovascularization.
  • the method comprises administering 0.5 ⁇ g-5 mg (e.g., 0.5 ⁇ g, 1 ⁇ g, 2 ⁇ g, 3 ⁇ g, 4 ⁇ g, 5 ⁇ g, 10 ⁇ g, 20 ⁇ g, 50 ⁇ g, 100 ⁇ g, 150 ⁇ g, 200 ⁇ g, 250 ⁇ g, 300 ⁇ g, 350 ⁇ g, 400 ⁇ g, 450 ⁇ g, 500 ⁇ g, 550 ⁇ g, 600 ⁇ g, 650 ⁇ g, 700 ⁇ g, 750 ⁇ g, 800 ⁇ g, 850 ⁇ g, 900 ⁇ g, 950 ⁇ g, 1 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 3.5 mg, 4 mg, 4.5 mg, 5 mg) of miRNA, modified miRNA, miRNA mimics, a carrier comprising miRNA or modified miRNA or miRNA mimics, or the above-mentioned drugs or pharmaceutical compositions to each eye.
  • 0.5 ⁇ g-5 mg e.g., 0.5
  • the method comprises administering to the intraocular space or cavity of the subject, such as one or more of the aqueous humor in the anterior chamber, the suspensory ligament, the ciliary body, the ciliary body and muscle, the lens or iris, the vitreous body, the retina, the choroid or the optic nerve.
  • the intraocular space or cavity of the subject such as one or more of the aqueous humor in the anterior chamber, the suspensory ligament, the ciliary body, the ciliary body and muscle, the lens or iris, the vitreous body, the retina, the choroid or the optic nerve.
  • modified miRNA for example, modified mir-15a-5p, whose antisense chain base modifications include cholesterol modification at the 3' end, two thio-skeleton modifications at the 5' end, four thio-skeleton modifications at the 3' end, and full-chain methoxy modification, i.e. Agomir-15a-5p
  • modified mir-15a-5p whose antisense chain base modifications include cholesterol modification at the 3' end, two thio-skeleton modifications at the 5' end, four thio-skeleton modifications at the 3' end, and full-chain methoxy modification, i.e. Agomir-15a-5p
  • pharmaceutically acceptable refers to a substance that neither significantly irritates an organism nor inhibits the biological activity and properties of the active substance of the administered product.
  • treating means slowing, interrupting, preventing, controlling, stopping, alleviating, or reversing the progression or severity of a sign, symptom, disorder, condition, or disease after the disease has begun to develop, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, or disorders.
  • the “effective amount” of the present invention refers to the amount or dosage of the miRNA, miRNA mimics, modified miRNA, drug or pharmaceutical composition of the present invention that provides the desired treatment or prevention after single or multiple doses are administered to an individual or organ.
  • prevention refers to a method implemented to prevent or delay the occurrence of a disease, disorder or symptom in an organism.
  • the "subject" described in the present invention can be a human or a non-human mammal, and the non-human mammal can be a wild animal, a zoo animal, an economic animal, a pet, an experimental animal, etc.
  • the non-human mammal includes but is not limited to pigs, cattle, sheep, horses, donkeys, foxes, raccoon dogs, minks, camels, dogs, cats, rabbits, mice (such as rats, mice, guinea pigs, hamsters, gerbils, chinchillas, squirrels) or monkeys, etc.
  • FIG. 1 After transfecting miR-15a-5p mimic control/mimic/inhibitor control/inhibitor into retinal vascular endothelial cells, the expression of miR-15a-5p in retinal vascular endothelial cells was detected. Mimics can increase the expression of miR-15a-5p in cells, while inhibitors can reduce the expression of miR-15a-5p in cells.
  • Figure 2 Effects of different concentrations of VEGF on cell proliferation in retinal endothelial cells. 10ng/ml VEGF can cause abnormal cell proliferation.
  • Figure 3 Effects of different concentrations of VEGF on the relative expression of miR-15a-5p in retinal endothelial cells. 10ng/ml VEGF did not affect the expression of miR-15a-5p in cells.
  • FIG. 4 CCK-8 kit was used to detect the effect of miR-15a-5p on cell proliferation.
  • miR-15a-5p mimics attenuated VEGF-induced pathological proliferation of retinal vascular endothelial cells.
  • Figure 5 Cell scratch assay to detect the effect of miR-15a-5p on cell proliferation ability.
  • Figure 6 Quantitative statistical analysis of cell scratches, miR-15a-5p mimics reduce VEGF-induced pathological proliferation of retinal endothelial cells.
  • Figure 7 Transwell assay to detect the effect of miR-15a-5p on cell migration ability.
  • FIG. 8 Quantitative statistical analysis of the number of cell migration in the Transwell experiment. miR-15a-5p mimics reduced VEGF-induced pathological migration of retinal vascular endothelial cells.
  • Figure 9 Tube formation experiment was used to detect the effect of miR-15a-5p on the tube-forming ability of cells in vitro.
  • Figure 10 Quantitative statistical analysis of the total length of the vascular lumen formed by cells in the tube formation experiment. miR-15a-5p mimics reduce VEGF-induced vascular tube formation in retinal endothelial cells.
  • Figure 11 Quantitative statistical analysis of the number of vascular tube nodes formed by cells in the tube formation experiment. miR-15a-5p mimics reduce VEGF-induced vascular tube formation in retinal endothelial cells.
  • Figure 12 Differences in mouse retinal uptake of modified (Agomir) and unmodified (mimic) miR-15a-5p mimics. * is the difference between the mimic group and the control group (P ⁇ 0.05, ** is p ⁇ 0.01); @ is the difference between the modified mimic (Agomir) and the control group (P ⁇ 0.05, @@@ is p ⁇ 0.001, @@@@ is P ⁇ 0.0001; # is the difference between the mimic group and the modified mimic (Agomir) (P ⁇ 0.05, #### is ⁇ 0.0001).
  • Figure 13 Flow chart of the oxygen-induced mouse retinal neovascularization model. P represents the day after birth of the mouse.
  • Figure 14 Expression levels of retinal miR-15a-5p during the development of oxygen-induced retinal neovascularization mice.
  • the normoxic group is the untreated group, and the hyperoxic group is the neovascularization model group.
  • Figure 15 Effects of different doses of miR-15a-5p mimics on oxygen-induced retinal neovascularization.
  • Normoxia is the untreated mice
  • hyperoxia is the neovascularization model group
  • the mimic is Agomir.
  • Figure 16 Statistical graph of the effect of different doses of miR-15a-5p mimics on oxygen-induced retinal neovascularization. Normoxia is untreated mice, hyperoxia is the neovascularization model group, the mimic is Agomir, and the mimic control is scrambled Agomir.
  • Figure 17 Effects of different doses of anti-VEGF drugs on oxygen-induced retinal neovascularization.
  • A is a staining image
  • B is a statistical graph.
  • normoxia is the untreated mouse
  • hyperoxia is the neovascularization model group.
  • Figure 18 Distribution and persistence of Agomir-15a-5p in the retina after administration.
  • A Representative images of CY3-labeled Agomir-15a-5p in the retina.
  • B Quantification of fluorescence intensity of CY3-labeled Agomir-15a-5p.
  • C Expression trend of Agomir-15a-5p in the OIR retina after injection.
  • Figure 19 Schematic diagram of the effect of 1 ⁇ g of miR-15a-5p mimics on oxygen-induced retinal neovascularization.
  • Normoxia (column 1) is untreated mice, hyperoxia (columns 2-4) is the neovascularization model group, the mimic is Agomir, the mimic control is scrambled Agomir, and the anti-VEGF is ranibizumab.
  • Figure 20 Statistical graph of the neovascularization area of retinal neovascularization induced by oxygen treatment with 1 ⁇ g of miR-15a-5p mimics.
  • Normoxia is untreated mice
  • hyperoxia is the neovascularization model group
  • the mimic is Agomir
  • the mimic control is scrambled Agomir
  • the VEGF monoclonal antibody is the thunder Bezoar monoclonal antibody.
  • Figure 21 Statistical graph of the non-perfused area of retinal neovascularization induced by oxygen after treatment with 1 ⁇ g of miR-15a-5p mimics.
  • Normoxia is untreated mice
  • hyperoxia is the neovascularization model group
  • the mimic is Agomir
  • the mimic control is scrambled Agomir
  • the VEGF monoclonal antibody is Ranibizumab.
  • FIG. 22 Schematic diagram of the structure of adeno-associated virus containing miR-15a-5p.
  • FIG. 23 Schematic diagram of infection of the retina with adeno-associated virus containing miR-15a-5p.
  • Figure 24 PCR results show the overexpression of retinal miR-15a-5p after adeno-associated virus infection of the retina, where OIR-AAV-NC is the control virus injection group, and OIR-AAV-15a is the adeno-associated virus injection containing miR-15a-5p.
  • Figure 25 Schematic diagram of the therapeutic effect of adeno-associated virus containing miR-15a-5p on retinal neovascularization and non-perfusion area, where OIR-AAV-NC is the control virus injection group, and OIR-AAV-15a is the adeno-associated virus injection containing miR-15a-5p.
  • FIG. 26 Statistical results of the therapeutic effect of adeno-associated virus containing miR-15a-5p on the non-perfused area.
  • FIG. 27 Statistical results of the therapeutic effect of adeno-associated virus containing miR-15a-5p on oxygen-induced retinal neovascularization.
  • Figure 29 Schematic diagram of the non-perfused area and neovascularization area of oxygen-induced retinal neovascularization treated with 1 ⁇ g of miR-15a-5p mimics.
  • Hyperoxia-normoxic mice are the normal mouse neovascularization model group, and hyperoxia-knockout mice are the miR-15a-5p knockout mouse neovascularization model group.
  • Figure 30 Statistical graph of the non-perfused area of retinal neovascularization induced by oxygen after treatment with 1 ⁇ g of miR-15a-5p mimics.
  • the hyperoxic-normal mice are the normal mouse neovascularization model group, and the hyperoxic-knockout mice are the miR-15a-5p knockout mouse neovascularization model group.
  • Figure 31 Statistical graph of the neovascularization cluster area of oxygen-induced retinal neovascularization treated with 1 ⁇ g of miR-15a-5p mimics.
  • the hyperoxic-normal mice are the normal mouse neovascularization model group, and the hyperoxic-knockout mice are the miR-15a-5p knockout mouse neovascularization model group.
  • Figure 32 Schematic diagram of the non-perfused area and neovascularization area of oxygen-induced retinal neovascularization treated with 1 ⁇ g of miR-15a-5p mimics.
  • Hyperoxia-normal mice are the normal mouse neovascularization model group
  • hyperoxia-knockout mice are the miR-15a-5p knockout mouse neovascularization model group
  • the mimic control is scrambled Agomir
  • the mimic is Agomir.
  • Figure 33 Statistical graph of the non-perfused area of retinal neovascularization induced by oxygen after treatment with 1 ⁇ g of miR-15a-5p mimics.
  • Normal is the normal mouse neovascularization model group
  • knockout is the miR-15a-5p knockout mouse neovascularization model group
  • mimic control is scrambled Agomir
  • mimic is Agomir.
  • Figure 34 Statistical graph of the neovascularization cluster area of oxygen-induced retinal neovascularization treated with 1 ⁇ g of miR-15a-5p mimics. Normal is the normal mouse neovascularization model group, knockout is the miR-15a-5p knockout mouse neovascularization model group, mimic control is scrambled Agomir, and mimic is Agomir.
  • FIG 35 Magnified images of retinal non-perfused areas show differences in activation of astrocytes and Müller glial cells (spot staining).
  • Hyperoxia-normal mice are normal mouse neovascularization model group
  • hyperoxia-knockout mice are miR-15a-5p knockout mouse neovascularization model group
  • the mimic control is scrambled Agomir
  • the mimic is Agomir.
  • Figure 36 Representative images of retinal tip cells and filopodia, as well as the interactions between tip cells, GFAP-positive astrocytes, and the end-foot regions of Müller cells are shown.
  • Hyperoxia-normal mice are normal mouse neovascularization model group
  • hyperoxia-knockout mice are miR-15a-5p knockout mouse neovascularization model group
  • the mimic control is scrambled Agomir
  • the mimic is Agomir.
  • Figure 37 Flowchart of the laser-induced choroidal neovascularization model.
  • FIG 38 Fundus fluorescein angiography (FFA) observed the inhibitory effect of miR-15a-5p on choroidal neovascularization.
  • Figure 40 Choroidal flat mounts were stained with IB4 to quantify the area of neovascularization clusters to observe the inhibitory effect of miR-15a-5p on choroidal neovascularization. IB4 positivity indicates neovascularization clusters.
  • FIG. 41 Quantitative analysis results of new blood vessel clusters.
  • Figure 42 Schematic diagram of fluorescence of retinal choroid infected with adeno-associated virus containing miR-15a-5p.
  • Figure 43 PCR results show the overexpression of miR-15a-5p in the retina and choroid after adeno-associated virus infection of the retina and choroid, where CNV-AAV-NC is a choroidal neovascularization group injected with a control virus, and CNV-AAV-15a is a choroidal neovascularization group injected with adeno-associated virus containing miR-15a-5p.
  • FIG44 Schematic diagram of IB4 staining of choroidal flat mounts to quantify the area of neovascular clusters and observe the inhibition of choroidal neovascularization by adeno-associated virus containing miR-15a-5p.
  • Figure 45 Statistical results of IB4 staining of choroidal flat mounts to quantify the area of neovascularization clusters and observe the inhibition of choroidal neovascularization by adeno-associated virus containing miR-15a-5p.
  • Figure 46 H&E staining of the retina shows the interlayer structure and cell morphology of the retina.
  • the asterisk indicates the outer plexiform layer.
  • GCL stands for ganglion cell layer
  • IPL stands for inner plexiform layer
  • INL stands for inner nuclear layer
  • OPL stands for outer plexiform layer
  • ONL stands for outer nuclear layer
  • RPE stands for pigment epithelium.
  • Normoxia represents untreated mice, and hyperoxia represents oxygen-induced retinal neovascularization model mice.
  • FIG. 47 Schematic diagram of quantification of retinal layer thickness using optical coherence tomography (OCT) at postnatal day 42 in mice.
  • IPL stands for inner plexiform layer
  • INL stands for inner nuclear layer
  • OPL stands for outer plexiform layer
  • ONL stands for outer nuclear layer.
  • Normoxia represents untreated mice
  • hyperoxia represents Representative rat model of oxygen-induced retinal neovascularization.
  • Figure 48 Topographic maps of retinal layer thickness quantified using optical coherence tomography (OCT) at postnatal day 42.
  • OCT optical coherence tomography
  • Figure 49 Statistical results of quantifying the thickness of each retinal layer using optical coherence tomography (OCT) on postnatal day 42 of mice to determine the protective effect of miR-15a-5p on retinal structure.
  • OCT optical coherence tomography
  • Normoxia represents untreated mice
  • hyperoxia represents oxygen-induced retinal neovascularization model mice.
  • Figure 50 Schematic diagram of the analysis of retinal function using retinal electrophysiological examination (ERG) on postnatal day 25 and 42 of mice.
  • Normoxia represents untreated mice, and hyperoxia represents oxygen-induced retinal neovascularization model mice.
  • Figure 51 Statistical results of retinal function analysis using retinal electrophysiological examination (ERG) at 25 and 42 days after birth, confirming the protective effect of miR-15a-5p on retinal function.
  • Normoxia represents untreated mice, and hyperoxia represents oxygen-induced retinal neovascularization model mice.
  • Figure 52 H&E staining of the retina and choroid shows the interlayer structure and cell morphology of the retina and choroid.
  • GCL stands for ganglion cell layer
  • IPL stands for inner plexiform layer
  • INL stands for inner nuclear layer
  • OPL stands for outer plexiform layer
  • ONL stands for outer nuclear layer
  • RPE stands for pigment epithelium.
  • Figure 53 Schematic diagram of analyzing retinal function in choroidal neovascularization model using retinal electrophysiological examination (ERG).
  • the mimic control is scrambled Agomir, the mimic is Agomir, and the VEGF monoclonal antibody is Ranibizumab.
  • Figure 54 Statistical results of analyzing retinal function in choroidal neovascularization model using retinal electrophysiological examination (ERG).
  • the mimic control was scrambled Agomir, the mimic was Agomir, and the VEGF monoclonal antibody was Ranibizumab.
  • Figure 55 miR-15a-5p inhibits glial proliferation in the retina of OIR.
  • A Representative images of GFAP immunostaining in retinal sections of normoxic and hyperoxic mice.
  • B Quantification and comparison of GFAP intensity in the above groups.
  • C Western blot showing GFAP expression in the retina of normoxic and hyperoxic mice.
  • D GFAP protein levels were quantified by densitometry with GAPDH levels as an internal reference.
  • FIG. 56 Enzyme-linked immunosorbent assay (ELISA) was used to detect the TNF ⁇ content in the retina of mice with oxygen-induced retinopathy at different time points.
  • ELISA Enzyme-linked immunosorbent assay
  • FIG 57 Western Blot showing the expression levels of intercellular adhesion molecule 1 (ICAM-1) in the retina of each group of mice with oxygen-induced retinopathy.
  • ICM-1 intercellular adhesion molecule 1
  • FIG 58 Western Blot shows the statistical results of the expression levels of intercellular adhesion molecule 1 (ICAM-1) in the retina of each group of mice with oxygen-induced retinopathy.
  • ICM-1 intercellular adhesion molecule 1
  • Figure 59 PCR results show the levels of laser-induced inflammatory factors in the retina of each group of mice.
  • A is TNF ⁇
  • B is ICAM-1
  • C is IL-1 ⁇ .
  • Figure 60 PCR results show that miR-15a-5p can reduce the mRNA level of VEGF in RPE cells induced by TGF- ⁇ 1.
  • Figure 61 Western Blot shows that miR-15a-5p can reduce the increased protein level of VEGF in RPE cells caused by TGF- ⁇ 1.
  • Figure 62 Western Blot showed that miR-15a-5p can reduce the increased protein level of VEGF in RPE cells caused by TGF- ⁇ 1.
  • Figure 64 Dual luciferase reporter assay shows that miR-15a-5p can target and bind to VEGF mRNA in vitro, where the pmirGLO vector is an empty plasmid, the wild type is the VEGF base sequence, and the mutant type is the VEGF base sequence with different bases in the binding region.
  • Figure 65 Intravitreal injection of Agomir-15a-5p mimetic can reverse the hyperoxia-induced increase in VEGF expression levels in the mouse retina.
  • FIG. 66 Intravitreal injection of Agomir-15a-5p mimics can reverse the hyperoxia-induced increase in VEGF expression levels in the mouse retina.
  • FIG. 67 Intravitreal injection of Agomir-15a-5p mimics can reverse laser-induced increase in VEGF expression levels in mouse retina.
  • Figure 68 miR-15a-5p mimics can inhibit the activation of retinal ERK phosphorylation signals for a longer period of time than VEGF monoclonal antibodies.
  • Figure A is a schematic diagram. After intravitreal injection on the 12th day after birth, the retina was collected at P13, P14, P15, P17, P20, and P25.
  • Figure B shows that at P12, the phosphorylation signal of ERK in the retina of the hyperoxia group of mice was enhanced.
  • Figure C shows that at P13, the phosphorylation signal of ERK in the retina of the VEGF monoclonal antibody group was reduced.
  • Figure D shows that at P14, the phosphorylation signal of ERK in the retina of the miR-15a-5p mimic group and the VEGF monoclonal antibody group was reduced.
  • Figure E shows that at P15, the phosphorylation signal of ERK in the retina of the miR-15a-5p mimic group was reduced.
  • Figure F shows that at P17, the phosphorylation signal of ERK in the retina of the miR-15a-5p mimic group was reduced.
  • Figure G shows that at P20, there was no significant difference in the phosphorylation signal of ERK in each retina.
  • Figure H shows that at P25, there was no significant difference in the phosphorylation signal of ERK in each retina.
  • Figures I, J, K, L, M, N, and O are the statistical results of P12, P13, P14, P15, P17, P20, and P25, respectively.
  • Figure P shows the relative expression of retinal phosphorylated ERK signal in the retina of each group of mice at different time points.
  • Figure 69 PCR results show that miR-15a-5p can reduce the mRNA level of Smad2 in retinal vascular endothelial cells.
  • Figure 70 Schematic diagram of Western Blot results showing that miR-15a-5p can reduce the protein level of Smad2 in retinal vascular endothelial cells.
  • Figure 71 Western Blot results showed that miR-15a-5p can reduce the protein level of Smad2 in retinal vascular endothelial cells.
  • Figure 72 Dual luciferase reporter assay shows that miR-15a-5p can target and bind to Smad2 mRNA in vitro.
  • the pmirGLO vector is an empty plasmid, the wild type is the Smad2 base sequence, and the mutant type is the Smad2 base sequence with different bases in the binding region.
  • Figure 73 Immunofluorescence staining results of fibrosis markers ⁇ -SMA and CD31 in retinal vascular endothelial cells stimulated by TGF- ⁇ 1 after transfection of miR-15a-5p mimics.
  • Figure 74 Immunofluorescence staining results of vimentin in retinal vascular endothelial cells stimulated with TGF- ⁇ 1 after transfection with miR-15a-5p mimics.
  • Figure 75 Western Blot diagram showing the changes in fibrosis markers vimentin, ⁇ -SMA, and CD31 in retinal vascular endothelial cells stimulated by TGF- ⁇ 1 after transfection with miR-15a-5p mimics.
  • Figure 76 Western Blot statistical results of the changes in fibrosis markers vimentin, ⁇ -SMA and CD31 in retinal vascular endothelial cells stimulated by TGF- ⁇ 1 after transfection with miR-15a-5p mimics.
  • Figure 77 Western Blot diagram of the changes in phosphorylated-Smad2 and total Smad2 in retinal vascular endothelial cells stimulated by TGF- ⁇ 1 after transfection of miR-15a-5p mimics.
  • Figure 78 Western Blot statistical results of phosphorylated-Smad2 and total Smad2 changes in retinal vascular endothelial cells stimulated by TGF- ⁇ 1 after transfection of miR-15a-5p mimics.
  • Figure 79 Western Blot results showing the expression levels of fibrosis-related proteins after Müller cells were stimulated by different concentrations of TGF- ⁇ 2.
  • Figure 80 Western Blot results show the statistical results of the expression levels of fibrosis-related proteins after Müller cells were stimulated by different concentrations of TGF- ⁇ 2.
  • Figure 81 Western Blot results showing the expression levels of fibrosis-related proteins in Müller cells stimulated by TGF- ⁇ 2 after transfection of miR-15a-5p mimics and mimic controls.
  • Figure 82 Western Blot results show the statistical results of the expression levels of fibrosis-related proteins after TGF- ⁇ 2 stimulation of Müller cells after transfection of miR-15a-5p mimics and mimic controls.
  • Figure 83 Immunofluorescence staining results of the cell marker GS in retinal Müller cells stimulated with TGF- ⁇ 2 after transfection of miR-15a-5p mimics.
  • Figure 84 Immunofluorescence staining results of fibrosis marker ⁇ -SMA and activation marker GFAP in retinal Müller cells stimulated with TGF- ⁇ 2 after transfection of miR-15a-5p mimics.
  • Figure 85 PCR results show the expression level of TNF- ⁇ after TGF- ⁇ 2 stimulation of Müller cells after transfection of miR-15a-5p mimics and mimic control.
  • FIG86 PCR results show the expression level of MCP-1 in Müller cells stimulated by TGF- ⁇ 2 after transfection of miR-15a-5p mimics and mimic control.
  • FIG 87 Western Blot results show the total protein and phosphorylation levels of Smad2 in Müller cells stimulated by TGF- ⁇ 2 after transfection of miR-15a-5p mimics and mimic controls.
  • P-Smad2 is phosphorylated Smad2
  • T-Smad2 is total Smad2.
  • Figure 88 Western Blot results show the statistical results of total Smad2 protein and phosphorylation levels after TGF- ⁇ 2 stimulation of Müller cells after transfection of miR-15a-5p mimics and mimic controls.
  • P-Smad2 is phosphorylated Smad2
  • T-Smad2 is total Smad2.
  • Figure 89 Retinal cryosections of each group of hyperoxia-induced mice show the expression and localization of ⁇ -SMA and retinal blood vessels.
  • Figure 90 Cryosections of the retinas of each group of mice induced by hyperoxia show the expression and localization of fibronectin and retinal blood vessels.
  • Figure 91 Retinal cryosections of each group of mice induced by hyperoxia show the expression and localization of ⁇ -SMA and activated Müller cells in the retina, among which GFAP indicates activated Müller cells.
  • Figure 92 Western Blot detection shows the expression of retinal fibrosis proteins.
  • Figure A shows the expression of retinal fibronectin in each group of hyperoxia-induced mice.
  • Figure B shows the statistical results of fibronectin expression levels.
  • Figure C shows the expression of retinal TGF ⁇ receptor 2 protein and ⁇ -SMA protein in each group of hyperoxia-induced mice.
  • Figure D shows the statistical results of TGF ⁇ receptor 2 protein and ⁇ -SMA protein expression levels.
  • Figure E shows the expression of phosphorylated Smad2 and total Smad2 in the retina of each group of hyperoxia-induced mice.
  • Figure F shows the statistical results of retinal phosphorylated Smad2 and total Smad2 expression levels.
  • Figure 93 Safety assessment of intraocular administration of miR-15a-5p on oxygen-induced mouse development.
  • Figure A shows the weight changes of mice from the day of administration P12 (12 days after birth) to the mice at P42 (42 days after birth).
  • Figure B shows the plasma color of each group.
  • Figure C shows the serum creatinine level of mice.
  • Figure D shows the serum urea level of mice.
  • Figure E shows the plasma triglyceride level of mice.
  • Figure F shows the total cholesterol level of mice.
  • Figures G and H show H&E staining of the liver and kidney structures of mice 17 days (P17), 25 days (P25), and 42 days (P42) after birth.
  • Figure 94 Safety assessment of intraocular administration of miR-15a-5p on normal mouse development and retina.
  • Figure A shows the weight change of mice from P12 (12 days after birth) on the day of administration to P42 (42 days after birth) when the mice are basically adults.
  • Figure B shows the serum creatinine level of mice.
  • Figure C shows the serum urea level of mice.
  • Figure D shows the plasma triglyceride level of mice.
  • Figure E shows the plasma total cholesterol level of mice.
  • Figure F shows the fluorescent sections of liver and kidney at different time points after injection.
  • Figures G and H show H&E staining of liver and kidney structures of mice 17 days (P12), 25 days (P25), and 42 days (P42) after birth.
  • Figures I, J, and K are OCT results showing the effects of intravitreal injection of miR-15a-5p mimics, mimic controls, and VEGF monoclonal antibodies on retinal thickness.
  • Figures L and M are retinal frozen sections showing the activation of retinal Müller cells after intravitreal injection of miR-15a-5p mimics, mimic controls, and VEGF monoclonal antibodies.
  • Figure 95 The therapeutic effect of intravitreal injection of miR-15a-5p mimics on retinal photoreceptor damage and retinal bipolar cell damage in diabetic mice (leptin receptor deficient model mice) was observed.
  • the a wave represents the response of photoreceptor cells to light stimulation
  • the b wave represents the response of bipolar cells to light stimulation
  • the op wave is a group of oscillating potentials in the b wave, which is generally believed to represent the response of bipolar cells-amarcine cells to light stimulation.
  • Figure 96 Statistical results of the therapeutic effect of intravitreal injection of miR-15a-5p mimics on retinal photoreceptor damage in diabetic mice (leptin receptor-deficient model mice), where the b wave represents the response of photoreceptor cells to light stimulation.
  • Figure 97 Statistical results of the therapeutic effect of intravitreal injection of miR-15a-5p mimics on retinal bipolar cell damage in diabetic mice (leptin receptor-deficient model mice), where the a wave represents the response of bipolar cells to light stimulation.
  • Figure 98 Statistical results of the therapeutic effect of intravitreal injection of miR-15a-5p mimics on retinal bipolar cell damage in diabetic mice (leptin receptor deficient model mice).
  • the op wave is a group of oscillatory potentials in the b wave, which is generally considered to represent the response of bipolar cells and amacrine cells to light stimulation.
  • Figure 99 There are differences in the growth of superficial retinal blood vessels along the astrocyte template between normal mice and knockout mice at P7.
  • A shows a representative image of superficial retinal blood vessels stained with IB4. The magnifications from left to right are 5X, 10X and 20X.
  • B shows the statistical results of the area covered by superficial blood vessels in the retina.
  • C shows the statistical results of the number of nodes generated by the intersection of superficial blood vessels.
  • D shows the statistical results of the total length of superficial blood vessels.
  • E shows the statistical results of the total branch length of superficial blood vessels.
  • F shows a representative image of superficial retinal blood vessels stained with IB4 and GFAP.
  • G shows the statistical results of the number of nodes generated by the intersection of superficial blood vessels in the GFAP-positive area.
  • H shows the statistical results of the total length of the lumen in the GFAP-positive area.
  • Area I shows the statistical results of the degree of overlap between superficial blood vessels and GFAP-positive areas.
  • Figure 100 There are differences in the growth of superficial and deep retinal vascular plexuses between normal mice and knockout mice at P9.
  • A shows a representative image of IB4-stained superficial retinal vessels. The magnifications from left to right are 5X, 10X, and 20X.
  • B shows a representative image of IB4-stained deep retinal vessels covering the retina.
  • C shows the statistical results of the number of nodes generated by the interlacing of superficial blood vessels.
  • D shows the statistical results of the number of superficial vascular meshes.
  • E shows the statistical results of the total length of superficial blood vessels.
  • F shows the statistical results of the total branch length of superficial blood vessels.
  • G shows the statistical results of the applied blood vessel coverage area.
  • Figure 101 Therapeutic effect of miR-15a-5p mutant on retinal neovascularization.
  • HRMEC Human retinal microvascular endothelial cells
  • ECM endothelial cell basal medium
  • ECGS endothelial cell growth supplement
  • CCK-8/WST-8 kit digest cells and make single cell suspension, inoculate at a density of 2000 cells/well in 96-well plates, set up 5 replicates for each group, change the medium after the cells adhere to the wall, add the target culture medium and detect cell activity for 24 hours. Before the test, add 10 ⁇ l CCK-8 reagent to each well, incubate at 37°C in the dark for 2h; then move the incubated culture medium to the ELISA plate, and measure the absorbance at 450nm using BIO-RAD ELISA reader.
  • HRMECs were seeded at a density of 2000 cells/well in a 96-well plate and cultured overnight in a cell culture incubator. 5 ⁇ l Opti-MEM medium was added to each of the two EP tubes, and 0.15 ⁇ l Lipofectamine 3000 reagent was added to the first tube, and 0.15 ⁇ l of mimic control/mimic/inhibitor control/inhibitor was added to the second tube. The two tubes of liquid were mixed, incubated at room temperature for 5 minutes, and then 10 ⁇ l of the mixture was added to one well. When preparing the liquid, it is generally necessary to calculate the total amount of liquid added to all wells, add it to each well after mixing, and do not prepare the liquid for each well separately to avoid errors. Other mimic controls, inhibitor controls, and inhibitors are also transfected into cells using the same method, but the inhibitor controls and inhibitors need to be added in twice the amount of the mimics.
  • reagents to a 200 ⁇ l EP tube according to the following ratios.
  • the volume of RNA varies according to the RNA concentration.
  • the total RNA volume is 500ng.
  • 1 ⁇ l of gDNA remover mix well, and store at room temperature for 5 minutes.
  • U6 is used as the internal reference for sEVs, and cel-miR-39 is used as the external reference for plasma and vitreous.
  • the expression level of miRNA is expressed as 2 ⁇ - ⁇ ct .
  • the expression of each miRNA Primer sequences are shown in Table 3.
  • HRMECs were digested and inoculated on 6-well plates. When the cell confluence reached 75%-80%, the mimic control, mimic, inhibitor control and inhibitor were transfected respectively. After 6 hours, the medium containing the transfection reagent was aspirated and a line was drawn in the center of each well with the same force using a 1ml pipette tip. Then, the cells were washed twice with PBS buffer to remove the detached cells. The photos were taken under a microscope and saved. The location of the photos was recorded to ensure that the photos were taken at the same location later. After 24 hours, the photos were taken again and saved. The pictures were analyzed and the area of the scratch was calculated using image J software.
  • Transwell cell migration assay was used to detect the effect of miR-15a-5p transfection on cell migration ability.
  • HRMECs were digested with trypsin, and 8000 cells were seeded into the upper chamber of the wells. After 24 hours of culture, they were washed three times with PBS. The cells were then fixed with PFA and stained with 0.01% crystal violet buffer. Five fields of view were randomly selected under a microscope, and the number of cells that migrated was counted.
  • Matrigel (Corning, Cat. No. 354234) was melted at 4°C for 12 hours, and an appropriate amount of Matrigel was spread on the surface of the 48-well cell plate to provide support for lumen formation. Then it was solidified at 37°C for 40 minutes. After pretreatment, HRMECs were seeded into a 48-well plate coated with Matrigel. After 3 hours, five areas of each well were collected using an inverted microscope. Image-J software was used to calculate the total length of the vascular lumen and the number of lumen nodes.
  • HRMECs were fixed in 4% paraformaldehyde for 15 minutes and soaked in PBS with 0.1 Triton-X100 (PBST) for 15 minutes. After blocking with 5% bovine serum albumin (BSA) at room temperature, the cells were incubated with primary antibodies vimentin (1:300, Abcam, ab45939), a-SMA (1:500, Abcam, ab124964), and CD31 (1:150, Abcam, ab24590) at 4°C overnight. After washing three times with PBST.
  • BSA bovine serum albumin
  • the cells were incubated with Alexa fluor 488-conjugated goat anti-rabbit IgG H&L (1:1000, Abcam) for vimentin, or with 'Alexa fluor 647-conjugated goat anti-mouse IgG H&L (1:1000, Abcam) at room temperature.
  • the nuclei were labeled with 4,6-diamino-2-phenylindole (1:500, Solarbio).
  • the cells were observed and photographed under a confocal laser scanning microscope (LSM800, Zeiss, Germany).
  • the target genes of the screened differential miRNAs were predicted in three databases: TargetScan, PITA, and microRNAorg.
  • Luciferase reporter assay was used to verify whether Smad2 is a target gene of miR-15a-5p.
  • Dual luciferase reporter plasmid pmIRGLO-Smad2 wild type and pmIRGLO-Smad2 mutant were constructed, and the empty plasmid was used as a control plasmid.
  • the reporter plasmid was co-transfected into HEK-293 cells with miR-15a-5p mimics or scrambled control. After 48 hours of transfection, the cells were lysed and the supernatant was collected according to the steps of the dual luciferase reporter assay system (Gene Pharma, Cat. No. G06001).
  • TECAN Infinite 200 (Germany) was used to detect the activity of the firefly luciferase reporter gene and the Renilla luciferase reporter gene. The ratio of firefly luciferase, with Renilla luciferase as the relative luciferase activity. Each experiment was repeated 3 times. The process of verifying whether VEGF is a target gene of miR-15a-5p is the same as above.
  • C57BL/6J mice were used to establish an oxygen-induced neovascular retinopathy model. Newborn mice and lactating female mice were exposed to 75% oxygen from day 7 (P7) to day 12 (P12) after birth. On day 12, the mice were removed from the oxygen chamber and placed in normoxia. Since hypoxia can cause neovascularization, retinal neovascularization peaks on day 17 (P17) after birth. This is accompanied by retinal inflammation, neural damage, and fibrotic changes.
  • a modified miR-15a-5p mimic (Agomir) at a concentration of 1 ⁇ g was injected into the vitreous cavity of mice using a 34-gauge needle (Hamilton, Reno, NV, United States). The same number of mice were injected with scrambled Agomir as a negative control group.
  • 1 ⁇ l of adeno-associated virus was injected into the vitreous cavity of mice at a titer of 1 ⁇ 10 12 .
  • Antibiotic gel was used to cover the ocular surface after injection to prevent corneal edema.
  • mice Male 6-week-old C57BL/6J mice were used for this experiment and were anesthetized and inserted into the scalpel using a 34-gauge needle (Hamilton, Reno, NV, United States). Unmodified miR-15a-5p mimics (mimics) or modified miR-15a-5p mimics (Agomir) at a concentration of 1 ⁇ g (Genepharma) were injected into the vitreous cavity of mice, and the same number of mice were injected with PBS as a negative control group. The retinas were harvested 8 hours, 24 hours, 48 hours, 5 days and 7 days after injection to detect the level of miR-15a-5p.
  • mice were killed at P17 and their eyeballs were removed. The retinas were peeled off, fixed with 4% paraformaldehyde, and cut into 4 radial flaps.
  • Goat serum was used for blocking for 2 hours, and retinal blood vessels were stained using IB4 (Thermo, Catalog No. I21411) or anti-glial fibrillary acidic protein (GFAP) antibody (Abcam, Catalog No. ab7260). After washing for 5 hours, goat anti-rabbit IgG (Abcam 150077) was used as a secondary antibody for staining, and anti-fluorescence attenuation mounting medium was used for mounting after washing for 5 hours. Retinal blood vessel images were taken using a confocal laser scanning microscope (LSM800, Zeiss, Germany). Photoshop was used for quantitative analysis of new blood vessels and non-perfused areas.
  • the eyeballs were dissected and quickly frozen in embedding gel.
  • the retina was sliced (8 ⁇ m) and fixed in 4% paraformaldehyde for 20 minutes at room temperature. Then they were incubated with GFAP antibody, ⁇ -SMA (Abcam, Catalog No. ab1224964), Fibronectin (Abcam, Catalog No. ab45688), and IB4 (Thermo, Catalog No. I21411) at 4°C overnight. After washing three times with PBS, the retinal sections were incubated with Alexa Fluor488-conjugated IgG (Abcam, Catalog No.
  • TUNEL assay was performed on retinal cryosections using the TUNEL system (Roche, Cat. No. 11684795910) according to the manufacturer's instructions.
  • Eyes were removed from mice sacrificed by cervical dislocation at P17, kidneys and livers were removed and fixed at P25 and P42, and the specimens were stained with hematoxylin and eosin (H&E) and imaged using light microscopy.
  • H&E hematoxylin and eosin
  • Mice were dark-adapted for 18 h and the light was emitted at a range of 0.01 to 1 cd-s/m according to the manufacturer's instructions (Phoenix Micron VI). Flash white light.
  • the German Heidelberg SPECTRALIS-OCT was used to observe the changes in the fundus structure of mice.
  • Tropicamide was used to dilate the pupils of both eyes of the mice with eye drops.
  • sodium hyaluronate gel was applied to the eyes of the mice for retinal scanning. The scanning image was centered on the mouse optic disc, and the full thickness of the retina was scanned.
  • mice Male 6-week-old C57BL/6J mice were used in this experiment. After anesthesia, the pupils were dilated and the Phoenix laser transmitter was used to irradiate the retina to destroy the retinal pigment epithelium and choroid. Seven days later, new blood vessels were generated from the choroid and invaded the retina.
  • mice Male 6-week-old C57BL/6J mice were used for this experiment. After anesthesia, the pupils were dilated and a 34-gauge needle (Hamilton, Reno, NV, United States) was used to insert the needle from the limbus of the cornea and sclera. The retina was lifted and 1 ⁇ l of adeno-associated virus was injected into the subretina of the mouse. The titer was 1 ⁇ 10 12 . After injection, antibiotic gel was used to cover the ocular surface to prevent corneal edema.
  • a 34-gauge needle Hemlton, Reno, NV, United States
  • OIR mice were killed by cervical dislocation 7 days after modeling, and their eyeballs were removed. The choroid was peeled off, fixed with 4% paraformaldehyde, and cut into 4 radial flaps. Goat serum was used for blocking for 2 hours, and choroidal blood vessels were stained using IB4 (Thermo, catalog number I21411). After washing for 5 hours, anti-fluorescence attenuation sealing agent was used for sealing. Choroidal blood vessel images were taken using a confocal laser scanning microscope (LSM800, Zeiss, Germany). Photoshop was used for quantitative analysis of new blood vessels.
  • Blood samples were collected from behind the eyeball, centrifuged at 2000 g for 15 minutes, serum was separated, and then stored at -80°C. The liver, spleen, brain, and kidneys were dissected out.
  • the major organ samples liver, spleen, kidney
  • the major organ samples were fixed with 4% neutral buffered paraformaldehyde for 24 h, and the samples were paraffin embedded, cut at 3 ⁇ m and stained with hematoxylin and eosin.
  • Micrographs were taken using an Olympus BX51 microscope and an Olympus DP71CCD camera (Olympus Corporation, Tokyo, Japan).
  • Serum concentrations of creatinine (Elabscience, Catalog No. EBCK188M), urea nitrogen (Elabscience, Catalog No. EBCK183M), triglycerides (Elabscience, Catalog No. EBCK126M), and total cholesterol (Elabscience, Catalog No. EBCK109S) were measured using commercially available test kits. The concentration of each parameter was calculated according to the manufacturer's instructions.
  • Mimics (miR-15a-5p mimics) were purchased from Shanghai Jima Pharmaceutical Technology Co., Ltd., catalog number B02001;
  • Agomir (Agomir-15a-5p mimetic) was purchased from Shanghai Jima Pharmaceutical Technology Co., Ltd., catalog number B06001; the structure is: The strand is SEQ ID NO: 1, and the antisense strand is SEQ ID NO: 6, which is modified with two thio backbones at the 5' end, four thio backbones at the 3' end, cholesterol at the 3' end, and methoxyl modifications throughout the strand.
  • Smad2-WT vector was purchased from Shanghai Jima Pharmaceutical Technology Co., Ltd., catalog number C09005-36176;
  • the Smad2-mut vector was purchased from Shanghai Jima Pharmaceutical Technology Co., Ltd., catalog number C09006-36176.
  • VEGF-WT vector was purchased from Shanghai Jima Pharmaceutical Technology Co., Ltd., catalog number C09005-56969;
  • VEGF-mut vector was purchased from Shanghai Jima Pharmaceutical Technology Co., Ltd., catalog number C09006-56969.
  • Adeno-associated virus was purchased from Shanghai Jima Pharmaceutical Technology Co., Ltd., catalog number D08001.
  • Example 1 Transfection of miR-15a-5p mimics or inhibitors into retinal vascular endothelial cells can increase and decrease the expression level of miR-15a-5p, respectively
  • MiRNA mimics and inhibitors can simulate its biological effects in vitro.
  • the miR-15a-5p mimics are the base sequences of miR-15a-5p synthesized in vitro
  • the miR-15a-5p inhibitors are the base complementary sequences of miR-15a-5p synthesized in vitro.
  • Antisense strand CACAAACCAUUAUGUGCUGCUA (SEQ ID NO: 6).
  • the retina and choroid contain a large number of blood vessels, among which endothelial cells are important cells for maintaining vascular permeability. Therefore, it is representative to select retinal endothelial cells (HRMECs) for experiments.
  • HRMECs retinal endothelial cells
  • Example 1 shows that miR-15a-5p mimics and inhibitors can be transfected into HRMECs in vitro to overexpress or downexpress miR-15a-5p, providing a research basis for subsequent observation of the effects of miR-15a-5p on cells.
  • Example 2 Optimal concentration of VEGF to stimulate pathological proliferation of retinal vascular endothelial cells
  • VEGF is a known potent factor that induces angiogenesis in vivo and in vitro, and has a strong pathogenic effect in retinopathy of prematurity, diabetic retinopathy, and choroidal neovascularization.
  • VEGF acting on HRMECs can establish a model of pathological proliferation. Therefore, it is necessary to determine the concentration of VEGF stimulation.
  • 0, 10, and 20 ng/mL of VEGF were selected to stimulate HRMECs, respectively.
  • the CCK-8 test results showed that concentrations above 10 ng/mL can cause significant proliferation of HRMECs (Figure 2), and this concentration will not cause changes in the expression of miR-15a-5p in HRMECs ( Figure 3).
  • Example 3 The therapeutic effect of miR-15a-5p on pathological proliferation of retinal vascular endothelial cells in vitro
  • VEGF can cause endothelial cell proliferation in vivo, thereby inducing the development of new blood vessels.
  • New blood vessels are different from physiological blood vessels.
  • the walls of new blood vessels lack tight junctions, and the substances inside leak into the retina and vitreous through the walls, causing fundus exudation and hemorrhage, which greatly affects normal visual function.
  • miR-15a-5p mimics After transfecting miR-15a-5p mimics into HRMECs in vitro, VEGF-induced abnormal cell proliferation, migration and lumen formation can be reduced. This shows that miR-15a-5p mimics have a significant therapeutic effect on the pathological proliferation of HRMECs in vitro.
  • Common miRNA mimics are mimics, which are composed of unmodified bases, and in vitro, cells have a good absorption effect on them.
  • unmodified bases are easily degraded by ubiquitous nucleases in the body. Therefore, this embodiment uses modified miRNA, such as adding cholesterol and other modifications.
  • this embodiment compares the efficiency of mice absorbing the same amount of miR-15a-5p mimics at different times. Among them, the modified mimic is Agomir. As shown in Figure 12, after 24 hours of injection of the mimic, there is a significant difference.
  • Agomir has good tissue compatibility, and the absorption effect of the retina on Agomir is 2.5 times that of mimic, and the high level continues until the 7th day after injection. Therefore, in subsequent animal experiments, Agomir (Agomir-15a-5p) was selected as a mimic of miR-15a-5p for in vivo injection.
  • Example 5 Therapeutic effect of miR-15a-5p on hyperoxia-induced retinal neovascularization in mice
  • OIR oxygen-induced retinopathy
  • VEGF monoclonal antibody was selected as a positive control.
  • the results showed that 2 ⁇ g of VEGF monoclonal antibody had the best inhibitory effect on retinal blood vessels, and then 2 ⁇ g was selected as the treatment concentration for further verification (Figure 17).
  • agomir-15a-5p labeled with CY3 was injected into the vitreous body of mice. It was diffusely distributed in the retina 8 hours after injection, and the fluorescence intensity reached a peak value 24 hours after injection (A, B in Figure 18).
  • the polymerase chain reaction was used to detect the increase in agomir in the retina.
  • Agomir-15a-5p increased by about 2-3 times 24 hours after injection, reached a peak concentration of about 3-5 times 48 hours after injection, and its high expression lasted for 5 days (C in Figure 18).
  • Agomir-15a-5p mimics can reduce retinal neovascularization to 65% of the control group ( Figures 19-20). Compared with VEGF monoclonal antibody, Agomir-15a-5p mimics can also promote the recovery of retinal non-perfusion areas, and the area of retinal non-perfusion areas is reduced to 73% of the control group ( Figures 19 and 21).
  • Adeno-associated virus is an efficient gene delivery tool. It can infect retinal cells and make them highly express miR-15a-5p.
  • the nucleic acid sequence carried by the adeno-associated virus is TAGCAGCACATAATGGTTTGTG (SEQ ID NO: 9), which can be transcribed into miR-15a-5p in vivo and exert a therapeutic effect.
  • adeno-associated viruses (1 ⁇ 10 12 ) containing miR-15a-5p and control viruses (1 ⁇ 10 12 ) were injected intraocularly.
  • the virus structure is shown in FIG22 .
  • the retina was sampled, and the site of adeno-associated virus infection was observed by frozen sections of the eyeball.
  • the results in FIG23 show that the adeno-associated virus containing miR-15a-5p infected the retina, and diffuse fluorescence appeared in the retina. Subsequently, the miR-15a-5p content of each group of retina was detected by PCR. The results are shown in FIG24 .
  • the miR-15a-5p in the retina of the group injected with adeno-associated viruses containing miR-15a-5p was 13 times that of the control group, indicating that the virus can successfully overexpress miR-15a-5p.
  • the retinal neovascularization of oxygen-induced mice was quantified, and the results showed that the retinal neovascularization and non-perfused areas in the group injected with adeno-associated viruses containing miR-15a-5p were significantly reduced (FIG25-FIG27).
  • adeno-associated virus containing miR-15a-5p can successfully deliver miR-15a-5p into mouse retinal cells, and has a therapeutic effect on hyperoxia-induced retinal neovascularization in mice and can promote the recovery of the non-perfused area.
  • Example 5 uses two methods to deliver miR-15a-5p, one is modified miR-15a-5p, and the other is adeno-associated virus.
  • the purpose of increasing the miR-15a-5p content in the retina was achieved by injecting two substances into the vitreous cavity.
  • the oxygen-induced retinopathy model adopted is a classic fundus disease model that can simulate the neovascularization phenotype of various fundus diseases, such as diabetic retinopathy and retinopathy of prematurity. This example observed the therapeutic effect of miR-15a-5p on the neovascularization and non-perfused areas of the model, and found that miR-15a-5p has a therapeutic effect on retinal neovascularization and can promote the recovery of non-perfused areas.
  • endothelial apical cells and astrocytes are essential for vascular remodeling in non-perfused areas.
  • vascular reconstruction in the non-perfused area we observed the relationship between astrocytes and vascular sprouts in the mouse retina on day 17.
  • Astrocytes in the vascular occlusion area of the OIR mouse retina degenerated, and the loss of astrocytes was accompanied by increased GFAP reactivity of Müller cells, which was manifested as spotted staining at the ends of Müller cells in the superficial vascular plexus ( Figure 28A, second column).
  • Figure 28A shows that retinal astrocytes in the normoxic group showed astrocyte stretching morphology, and the hyperoxia control treatment group lacked stretched astrocyte morphology. Instead, the spotted irregular cell morphology is the footplate of activated Müller cells, representing the increased inflammatory reactivity of Müller cells.
  • Figure 28B shows the filopodia emitted by the endothelial tip at the edge of the non-perfused area, indicating the reconstruction of the degenerated blood vessels.
  • the miR-15a-5p-mediated vascular rescue in the non-perfused area of the retina is related to the protection of endogenous astrocytes in the vascular occlusion area.
  • Example 7 Effects of miR-15a-5p deficiency on hyperoxia-induced retinal neovascularization and non-perfused areas in mice
  • the inhibitory effect of miR-15a-5p on neovascularization was verified in miR-15a-5p knockout mice.
  • the oxygen-induced retinopathy (OIR) model was selected to further verify the role of miR-15a-5p in retinal angiogenesis.
  • Normal mice and knockout mice were placed in a hyperoxic environment to induce pathological retinal neovascularization ( Figure 29).
  • Retinal neovascularization clusters and vascular occlusion areas were analyzed by IB4 staining on the 17th day after birth. The results showed that miR-15a-5p deficiency significantly increased the non-perfused area and neovascularization area of the OIR retina ( Figure 30- Figure 31).
  • Example 8 Therapeutic effect of miR-15a-5p on laser-induced choroidal neovascularization in mice
  • the inhibitory effect of MiR-15a-5p on neovascularization was also verified in the choroidal neovascularization model.
  • a laser was used to induce a choroidal neovascularization model.
  • the model construction process is shown in Figure 37, and the Agomir-15a-5p mimic, mimic control and VEGF monoclonal antibody were injected into the mouse vitreous cavity.
  • fundus fluorescein angiography observed that the fluorescence leakage area of the Agomir-15a-5p mimic and VEGF monoclonal antibody groups was significantly reduced compared with the control group, indicating that both can inhibit the formation of choroidal neovascularization ( Figure 38- Figure 39).
  • mice with choroidal neovascularization was taken for IB4 staining to quantify the area of the neovascularization clusters ( Figure 40).
  • the Agomir-15a-5p mimic and VEGF monoclonal antibody groups can significantly inhibit the formation of choroidal neovascularization clusters ( Figure 41).
  • the virus shown in Figure 22 was also used to deliver miR-15a-5p.
  • the nucleic acid sequence carried by the adeno-associated virus is TAGCAGCACATAATGGTTTGTG (SEQ ID NO: 9), which can exert a therapeutic effect after being transcribed into miR-15a-5p in vivo.
  • adeno-associated viruses containing miR-15a-5p (1 ⁇ 10 12 ) and control viruses (1 ⁇ 10 12 ) were injected subretinaly.
  • the retina was harvested, and the site of adeno-associated virus infection was observed by frozen sections of the eyeball.
  • the results in Figure 42 show that adeno-associated viruses containing miR-15a-5p infected the retina and choroid, and diffuse fluorescence appeared in the retinal pigment epithelium and choroid.
  • the miR-15a-5p content in the retina and choroid of each group was detected by PCR. The results are shown in Figure 43.
  • the miR-15a-5p in the retina and choroid of the group injected with adeno-associated viruses containing miR-15a-5p was 7 times that of the control group, indicating that the virus can successfully infect the retina and choroid and overexpress miR-15a-5p.
  • the retinal neovascularization of mice was quantified, and the results showed that the retinal neovascularization of the group injected with adeno-associated virus containing miR-15a-5p was significantly reduced ( Figure 44- Figure 45).
  • the above results show that adeno-associated virus containing miR-15a-5p can successfully deliver miR-15a-5p to mouse retinal choroid cells and has a therapeutic effect on laser-induced retinal neovascularization in mice.
  • Example 8 also uses two methods to deliver miR-15a-5p, one is modified miR-15a-5p, and the other is adeno-associated virus.
  • the purpose of increasing the content of miR-15a-5p in the retinal choroid is achieved by intraocular injection of two substances.
  • the laser-induced choroidal neovascularization model adopted can simulate choroidal neovascularization. This example observed the therapeutic effect of miR-15a-5p on the neovascularization of the model, and found that miR-15a-5p has a therapeutic effect on choroidal neovascularization.
  • Example 5 shows that miR-15a-5p can restore the early blood perfusion of the OIR retina, which has a great impact on the nutrition and development of retinal neurons. Education is crucial.
  • This example continues to evaluate the protective effect of miR-15a-5p on the retina from two aspects: retinal thickness and optic nerve function.
  • H&E staining can be used to visually observe the structural changes of the retina.
  • Figure 46 shows that the retinal structure of mice in the Agomir-15a-5p mimics treatment group is intact, and the thickness of the outer plexiform layer (the layer indicated by the star mark) is normal, while the outer plexiform layer of the VEGF monoclonal antibody group mice is significantly thinner.
  • Agomir-15a-5p mimics can significantly improve the damage to the structure and function of the retina of oxygen-induced mice.
  • optical coherence tomography OCT was used to quantify the thickness of each layer of the retina ( Figures 47-48).
  • the results showed that Agomir-15a-5p mimics can significantly improve the retinal thinning of oxygen-induced mice, while the retinal thickness of the VEGF monoclonal antibody group was not significantly different from that of untreated mice, and the retinal thinning of oxygen-induced mice could not be improved (Figure 49).
  • Retinal electrophysiological detection (ERG) is widely used to evaluate retinal function.
  • the retina is part of the nervous tissue, and its main function is to convert light signals into electrical signals and transmit them to the brain.
  • the structure of the retina is the basis for normal visual function, and visual function directly determines the patient's vision.
  • Various fundus lesions are accompanied by the loss of retinal structure and function.
  • the oxygen-induced retinopathy model adopted in this embodiment showed obvious retinal thinning and weakened electrophysiological function after the onset of the disease, and after intraocular administration of miR-15a-5p, the retinal structure and neural function were significantly improved, and were better than the VEGF monoclonal antibody group.
  • Example 10 Therapeutic effects of miR-15a-5p on laser-induced choroidal retinal structural and functional damage in mice
  • H&E staining was used to evaluate the retinal interlayer structure, cell morphology and lesions of choroidal neovascularization.
  • the choroid sent new blood vessels to grow under the retinal layer, and the retina was locally bulged.
  • the Agomir-15a-5p mimetic treatment group had less bulges, the lesions basically disappeared, and there were a few lesions remaining in the VEGF monoclonal antibody group.
  • ERG was used to evaluate the electrophysiological function of the retina of choroidal neovascularization mice. The results were shown in Figures 53-54.
  • the Agomir-15a-5p mimetic treatment group greatly improved the retinal function damage of choroidal neovascularization mice, and the VEGF monoclonal antibody group had no significant therapeutic effect on retinal function damage.
  • gliosis will occur in the retina, and GFAP is an indicator of glial cell activation, representing an increase in the level of retinal inflammation.
  • GFAP is an indicator of glial cell activation, representing an increase in the level of retinal inflammation.
  • the activation of GFAP in the OIR retina was significantly reduced (A, B, C, D in Figure 55), indicating that miR-15a-5p inhibits the proliferation of glial cells in the OIR retina.
  • VEGF monoclonal antibody treatment did not inhibit GFAP activation, and the trend was not statistically significant.
  • the laser-induced choroidal retinal injury model adopted in this example also showed obvious retinal thinning and weakened electrophysiological function after the onset of the disease. After intraocular administration of miR-15a-5p, the retinal structure and neural function were significantly improved, and were better than the VEGF monoclonal antibody group.
  • Example 11 Therapeutic effect of miR-15a-5p on hyperoxia-induced and laser-induced retinal inflammation in mice
  • Oxygen-induced mouse retinopathy is accompanied by an increase in the level of inflammation.
  • the retina was taken at P17, P25, and P42 to detect the expression level of TNF ⁇ .
  • the results in Figure 56 show that the expression level of TNF ⁇ protein in the mice injected with Agomir-15a-5p mimics was reduced at P17, and the VEGF monoclonal antibody group had no therapeutic effect on the increased level of retinal TNF ⁇ .
  • IAM-1 Intercellular adhesion molecule 1
  • TNF ⁇ and IL-1 ⁇ are often classic inflammatory factors that recruit inflammatory cells to aggregate.
  • ICAM-1 is an endothelial cell adhesion molecule that can recruit leukocytes to adhere to endothelial cells and increase vascular permeability. This example detected the levels of inflammatory factors in the retina at different treatment nodes, and the results showed that intraocular administration of miR-15a-5p can significantly reduce the expression levels of inflammatory factors in the retina and choroid.
  • the VEGF monoclonal antibody group did not show a significant effect in improving retinal inflammation.
  • Example 12 miR-15a-5p targeted regulation of VEGF
  • MiR-15a-5p is a base sequence that regulates downstream genes by binding to the RNA of the target gene through complementary base pairing, thereby hindering the translation of the target gene into protein.
  • This embodiment observes the targeted regulatory effect of miR-15a-5p on VEGF through in vitro and in vivo experiments. Unmodified miR-15a-5p mimics were used in the in vitro experiments, and modified miR-15a-5p mimics (Agomir) were used in the in vivo experiments. It was found through complementary base pairing that miR-15a-5p can specifically bind to the mRNA of VEGF, so the subsequent embodiments are verified in cells and animals respectively.
  • Retinal pigment epithelial (RPE) cells are one of the sources of intraocular VEGF.
  • TGF- ⁇ 1 can cause an increase in the level of VEGF secreted by RPE cells in vitro. Therefore, miR-15a-5p mimics were transfected into RPE cells, and TGF- ⁇ 1 was used to induce VEGF secretion. The results showed that miR-15a-5p could reduce the increase in VEGF expression induced by TGF- ⁇ 1.
  • Figure 60 shows the mRNA level of VEGF
  • Figures 61-62 show the protein level of VEGF
  • Figure 63 shows the protein level of VEGF in the supernatant of RPE cells.
  • Figure 64 shows a dual luciferase reporter experiment.
  • Wild VEGF mRNA can bind to miR-15a-5p, and mutant VEGF cannot bind to miR-15a-5p. Therefore, the fluorescence is quenched only in the bound group, indicating that miR-15a-5p can specifically regulate VEGF transcription.
  • Figures 65 and 66 show that intravitreal injection of Agomir-15a-5p mimics can reverse the hyperoxia-induced increase in VEGF expression in the mouse retina.
  • Figure 67 shows that intravitreal injection of Agomir-15a-5p mimics can reverse the laser-induced increase in VEGF mRNA expression in the mouse retina.
  • Example 13 miR-15a-5p can inhibit retinal ERK phosphorylation signal activation for a longer period of time than anti-VEGF
  • miR-15a-5p specifically binds to VEGF mRNA and inhibits VEGF transcription.
  • VEGF monoclonal antibody antagonizes VEGF protein. The mechanism of action is different. Therefore, when comparing the efficacy of the two, the activation of the VEGF downstream signaling pathway can be selected for observation.
  • VEGF specifically binds to its receptor VEGFR2, it activates endothelial cell proliferation by phosphorylating ERK and other signaling pathways. Therefore, the ERK phosphorylation signaling pathway is selected as the observation indicator.
  • FIG. 68A The schematic diagram of injection and sampling is shown in Figure 68A.
  • the results showed that on the day of injection, P12, the retina of mice in the hyperoxia-induced group showed an enhanced phosphorylated ERK signal (Figure 68B and Figure 68I); on the first day after injection, P13, the phosphorylated ERK signal in the retina of mice in the VEGF monoclonal antibody group first showed a downward trend ( Figure 68C and Figure 68J); on the second day after injection, P14, the phosphorylated ERK signal in both the VEGF monoclonal antibody and Agomir-15a-5p mimetic groups showed a downward trend (Figure 68D and Figure 68K); on the fourth day after injection, P15, the ERK phosphorylation signal in the VEGF monoclonal antibody group returned to a high level, while A The Agomir-15a-5p mimetic group maintained a decreasing trend in phosphorylated ERK signals, and there was a statistical difference between the
  • the VEGF monoclonal antibody group inhibited ERK phosphorylation to a higher degree than the Agomir-15a-5p mimic at P13, but since P14, the Agomir-15a-5p mimic inhibited ERK phosphorylation to a higher degree than the VEGF monoclonal antibody, and compared with the VEGF monoclonal antibody group at P15 and P17, the difference was statistically significant. The difference between the two reached a peak at P17, and there was no inhibitory effect on ERK phosphorylation at P20 and P25. This shows that miR-15a-5p can inhibit the ERK signaling pathway more persistently by binding to VEGF mRNA, thereby reducing the formation of pathological neovascularization.
  • Example 14 miR-15a-5p targeted regulation of Smad2 reduces retinal endothelial cell mesenchymal transition
  • miR-15a-5p can specifically bind to the mRNA of Smad2, so the subsequent examples were verified in cells and animals respectively.
  • Unmodified miR-15a-5p mimics were used in the in vitro experiment, and modified miR-15a-5p mimics (Agomir) were used in the in vivo experiment.
  • miR-15a-5p mimics and inhibitors were transfected in retinal endothelial cells.
  • PCR Figure 69
  • Western Blot Figure 70- Figure 71
  • miR-15a-5p can regulate the expression of Smad2.
  • Smad2 In order to determine the direct binding of miR-15a-5p to Smad2, it was verified by dual luciferase reporter ( Figure 72). The results showed that compared with the control group, miR-15a-5p mimics significantly reduced the luciferase activity of the Smad2 wild-type vector and had no effect on the luciferase activity of the mutant Smad2. Therefore, miR-15a-5p inhibits the transcription and expression of Smad2 by binding to its mRNA, that is, Smad2 is a direct regulatory target of miR-15a-5p.
  • Smad2 is a typical profibrotic pathway protein.
  • TGF- ⁇ 1 was used to stimulate retinal endothelial cells to induce endothelial-mesenchymal transition (EndoMT) model to simulate retinal fibrosis changes, and miR-15a-5p mimics were transfected into cells to observe the inhibitory effect of miR-15a-5p mimics on EndoMT.
  • Example 15 miR-15a-5p targeted regulation of Smad2 reduces retinal Müller cell fibrosis
  • Müller cells are specialized glial cells throughout the retina. They have the function of maintaining the normal structure and function of the retina and are also involved in a variety of pathological processes, especially proliferative fundus lesions, such as retinal neovascularization, diabetic retinopathy, etc.
  • TGF- ⁇ 2 can stimulate Müller cells to show a fibrotic phenotype in vitro.
  • the results of Figures 79 and 80 show that 1 ng/mL of TGF- ⁇ 2 can cause Müller cell activation accompanied by increased expression levels of fibrosis-related proteins. Therefore, this example uses 1 ng/mL of TGF- ⁇ 2 for modeling.
  • Müller cells were transfected with miR-15a-5p mimics and mimic controls, and then 1 ng/mL of TGF- ⁇ 2 was added for co-culture.
  • the results showed that the GFAP expression level of Müller cells transfected with miR-15a-5p mimics was reduced, and the expression level of fibrosis-related proteins was reduced ( Figures 81 and 82). Further cell immunofluorescence observation was also performed.
  • the Müller cell marker GS was used to identify Müller cells (Figure 83). The activation and fibrosis of Müller cells could be reversed after transfection with the mimic ( Figure 84).
  • the expression of inflammatory factors including TNF- ⁇ and MCP1 in Müller cells was reduced ( Figures 85 and 86).
  • Example 16 miR-15a-5p targeted regulation of Smad2 reduces the trend of hyperoxia-induced retinal fibrosis in mice
  • Agomir-15a-5p mimics, mimic controls and VEGF monoclonal antibodies were injected into the vitreous cavity of hyperoxia-induced mice.
  • the eyeballs of mice were frozen and sectioned at P17, and the proteins were extracted for Western Blot detection.
  • Figure 89 shows the co-localization of ⁇ -SMA and retinal blood vessels in the retina. It can be seen that the expression level of ⁇ -SMA in the hyperoxia control group of mice was higher and co-localized with blood vessels, while in the vitreous cavity The expression level of ⁇ -SMA in the group injected with Agomir-15a-5p mimics was lower, and there was less retinal co-localization.
  • Figure 90 shows the expression and localization of fibronectin and retinal blood vessels.
  • FIG. 91 shows the expression and localization of ⁇ -SMA and retinal activated Müller cells, among which GFAP shows activated Müller cells. The results showed that the co-localization of ⁇ -SMA and retinal activated Müller cells in the hyperoxic mouse control group and VEGF monoclonal antibody group was more significant.
  • Example 5 and Example 9 determined the therapeutic effect of intravitreal injection of Agomir-15a-5p mimics on oxygen-induced neovascularization in mice, the drug safety should be evaluated to determine whether this treatment regimen has adverse effects on the growth, development, metabolism and important organs of mice.
  • Figure 93 A shows the weight changes of mice from P12 (12th day after birth) on the day of administration to P42 (42 days after birth) when the mice are basically adults.
  • Diabetic patients have high blood sugar levels, which cause irreversible damage to the nervous system.
  • the visual function of diabetic patients decreases significantly.
  • spontaneous diabetic model mice (leptin knockout mice) are selected as a model of retinal neurodegeneration for research, and the protective effect of miR-15a-5p intraocular injection on neurodegeneration is observed.
  • Agomir-15a-5p mimics were injected into the vitreous cavity of 12-week-old mice, and the same number of mice were injected into the vitreous cavity with mimics for control. Retinal electrophysiology was used to detect the retinal function of diabetic mice at 16 weeks.
  • Agomir-15a-5p mimics can significantly improve the amplitude intensity of diabetic mouse retinal photoreceptor cells (Figure 95, Figure 96 and Figure 98) and bipolar cells ( Figure 95 and Figure 97) compared with their controls, that is, Agomir-15a-5p mimics have a protective effect on retinal neurological damage in diabetic mice.
  • Figure F in Figure 99 is a representative image of the co-staining of blood vessels and astrocytes. Although there is no vascular perfusion in the peripheral area of normal mice, there is still a reticular structure composed of a large number of astrocytes, but the peripheral area of the knockout mice lacks astrocytes. According to statistics, the number of nodes and total length of the network structure composed of GFAP-positive astrocytes in the knockout mouse retina were less than those in normal mice ( Figure 99 G and H). The overlap between the vascular network and the network structure composed of astrocytes in the knockout mouse was lower than that in the normal mouse (Figure 99 I).
  • the superficial vascular network basically covers the retina, but the superficial vascular network of the knockout mouse is less regular (Figure 100, A), the number of superficial vascular nodes, the number of superficial vascular meshes, the total length of superficial blood vessels and the total length of superficial blood vessels are all lower than those of normal mice ( Figure 100, C, D, E and F).
  • the development of deep blood vessels is shown in Figure 100, B.
  • the deep vascular network of the knockout mouse covers the retina less than that of normal mice ( Figure 100, G). Therefore, the absence of miR-15a-5p will lead to slow development of superficial and deep retinal blood vessels, abnormal development of astrocytes, and then lead to the lack of connection between the vascular network and the astrocyte network. That is, miR-15a-5p has an important regulatory effect on vascular development.
  • Example 1 and Example 4 show that unmodified miR-15a-5p or modified miR-15a-5p has biological activity, can be absorbed by cells in vitro, or absorbed by retinal cells in vivo, and quickly exert biological effects.
  • Example 2 and Example 3 show that in vitro, miR-15a-5p can inhibit pathological angiogenesis.
  • Examples 5-11 show that in vivo, by intraocular administration, miR-15a-5p can inhibit pathological angiogenesis, promote recovery of non-perfused areas, reduce the expression of retinal inflammatory factors and promote nerve damage repair.
  • VEGF is a classic angiogenesis factor that participates in the progression of a variety of fundus diseases.
  • Examples 6 and 7 show that miR-15a-5p can rescue astrocytes and provide a template for vascular apical cells. Compared with VEGF monoclonal antibodies, it can significantly promote the recovery of non-perfused areas, and vascular perfusion is essential for the survival of neurons.
  • Example 12 shows that miR-15a-5p can directly target and regulate the mRNA expression level of VEGF.
  • Example 13 further compares the persistence of miR-15a-5p and VEGF monoclonal antibody in inhibiting the retinal neovascularization signaling pathway. The results show that miR-15a-5p can exert a more lasting inhibitory effect.
  • Smad2 is a typical pro-fibrotic pathway protein.
  • Examples 14-16 show that miR-15a-5p inhibits the occurrence of retinal fibrosis by inhibiting Smad2.
  • Examples 17-18 evaluated the effects of intraocular injection of miR-15a-5p and VEGF monoclonal antibody on oxygen-induced mouse development and normal mouse development.
  • VEGF monoclonal antibody can cause weight loss and dyslipidemia in normal mice, while miR-15a-5p has no adverse effects on mouse development.
  • Example 19 shows It is clear that miR-15a-5p can alleviate retinal neurodegeneration in diabetic mice.
  • Example 20 shows that the absence of miR-15a-5p will lead to delayed development of superficial and deep retinal blood vessels in mice, abnormal development of astrocytes, and then lead to the loss of connection between the vascular network and the astrocyte network, affecting retinal development, that is, miR-15a-5p has an important regulatory effect on vascular development.
  • miR-15a-5p or modified miR-15a-5p has a clear effect in the treatment of fundus diseases and has very good application and research value in the field of biomedicine.
  • Example 21 Therapeutic effect of miR-15a-5p mutant on retinal neovascularization
  • Examples 1-20 demonstrate that miR-15a-5p or modified miR-15a-5p has a clear effect in treating fundus diseases.
  • This example further mutates miR-15a-5p to determine the role of miR-15a-5p mutants in treating fundus diseases.
  • the mutation sites are shown in Table 4, where the bold sequence is the seed sequence (a sequence that must be included to treat the disease), and the underlined sequence is the mutation sequence, which mutates 1, 2, 3, and 5 sites, respectively.
  • the mutant sequence was then used to treat the retinal neovascularization model, and the results are shown in Table 4 and Figure 101.
  • the wild-type sequence can inhibit neovascularization by 74.8%, and the non-seed region mutant sequence can still achieve a similar effect of inhibiting neovascularization. Therefore, this example confirms that the miR-15a-5p seed sequence has a regulatory effect on both.

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Abstract

本发明属于生物医药技术领域，具体涉及miRNA或修饰的miRNA在治疗眼底疾病中的应用。所述miRNA或修饰的miRNA具有生物活性，可在体外被细胞吸收，或者在体内被视网膜细胞吸收，并迅速发挥生物学作用。通过局部给药的方式，所述miRNA或修饰的miRNA可以抑制病理性新生血管、促进无灌注区恢复、减少视网膜炎症因子表达、减轻视网膜纤维化并促进神经损伤修复。在生物医药领域中具有非常好的应用和研究价值。

Description

miR-15a-5p在治疗眼底疾病中的应用 技术领域
本发明涉及生物医药技术领域，具体涉及miRNA或修饰的miRNA(尤其是miR-15a-5p或修饰的miR-15a-5p)在治疗眼底疾病中的应用。
背景技术
眼底是眼球最重要的生理结构，包括脉络膜、视网膜和玻璃体等。其中视网膜遍布了血管及神经，是承担视觉功能的主要结构，具有重要的生理作用。血糖升高或者缺氧等致病因素会导致视网膜损伤，造成视力的下降。例如早产儿视网膜病变(ROP)是常见于早产儿的致盲性眼底疾病，其病理机制为新生儿吸氧导致的视网膜血管退化以及持续大量的新生血管生成。在新生儿视网膜发育过程中血管的异常同时导致神经的变性及退化，极大的影响了婴幼儿的视网膜发育。目前的治疗方法为玻璃体腔注射抗血管内皮生长因子(VEGF)的单抗类药物，但是其靶点单一，无法改善视网膜的神经变性以及降低视网膜炎症水平；而且由于VEGF是重要的神经营养因子，使用抗VEGF治疗反而可导致视网膜神经细胞进一步损害和视网膜功能下降。而在劳动年龄发病率较高的糖尿病视网膜病变(DR)也是以新生血管和神经变性为主要特征的微血管疾病。其致病因素为持续升高的血糖，但后续病理机制较为复杂。现有的治疗方法为在非增殖期采取激光光凝破坏视网膜周边部以减少血管渗漏以及降低视网膜耗氧量，但是激光疗法具有一定的破坏性，并不能完全阻止新生血管的发生。而在增殖期一般采取玻璃体腔注射抗VEGF单抗类药物抑制新生血管或者进行玻璃体切割手术。如前所述，抗VEGF治疗无法改善视网膜神经损伤和功能。
MicroRNA(miRNA)是一类由内源基因编码的长度约为22个核苷酸的非编码单链RNA分子，它们在动植物中参与转录后基因表达调控。主要特点为人体天然存在，多靶向调控，生物学功能丰富。其中，miR-15a-5p与多种疾病的发生和发展密切相关。
专利文献：CN112575088B公开了一种血浆外泌体miRNA生物标记物在制备子宫内膜癌筛查诊断的试剂盒中的应用，所述生物标记物为miR-15a-5p，miR-106b-5p和miR-107的组合。
专利文献：CN109414459B公开了包含miR-15a-5p等miRNA的外泌体可以促进伤口愈合。
专利文献：CN113943800A公开了检测外泌体miR-15a-5p的试剂在制备甲状腺癌碘抵抗筛查试剂盒中的用途。
专利文献：CN110205377A公开了基于miRNA分子标记物进行提前评估川崎病风险的方法，miRNA分子标记物为miR-30c-5p、miR-26a-5p、miR-27a-3p、miR-15a-5p、miR-186-5p、let-7g-5p、miR-941、miR-92a-3p、miR-22-3p、miR-151a-3p、miR-140-3p、miR-199a-3p、miR-4433b-5p。
目前现有技术对单一生物成分的miR-15a-5p在眼底疾病中的应用及机理研究较少。
发明内容
为弥补现有技术的不足，本申请展示了miRNA在治疗眼底疾病的有效性，尤其是miR-15a-5p或修饰的miR-15a-5p具有生物活性，可在体外被细胞吸收，或者在体内被视网膜细胞吸收，并迅速发挥生物学作用。通过局部给药的方式，所述miR-15a-5p或修饰的miR-15a-5p可以抑制病理性新生血管、促进无灌注区恢复、减少视网膜炎症因子表达、减轻视网膜纤维化并促进神经损伤修复。另外，经实验确定miRNA中实际发挥作用的片段为ACGACGAU(SEQ ID NO：10)，且经突变或修饰的miR-15a-5p依然具有很好的效果。该成果在生物医药领域中具有非常好的应用和研究价值。
具体方案如下：
本发明的第一方面，提供了一种miRNA和/或修饰的miRNA和/或包含转录为miRNA的核酸的载体在制备治疗和/或预防眼底疾病的产品中的应用。
所述的miRNA的核苷酸序列包括ACGACGAU(SEQ ID NO：10)。
优选的，所述的miRNA的核苷酸序列包括SEQ ID NO：10，并且与SEQ ID NO：1的核苷酸序列具有60％、65％、70％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、95％或99％以上的同一性。
优选的，所述的miRNA的核苷酸序列包括SEQ ID NO：10，并且与SEQ ID NO：1的核苷酸序列具有包含一个或多个核苷酸的取代、缺失或插入的核苷酸序列，优选包含不超过十、九、八、七、六、五、四、三、二或一个核苷酸的取代、缺失或插入的核苷酸序列。
优选的，所述的miRNA为miR-15a-5p。
在本发明的一个具体实施方式中，所述的miRNA的核苷酸序列为SEQ ID NO：1。
所述的眼底疾病为通过抑制VEGF和/或TGF-β1有益于预防和/或治疗的眼底疾病。优选同时抑制VEGF和TGF-β1有益于预防和/或治疗的眼底疾病。
所述的眼底疾病选自玻璃体病变、视网膜病变、视神经病变或脉络膜病变中的一种或两种以上。
优选的，所述的眼底疾病选自早产儿视网膜疾病、视网膜新生血管疾病、脉络膜新生血管疾病或糖尿病视网膜病变中的一种或两种以上。
所述的miRNA和/或修饰的miRNA和/或包含转录为miRNA的核酸的载体靶向和/或调节VEGF和/或TGF-β1，优选同时靶向VEGF和TGF-β1。所述的调节为上调或下调。优选的，所述调节TGF-β1包括或通过抑制Smad2。优选的，所述的调节VEGF包括抑制VEGF。
优选的，所述的miRNA和/或修饰的miRNA和/或包含转录为miRNA的核酸的载体包含靶向VEGF和/或TGF-β1的结构域(例如SEQ ID NO：10)。
优选的，所述miRNA的序列包含修饰，例如在碱基上进行的修饰。
其中，SEQ ID NO：1包含或为UAGCAGCACAUAAUGGUUUGUG。
所述的miRNA或其模拟物包含正义链和反义链。正义链包含或为SEQ ID NO：1或与SEQ ID NO：1的核苷酸序列具有60％、65％、70％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、95％或99％以上同源性或包含一个或多个核苷酸的取代、缺失或插入的核苷酸序列，反义链包含或为CACAAACCAUUAUGUGCUGCUA(SEQ ID NO：6)或与SEQ ID NO：6的核苷酸序列具有60％、65％、70％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、95％或99％以上同源性或包含一个或多个核苷酸的取代、缺失或插入的核苷酸序列。
所述的产品包含miRNA和/或修饰的miRNA和/或miRNA模拟物，或者，包含miRNA和/或修饰的miRNA和/或miRNA模拟物的载体(所述的载体可以为包裹或转录或表达miRNA的任何载体)。
所述的载体为病毒载体或非病毒载体。
优选的，所述病毒载体包括慢病毒载体、逆转录病毒载体、腺病毒载体、腺相关病毒载体、痘病毒载体或疱疹病毒载体中的一种或两种以上。
优选的，所述的非病毒载体包括脂质体、脂质纳米颗粒、聚合物、多肽、抗体、适配体或N-乙酰半乳糖胺中的一种或两种以上。
所述miRNA的序列包含修饰，例如在碱基上进行的修饰。
修饰的miRNA包括在碱基上进行的修饰。
所述碱基的修饰位于正义链和/或反义链。
优选的，所述碱基上进行的修饰包括3'端进行胆固醇修饰、5'端两个硫代骨架修饰、3'端四个硫代骨架修饰或全链甲氧基修饰中的一种或两种以上。
所述的产品包含正义链和反义链。
优选的，所述的正义链和/或反义链上包含悬挂碱基。
所述的悬挂碱基位于正义链和/或反义链的3'末端。
所述的悬挂碱基为脱氧核苷。优选的，所述的悬挂碱基为dTdT、dTdC或dUdU。例如，所述的miRNA或其模拟物包含的正义链可以为UAGCAGCACAUAAUGGUUUGUGdTdT(SEQ ID NO：7)，反义链可以为CACAAACCAUUAUGUGCUGCUAdTdT(SEQ ID NO：8)。
miRNA或其模拟物包含的正义链和/或反义链经过全链甲氧基修饰、3'端胆固醇修饰、5'端硫代骨架修饰或3'端硫代骨架修饰中的一种或两种以上。
在本发明的一个具体实施方式中，miRNA或其模拟物的反义链经过全链甲氧基修饰、3'端胆固醇修饰、5'端2个硫代骨架修饰和3'端4个硫代骨架修饰。
在本发明的一个具体实施方式中，所述的正义链包含或为SEQ ID NO：1或与SEQ ID NO：1的核苷酸序列具有60％、65％、70％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、95％或99％以上同源性或包含一个或多个核苷酸的取代、缺失 或插入的核苷酸序列，所述的反义链包含经5'端2个硫代骨架修饰，3'端4个硫代骨架修饰，3'端胆固醇修饰以及全链甲氧基修饰的SEQ ID NO：6或与SEQ ID NO：6的核苷酸序列具有60％、65％、70％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、95％或99％以上同源性或包含一个或多个核苷酸的取代、缺失或插入的核苷酸序列。
所述的治疗和/或预防眼底疾病选自抑制视网膜血管内皮细胞(HRMECs)活力、抑制HRMECs增殖(尤其是减弱VEGF诱导的HRMECs的增殖)、抑制Smad2的磷酸化、抑制总Smad2的表达、抑制纤维化、抑制VEGF的表达、抑制视网膜的炎症、抗新生血管或促进无灌注区的恢复、改善视网膜变薄、恢复视功能或促进神经损伤修复中的一种或两种以上。
优选的，所述的抗新生血管包括抑制视网膜新生血管形成和/或脉络膜新生血管形成。
所述的治疗和/或预防眼底疾病包括向需要的受试者施用miRNA和/或修饰的miRNA或miRNA模拟物和/或施用包含miRNA和/或修饰的miRNA或miRNA模拟物的载体(例如包含转录为miRNA的核酸的载体)。
所述的施用部位可以为受试者的眼内空间或腔。例如前房中的房水、悬吊韧带、睫状体、睫状体内和肌肉、晶状体或虹膜、玻璃体、视网膜、脉络膜或视神经等中的一种或两种以上。
本发明的第二方面，提供了一种包含转录为miRNA或其模拟物的核酸的载体。
所述的miRNA的核苷酸序列包括SEQ ID NO：10。
优选的，所述的miRNA的核苷酸序列包括SEQ ID NO：10，并且与SEQ ID NO：1的核苷酸序列具有60％、65％、70％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、95％或99％以上的同一性。
优选的，所述的miRNA的核苷酸序列包括SEQ ID NO：10，并且与SEQ ID NO：1的核苷酸序列具有包含一个或多个核苷酸的取代、缺失或插入的核苷酸序列，优选包含不超过十、九、八、七、六、五、四、三、二或一个核苷酸的取代、缺失或插入的核苷酸序列。
优选的，所述的miRNA为miR-15a-5p。
在本发明的一个具体实施方式中，所述的miRNA的核苷酸序列为SEQ ID NO：1。
所述miRNA的序列包含修饰，例如碱基上的修饰。所述碱基的修饰位于正义链和/或反义链，优选包括3'端进行胆固醇修饰、5'端两个硫代骨架修饰、3'端四个硫代骨架修饰或全链甲氧基修饰中的一种或两种以上。
所述转录为miRNA的核酸包含TAGCAGCA(SEQ ID NO：11)。
优选的，所述转录为miRNA的核酸包含SEQ ID NO：11，且与SEQ ID NO：9的核苷酸序列具有60％、65％、70％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、95％或99％以上的同一性。
优选的，所述转录为miRNA的核酸包含SEQ ID NO：11，且与SEQ ID NO：9的核苷酸序列具有包含一个或多个核苷酸的取代、缺失或插入的核苷酸序列，优选包含不超过十、九、八、七、六、五、四、三、二或一个核苷酸的取代、缺失或插入的核苷酸序列。
在本发明的一个具体实施方式中，所述转录为miRNA的核酸为SEQ ID NO：9。
所述的miRNA或其模拟物包含正义链和反义链。所述的正义链包含SEQ ID NO：1或与SEQ ID NO：1的核苷酸序列具有60％、65％、70％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、95％或99％以上同源性或包含一个或多个核苷 酸的取代、缺失或插入的核苷酸序列，所述的反义链包含SEQ ID NO：6或与SEQ ID NO：6的核苷酸序列具有60％、65％、70％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、95％或99％以上同源性或包含一个或多个核苷酸的取代、缺失或插入的核苷酸序列。
所述的正义链和/或反义链上包含悬挂碱基。
所述的悬挂碱基位于正义链和/或反义链的3'末端。
所述的悬挂碱基为脱氧核苷。
所述的悬挂碱基为dTdT、dTdC或dUdU。
所述的载体为病毒载体或非病毒载体。
所述病毒载体包括慢病毒载体、逆转录病毒载体、腺病毒载体、腺相关病毒载体、痘病毒载体或疱疹病毒载体中的一种或两种以上。
所述的非病毒载体包括脂质体、脂质纳米颗粒、聚合物、多肽、抗体、适配体或N-乙酰半乳糖胺中的一种或两种以上。
本发明的第三方面，提供了一种修饰的miR-15a-5p或miR-15a-5p模拟物。
所述的修饰的miR-15a-5p包括在碱基上进行的修饰。
所述碱基的修饰位于反义链。优选包括3'端进行胆固醇修饰、5'端两个硫代骨架修饰、3'端四个硫代骨架修饰或全链甲氧基修饰中的一种或两种以上。
所述的miR-15a-5p或其模拟物包含的正义链和/或反义链经过全链甲氧基修饰、3'端胆固醇修饰、5'端硫代骨架修饰或3'端硫代骨架修饰中的一种或两种以上。
在本发明的一个具体实施方式中，miR-15a-5p或其模拟物的反义链经过全链甲氧基修饰、3'端胆固醇修饰、5'端2个硫代骨架修饰和3'端4个硫代骨架修饰。
在本发明的一个具体实施方式中，所述的正义链包含SEQ ID NO：1或与SEQ ID NO：1的核苷酸序列具有60％、65％、70％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、95％或99％以上同源性或包含一个或多个核苷酸的取代、缺失或插入的核苷酸序列，所述的反义链包含经5'端2个硫代骨架修饰，3'端4个硫代骨架修饰，3'端胆固醇修饰以及全链甲氧基修饰的SEQ ID NO：6或与SEQ ID NO：6的核苷酸序列具有60％、65％、70％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、95％或99％以上同源性或包含一个或多个核苷酸的取代、缺失或插入的核苷酸序列。
本发明的第四方面，提供了一种药物或药物组合物，所述的药物或药物组合物包括上述miRNA和/或修饰的miRNA和/或miRNA模拟物和/或上述的包含转录为miRNA或miRNA模拟物的核酸的载体，以及药学上可接受的辅料。
所述的miRNA的核苷酸序列包括ACGACGAU(SEQ ID NO：10)。
优选的，所述的miRNA的核苷酸序列包括SEQ ID NO：10，并且与SEQ ID NO：1的核苷 酸序列具有60％、65％、70％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、95％或99％以上的同一性。
优选的，所述的miRNA的核苷酸序列包括SEQ ID NO：10，并且与SEQ ID NO：1的核苷酸序列具有包含一个或多个核苷酸的取代、缺失或插入的核苷酸序列，优选包含不超过十、九、八、七、六、五、四、三、二或一个核苷酸的取代、缺失或插入的核苷酸序列。
优选的，所述的miRNA为miR-15a-5p。
在本发明的一个具体实施方式中，所述的miRNA的核苷酸序列为SEQ ID NO：1。
修饰的miRNA包括在碱基上进行的修饰。
优选的，所述碱基上进行的修饰包括3'端进行胆固醇修饰、5'端两个硫代骨架修饰、3'端四个硫代骨架修饰或全链甲氧基修饰中的一种或两种以上。
所述的药物或药物组合物可以治疗眼底疾病。
所述的药物组合物还可以包含其他治疗或预防眼底疾病的核酸、多肽、蛋白、化合物等或者降低副作用的核酸、多肽、蛋白、化合物等。
所述的药物或药物组合物可以采用任何合适的给药途径，例如胃肠道给药(例如口服)或非胃肠道给药(例如，静脉内、肌内、皮下、皮内、器官内、鼻内、眼内、滴注、脑内、鞘内、透皮、直肠内等)途径。
所述的药物或药物组合物可以为任何合适的剂型，例如经胃肠道给药剂型或非经胃肠道给药剂型，优选包括但不限于片剂、丸剂、粉剂、颗粒剂、胶囊剂、锭剂、糖浆剂、液体、乳剂、微乳剂、混悬剂、注射剂、喷雾剂、气雾剂、粉雾剂、洗剂、软膏剂、硬膏剂、糊剂、贴剂、滴眼剂、滴鼻剂、舌下片剂、栓剂、气雾剂、泡腾片、滴丸剂、凝胶剂等等。
所述药物或药物组合物的各种剂型可以按照药学领域的常规生产方法制备。
所述的药物或药物组合物可以含有重量比为0.01-99.5％(具体如，0.01％、0.1％、0.5％、1％、2％、3％、4％、5％、6％、7％、8％、9％、10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、99％、99.5％)的所述miRNA、修饰的miRNA、包含miRNA或修饰的miRNA的载体。
所述的药物或药物组合物可以为人用药或兽用药。
本发明的第五方面，提供了一种治疗和/或预防眼底疾病的方法，所述的方法包括向有需要的受试者施用有效量的miRNA和/或修饰的miRNA和/或miRNA模拟物和/或包含miRNA或修饰的miRNA或miRNA模拟物的载体和/或药物组合物。
所述的miRNA的核苷酸序列包括ACGACGAU(SEQ ID NO：10)。
优选的，所述的miRNA的核苷酸序列包括SEQ ID NO：10，并且与SEQ ID NO：1的核苷酸序列具有60％、65％、70％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、95％或99％以上的同一性。
优选的，所述的miRNA的核苷酸序列包括SEQ ID NO：10，并且与SEQ ID NO：1的核苷酸序列具有包含一个或多个核苷酸的取代、缺失或插入的核苷酸序列，优选包含不超过十、九、八、七、六、五、四、三、二或一个核苷酸的取代、缺失或插入的核苷酸序列。
优选的，所述的miRNA为miR-15a-5p。
在本发明的一个具体实施方式中，所述的miRNA的核苷酸序列为SEQ ID NO：1。
所述的眼底疾病为通过抑制VEGF和/或TGF-β1有益于预防和/或治疗的眼底疾病。
所述的眼底疾病选自玻璃体病变、视网膜病变、视神经病变或脉络膜病变中的一种或两种以上。
所述的眼底疾病包括但不限于早产儿视网膜疾病、视网膜新生血管疾病、脉络膜新生血管疾病或糖尿病视网膜病变中的一种或两种以上。
所述的miRNA或其模拟物包含正义链和反义链，所述的正义链包含SEQ ID NO：1或与SEQ ID NO：1的核苷酸序列具有60％、65％、70％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、95％或99％以上同源性或包含一个或多个核苷酸的取代、缺失或插入的核苷酸序列，所述的反义链包括SEQ ID NO：6或与SEQ ID NO：6的核苷酸序列具有60％、65％、70％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、95％或99％以上同源性或包含一个或多个核苷酸的取代、缺失或插入的核苷酸序列。
修饰的miRNA包括在碱基上进行的修饰。所述碱基的修饰包括3'端进行胆固醇修饰、5'端两个硫代骨架修饰、3'端四个硫代骨架修饰或全链甲氧基修饰中的一种或两种以上。
所述的治疗和/或预防眼底疾病选自抑制HRMECs活力、抑制HRMECs增殖(尤其是减弱VEGF诱导的HRMECs的增殖)、抑制Smad2的磷酸化、抑制总Smad2的表达、抑制纤维化、抑制VEGF的表达、抑制视网膜的炎症、抗新生血管或促进无灌注区的恢复、改善视网膜变薄、恢复视功能或促进神经损伤修复中的一种或两种以上。
优选的，所述的抗新生血管包括抑制视网膜新生血管形成和/或脉络膜新生血管形成。
优选的，所述的方法包括每只眼施用0.5μg-5mg(例如0.5μg、1μg、2μg、3μg、4μg、5μg、10μg、20μg、50μg、100μg、150μg、200μg、250μg、300μg、350μg、400μg、450μg、500μg、550μg、600μg、650μg、700μg、750μg、800μg、850μg、900μg、950μg、1mg、1.5mg、2mg、2.5mg、3mg、3.5mg、4mg、4.5mg、5mg)的miRNA、修饰的miRNA、miRNA模拟物、包含miRNA或修饰的miRNA或miRNA模拟物的载体或上述的药物或药物组合物。
优选的，所述的方法包括施用至受试者的眼内空间或腔。例如前房中的房水、悬吊韧带、睫状体、睫状体内和肌肉、晶状体或虹膜、玻璃体、视网膜、脉络膜或视神经等中的一种或两种以上。
修饰的miRNA(例如修饰的mir-15a-5p，其反义链碱基上进行的修饰包括3'端进行胆固醇修饰、5'端两个硫代骨架修饰、3'端四个硫代骨架修饰和全链甲氧基修饰，即Agomir-15a-5p)在体内实验的验证均与临床上经典的Anti-VEGF药物雷珠单抗雷珠单抗做对比。结果表明其抑制视网膜及脉络膜新生血管的作用与雷珠单抗相当，优于雷珠单抗在于以下四点，一是抑制新生血管的同时促进视网膜血运恢复，减少视网膜无灌注区面积；二是具有视神经保护作用；三是具有抑制视网膜炎症因子及炎症 介质的作用；四是有减少视网膜纤维化改变的作用。
本发明所述的“药学上可接受的”是指既不显著刺激生物体也不抑制所施用的产品的活性物质的生物学活性及特性。
本发明所述的“药学上可接受的辅料”，包括但不限于载体、赋形剂、稀释剂、润湿剂、填充剂、粘合剂、润滑剂、崩解剂、抗氧化剂、缓冲剂、助悬剂、增溶剂、增稠剂、稳定剂、矫味剂或防腐剂等中的一种或两种以上。
本发明所述的“治疗”表示在疾病已开始发展后减缓、中断、阻止、控制、停止、减轻、或逆转一种体征、症状、失调、病症、或疾病的进展或严重性，但不一定涉及所有疾病相关体征、症状、病症、或失调的完全消除。
本发明所述“有效量”是指在以单个或多个剂量给予至个体或器官之后提供所希望的治疗或预防的本发明的miRNA、miRNA模拟物、修饰的miRNA、药物或药物组合物的量或剂量。
本发明所述的“预防”表示为了阻止或延迟疾病或病症或症状在机体内的发生而实施的方式。
本发明所述的“受试者”可以为人或非人哺乳动物，所述的非人哺乳动物可以为野生动物、动物园动物、经济动物、宠物、实验动物等等。优选的，所述的非人哺乳动物包括但不限于猪、牛、羊、马、驴、狐、貉、貂、骆驼、狗、猫、兔、鼠(例如大鼠、小鼠、豚鼠、仓鼠、沙鼠、龙猫、松鼠)或猴等等。
附图说明
以下，结合附图来详细说明本发明的实施例，其中：
图1：向视网膜血管内皮细胞分别转染miR-15a-5p的模拟物对照/模拟物/抑制剂对照/抑制剂后，检测视网膜血管内皮细胞中miR-15a-5p的表达量。模拟物可以增加细胞内miR-15a-5p的表达量，抑制剂可以减少细胞内miR-15a-5p的表达量。
图2：不同浓度的VEGF刺激视网膜血管内皮细胞对细胞增殖的影响。10ng/ml的VEGF即可引起细胞异常增殖。
图3：不同浓度的VEGF刺激视网膜血管内皮细胞对miR-15a-5p相对表达量的影响。10ng/ml的VEGF不会影响细胞内miR-15a-5p的表达量。
图4：CCK-8试剂盒检测miR-15a-5p对细胞增殖活力的影响。miR-15a-5p模拟物减弱VEGF诱导的视网膜血管内皮细胞病理性增殖。
图5：细胞划痕实验检测miR-15a-5p对细胞增殖能力的影响。
图6：细胞划痕定量统计分析，miR-15a-5p模拟物减少VEGF诱导的视网膜内皮细胞病理性增殖。
图7：Transwell实验检测miR-15a-5p对细胞迁移能力的影响。
图8：对Transwell实验细胞迁移数量的定量统计分析，miR-15a-5p模拟物减少VEGF诱导的视网膜血管内皮细胞病理性迁移。
图9：管腔形成实验检测miR-15a-5p对细胞体外成管能力的影响。
图10：对管腔形成实验细胞形成的血管管腔总长度的定量统计分析，miR-15a-5p模拟物减少VEGF诱导的视网膜血管内皮细胞血管管腔生成。
图11：对管腔形成实验细胞形成的血管管腔节点数的定量统计分析，miR-15a-5p模拟物减少VEGF诱导的视网膜血管内皮细胞血管管腔生成。
图12：小鼠视网膜吸收修饰(Agomir)及未修饰(mimic)的miR-15a-5p模拟物的差异。*为模拟物(mimic)组与对照组比P<0.05，**为p<0.01；@为修饰的模拟物(Agomir)与对照组比P<0.05，@@@为p<0.001，@@@@为P<0.0001；#为模拟物(mimic)组与修饰的模拟物(Agomir)比P<0.05，####为<0.0001。
图13：氧诱导的小鼠视网膜新生血管模型流程图。P代表小鼠出生后的天数。
图14：氧诱导的视网膜新生血管小鼠发育过程中视网膜miR-15a-5p的表达水平。常氧为未处理鼠，高氧为新生血管造模组。
图15：不同剂量miR-15a-5p模拟物治疗氧诱导的视网膜新生血管的作用。常氧为未处理鼠，高氧为新生血管造模组，模拟物为Agomir。
图16：不同剂量miR-15a-5p模拟物治疗氧诱导的视网膜新生血管的作用统计图。常氧为未处理鼠，高氧为新生血管造模组，模拟物为Agomir，模拟物对照为乱序的Agomir。
图17：不同剂量抗VEGF药物治疗氧诱导的视网膜新生血管的作用。A为染色图，B为统计图。其中，常氧为未处理鼠，高氧为新生血管造模组。
图18：给药后视网膜中Agomir-15a-5p的分布和持续时间。(A)视网膜中CY3标记的Agomir-15a-5p的代表性图像。(B)CY3标记的Agomir-15a-5p的荧光强度的定量。(C)Agomir-15a-5p注射后在OIR视网膜中的表达趋势。
图19：1μg的miR-15a-5p模拟物治疗氧诱导的视网膜新生血管的作用示意图。常氧(第1列)为未处理鼠，高氧(第2-4列)为新生血管造模组，模拟物为Agomir，模拟物对照为乱序的Agomir，抗-VEGF为雷珠单抗。
图20：1μg的miR-15a-5p模拟物治疗氧诱导的视网膜新生血管的新生血管面积统计图。常氧为未处理鼠，高氧为新生血管造模组，模拟物为Agomir，模拟物对照为乱序的Agomir，VEGF单抗为雷 珠单抗。
图21：1μg的miR-15a-5p模拟物治疗氧诱导的视网膜新生血管的无灌注区面积统计图。常氧为未处理鼠，高氧为新生血管造模组，模拟物为Agomir，模拟物对照为乱序的Agomir，VEGF单抗为雷珠单抗。
图22：包含miR-15a-5p的腺相关病毒的结构示意图。
图23：包含miR-15a-5p的腺相关病毒感染视网膜的示意图。
图24：PCR结果显示腺相关病毒感染视网膜后，视网膜miR-15a-5p过表达倍数。其中OIR-AAV-NC为注射对照病毒组，OIR-AAV-15a为注射包含miR-15a-5p的腺相关病毒。
图25：包含miR-15a-5p的腺相关病毒对视网膜新生血管和无灌注区的治疗效果示意图。其中OIR-AAV-NC为注射对照病毒组，OIR-AAV-15a为注射包含miR-15a-5p的腺相关病毒。
图26：包含miR-15a-5p的腺相关病毒对无灌注区的治疗效果统计结果。
图27：包含miR-15a-5p的腺相关病毒对氧诱导的视网膜新生血管的治疗效果统计结果。
图28：A：视网膜无灌注区域放大的图像显示星形胶质细胞和Müller胶质细胞活化的差异(斑点染色)。B：显示了视网膜尖端细胞和丝状足的代表性图像。原始放大倍数，×40。C：视网膜丝状伪足的统计分析。(n＝6只小鼠视网膜)。D：代表性图像显示了尖端细胞、GFAP阳性星形胶质细胞和Müller细胞的末端足区之间的相互作用。其中，常氧为未处理鼠，高氧为新生血管造模组，模拟物为Agomir，模拟物对照为乱序的Agomir，VEGF单抗为雷珠单抗。
图29：1μg的miR-15a-5p模拟物治疗氧诱导的视网膜新生血管的无灌注区和新生血管面积示意图。高氧-正常鼠为正常鼠新生血管造模组，高氧-敲除鼠为miR-15a-5p敲除鼠新生血管造模组。
图30：1μg的miR-15a-5p模拟物治疗氧诱导的视网膜新生血管的无灌注区面积统计图。高氧-正常鼠为正常鼠新生血管造模组，高氧-敲除鼠为miR-15a-5p敲除鼠新生血管造模组。
图31：1μg的miR-15a-5p模拟物治疗氧诱导的视网膜新生血管的新生血管簇面积统计图。高氧-正常鼠为正常鼠新生血管造模组，高氧-敲除鼠为miR-15a-5p敲除鼠新生血管造模组。
图32：1μg的miR-15a-5p模拟物治疗氧诱导的视网膜新生血管的无灌注区和新生血管面积示意图。高氧-正常鼠为正常鼠新生血管造模组，高氧-敲除鼠为miR-15a-5p敲除鼠新生血管造模组，模拟物对照为乱序的Agomir，模拟物为Agomir。
图33：1μg的miR-15a-5p模拟物治疗氧诱导的视网膜新生血管的无灌注区面积统计图。正常为正常鼠新生血管造模组，敲除为miR-15a-5p敲除鼠新生血管造模组，模拟物对照为乱序的Agomir，模拟物为Agomir。
图34：1μg的miR-15a-5p模拟物治疗氧诱导的视网膜新生血管的新生血管簇面积统计图。正常为正常鼠新生血管造模组，敲除为miR-15a-5p敲除鼠新生血管造模组，模拟物对照为乱序的Agomir，模拟物为Agomir。
图35：视网膜无灌注区域放大的图像显示星形胶质细胞和Müller胶质细胞活化的差异(斑点染色)。高氧-正常鼠为正常鼠新生血管造模组，高氧-敲除鼠为miR-15a-5p敲除鼠新生血管造模组，模拟物对照为乱序的Agomir，模拟物为Agomir。
图36：显示了视网膜尖端细胞和丝状足的代表性图像，以及尖端细胞、GFAP阳性星形胶质细胞和Müller细胞的末端足区之间的相互作用。原始放大倍数×40。高氧-正常鼠为正常鼠新生血管造模组，高氧-敲除鼠为miR-15a-5p敲除鼠新生血管造模组，模拟物对照为乱序的Agomir，模拟物为Agomir。
图37：激光诱导的脉络膜新生血管模型流程图。
图38：眼底荧光造影(FFA)观察miR-15a-5p抑制脉络膜新生血管作用。
图39：对渗漏的荧光进行定量分析结果。
图40：脉络膜铺片行IB4染色以定量新生血管簇的面积察miR-15a-5p抑制脉络膜新生血管作用。IB4阳性示新生血管团簇。
图41：对新生血管团簇进行定量分析结果。
图42：包含miR-15a-5p的腺相关病毒感染视网膜脉络膜的荧光示意图。
图43：PCR结果显示腺相关病毒感染视网膜脉络膜后，视网膜和脉络膜miR-15a-5p过表达倍数。其中CNV-AAV-NC为脉络膜新生血管并且注射对照病毒组，CNV-AAV-15a为脉络膜新生血管并且注射包含miR-15a-5p的腺相关病毒组。
图44：脉络膜铺片行IB4染色以定量新生血管簇的面积观察包含miR-15a-5p的腺相关病毒抑制脉络膜新生血管的示意图。
图45：脉络膜铺片行IB4染色以定量新生血管簇的面积观察包含miR-15a-5p的腺相关病毒抑制脉络膜新生血管的统计结果图。
图46：视网膜H&E染色示视网膜层间结构及细胞形态。星号表示外丛状层。GCL代表神经节细胞层，IPL代表内丛状层，INL代表内核层，OPL代表外丛状层，ONL代表外核层，RPE代表色素上皮层。常氧代表未处理小鼠，高氧代表氧诱导的视网膜新生血管模型鼠。
图47：在小鼠出生后第42天使用光学相干断层成像(OCT)量化视网膜各层厚度的示意图，IPL代表内丛状层，INL代表内核层，OPL代表外丛状层，ONL代表外核层。常氧代表未处理小鼠，高氧 代表氧诱导的视网膜新生血管模型鼠。
图48：在小鼠出生后第42天使用光学相干断层成像(OCT)量化视网膜各层厚度的地形图。常氧代表未处理小鼠，高氧代表氧诱导的视网膜新生血管模型鼠。
图49：在小鼠出生后第42天使用光学相干断层成像(OCT)量化视网膜各层厚度的统计结果，确定miR-15a-5p对视网膜结构的保护作用。常氧代表未处理小鼠，高氧代表氧诱导的视网膜新生血管模型鼠。
图50：在小鼠出生后25天及42天使用视网膜电生理检查(ERG)分析视网膜功能的示意图。常氧代表未处理小鼠，高氧代表氧诱导的视网膜新生血管模型鼠。
图51：在小鼠出生后25天及42天使用视网膜电生理检查(ERG)分析视网膜功能的统计结果图，确认miR-15a-5p对视网膜功能的保护作用。常氧代表未处理小鼠，高氧代表氧诱导的视网膜新生血管模型鼠。
图52：视网膜脉络膜H&E染色示视网膜脉络膜层间结构及细胞形态。GCL代表神经节细胞层，IPL代表内丛状层，INL代表内核层，OPL代表外丛状层，ONL代表外核层，RPE代表色素上皮层。
图53：使用视网膜电生理检查(ERG)分析脉络膜新生血管模型视网膜功能的示意图。模拟物对照为乱序的Agomir，模拟物为Agomir，VEGF单抗为雷珠单抗。
图54：使用视网膜电生理检查(ERG)分析脉络膜新生血管模型视网膜功能的统计结果图。模拟物对照为乱序的Agomir，模拟物为Agomir，VEGF单抗为雷珠单抗。
图55：miR-15a-5p会抑制OIR视网膜的神经胶质细胞增殖。其中，(A)常氧小鼠和高氧小鼠视网膜切片中GFAP免疫染色的代表性图像。(B)对上述各组的GFAP强度进行量化和比较。(C)Western印迹显示GFAP在常氧小鼠和高氧小鼠的视网膜中的表达。(D)GFAP的蛋白质水平通过密度测定法进行定量，内参为GAPDH水平。
图56：酶联免疫吸附实验(ELISA)检测不同时间点氧诱导的视网膜病变小鼠视网膜中TNFα的含量。
图57：Western Blot示氧诱导视网膜病变各组小鼠视网膜细胞间粘附分子1(ICAM-1)表达量示意图。
图58：Western Blot示氧诱导视网膜病变各组小鼠视网膜细胞间粘附分子1(ICAM-1)表达量统计结果。
图59：PCR结果示激光诱导的各组小鼠视网膜炎症因子水平。A图为TNFα，B图为ICAM-1，C图为IL-1β。
图60：PCR结果显示miR-15a-5p可以减少TGF-β1引起的RPE细胞中VEGF的mRNA水平。
图61：Western Blot示miR-15a-5p可以减少TGF-β1引起的RPE细胞中VEGF的蛋白水平升高示意图。
图62：Western Blot示miR-15a-5p可以减少TGF-β1引起的RPE细胞中VEGF的蛋白水平升高统计结果。
图63：Elisa示miR-15a-5p可以减少TGF-β1引起的RPE细胞上清中VEGF的蛋白水平升高。
图64：双荧光素酶报告实验显示miR-15a-5p可以在体外与VEGF的mRNA靶向结合。其中pmirGLO载体为空载质粒，野生型为VEGF碱基序列，突变型为结合区碱基不同的VEGF碱基序列。
图65：玻璃体腔注射Agomir-15a-5p模拟物可以逆转高氧诱导的小鼠视网膜VEGF表达水平增加。
图66：玻璃体腔注射Agomir-15a-5p模拟物可以逆转高氧诱导的小鼠视网膜VEGF表达水平增加。
图67：玻璃体腔注射Agomir-15a-5p模拟物可以逆转激光诱导的小鼠视网膜VEGF表达水平增加。
图68：miR-15a-5p模拟物相较VEGF单抗可以更长时间抑制视网膜ERK磷酸化信号激活。其中，A图为示意图，在出生后第12天进行玻璃体腔注射后，在P13、P14、P15、P17、P20、P25取材视网膜。B图为P12时，高氧组小鼠视网膜ERK的磷酸化信号增强。C图为P13时，VEGF单抗组视网膜ERK的磷酸化信号降低。D图为P14时，miR-15a-5p模拟物组和VEGF单抗组视网膜ERK的磷酸化信号降低。E图为P15时，miR-15a-5p模拟物组视网膜ERK的磷酸化信号降低。F图为P17时，miR-15a-5p模拟物组视网膜ERK的磷酸化信号降低。G图为P20时，各视网膜ERK的磷酸化信号无明显差异。H图为P25时，各视网膜ERK的磷酸化信号无明显差异。I图、J图、K图、L图、M图、N图、O图分别为P12、P13、P14、P15、P17、P20、P25的统计结果。P图为各组小鼠视网膜在不同时间点视网膜磷酸化ERK信号相对表达量。
图69：PCR结果显示miR-15a-5p可以降低视网膜血管内皮细胞中Smad2的mRNA水平。
图70：Western Blot结果显示miR-15a-5p可以降低视网膜血管内皮细胞中Smad2的蛋白水平示意图。
图71：Western Blot结果显示miR-15a-5p可以降低视网膜血管内皮细胞中Smad2的蛋白水平统计结果。
图72：双荧光素酶报告实验显示miR-15a-5p可以在体外与Smad2的mRNA靶向结合，其中， pmirGLO载体为空载质粒，野生型为Smad2碱基序列，突变型为结合区碱基不同的Smad2碱基序列。
图73：转染miR-15a-5p模拟物后，经TGF-β1刺激的视网膜血管内皮细胞纤维化标志物α-SMA和CD31的免疫荧光染色结果图。
图74：转染miR-15a-5p模拟物后，经TGF-β1刺激的视网膜血管内皮细胞波形蛋白的免疫荧光染色结果图。
图75：转染miR-15a-5p模拟物后，经TGF-β1刺激的视网膜血管内皮细胞纤维化标志物波形蛋白、α-SMA以及CD31的变化Western Blot示意图。
图76：转染miR-15a-5p模拟物后，经TGF-β1刺激的视网膜血管内皮细胞纤维化标志物波形蛋白、α-SMA以及CD31的变化Western Blot统计结果图。
图77：转染miR-15a-5p模拟物后，经TGF-β1刺激的视网膜血管内皮细胞磷酸化-Smad2，总Smad2变化Western Blot示意图。
图78：转染miR-15a-5p模拟物后，经TGF-β1刺激的视网膜血管内皮细胞磷酸化-Smad2，总Smad2变化Western Blot统计结果图。
图79：Western Blot结果示不同浓度的TGF-β2刺激Müller细胞后纤维化相关蛋白表达水平示意图。
图80：Western Blot结果示不同浓度的TGF-β2刺激Müller细胞后纤维化相关蛋白表达水平统计结果。
图81：Western Blot结果示转染miR-15a-5p模拟物及模拟物对照后，TGF-β2刺激Müller细胞后纤维化相关蛋白表达水平示意图。
图82：Western Blot结果示转染miR-15a-5p模拟物及模拟物对照后，TGF-β2刺激Müller细胞后纤维化相关蛋白表达水平统计结果。
图83：转染miR-15a-5p模拟物后，经TGF-β2刺激的视网膜Müller细胞后细胞标志物GS的免疫荧光染色结果图。
图84：转染miR-15a-5p模拟物后，经TGF-β2刺激的视网膜Müller细胞后纤维化标志物α-SMA和活化标志物GFAP的免疫荧光染色结果图。
图85：PCR结果示转染miR-15a-5p模拟物及模拟物对照后，TGF-β2刺激Müller细胞后TNF-α表达水平。
图86：PCR结果示转染miR-15a-5p模拟物及模拟物对照后，TGF-β2刺激Müller细胞后MCP-1表达水平。
图87：Western Blot结果示转染miR-15a-5p模拟物及模拟物对照后，TGF-β2刺激Müller细胞后Smad2总蛋白及磷酸化水平示意图。其中P-Smad2为磷酸化Smad2，T-Smad2为总Smad2。
图88：Western Blot结果示转染miR-15a-5p模拟物及模拟物对照后，TGF-β2刺激Müller细胞后Smad2总蛋白及磷酸化水平统计结果。其中P-Smad2为磷酸化Smad2，T-Smad2为总Smad2。
图89：高氧诱导小鼠各组视网膜冰冻切片示α-SMA和视网膜血管的表达定位。
图90：高氧诱导小鼠各组视网膜冰冻切片示纤维连接蛋白和视网膜血管的表达定位。
图91：高氧诱导小鼠各组视网膜冰冻切片示α-SMA和视网膜活化的Müller细胞表达定位。其中GFAP示活化的Müller细胞。
图92：Western Blot检测示视网膜纤维化蛋白的表达。A图示高氧诱导小鼠各组视网膜纤维连接蛋白表达情况。B图为纤维蛋白表达水平统计结果。C图示高氧诱导小鼠各组视网膜TGFβ受体2蛋白和α-SMA蛋白表达情况。D图为TGFβ受体2蛋白和α-SMA蛋白表达水平统计结果。E图为高氧诱导小鼠各组视网膜磷酸化Smad2和总Smad2表达情况。F图为视网膜磷酸化Smad2和总Smad2表达水平统计结果。
图93：眼内给药miR-15a-5p对氧诱导小鼠发育的安全性评估。其中，A图示自给药当天P12(出生后第12天)至小鼠基本成年P42(出生后42天)，小鼠的体重变化。B图示各组血浆颜色。C图示小鼠血清肌酐水平。D图示小鼠血清尿素水平。E图示小鼠血浆甘油三酯水平。F图示小鼠血浆总胆固醇水平。G图和H图示小鼠的出生后17天(P17)，25天(P25)，42天(P42)的肝脏及肾脏结构H&E染色。
图94：眼内给药miR-15a-5p对正常小鼠发育及视网膜的安全性评估。其中，A图示自给药当天P12(出生后第12天)至小鼠基本成年P42(出生后42天)，小鼠的体重变化。B图示小鼠血清肌酐水平。C图示小鼠血清尿素水平。D图示小鼠血浆甘油三酯水平。E图示小鼠血浆总胆固醇水平。F图示注药后不同时间点肝肾的荧光切片。G图和H图示小鼠的出生后17天(P12)，25天(P25)，42天(P42)的肝脏及肾脏结构H&E染色。I图、J图、K图为OCT结果示玻璃体腔注射miR-15a-5p模拟物、模拟物对照以及VEGF单抗对视网膜厚度的影响。L图、M图为视网膜冰冻切片，示玻璃体腔注射miR-15a-5p模拟物、模拟物对照以及VEGF单抗后视网膜Müller细胞的激活情况。
图95：在糖尿病鼠(瘦素受体缺乏模型鼠)中观察了玻璃体腔注射miR-15a-5p模拟物对糖尿病鼠视网膜光感受器损伤和视网膜双极细胞损伤的治疗作用。其中a波代表光感受器细胞对光刺激的反应，b波代表双极细胞对光刺激的反应，op波是b波中的一组振荡电位，一般认为代表双极细胞-无长突细胞对光刺激的反应。
图96：在糖尿病鼠(瘦素受体缺乏模型鼠)中观察了玻璃体腔注射miR-15a-5p模拟物对糖尿病鼠视网膜光感受器损伤的治疗作用统计结果。其中b波代表光感受器细胞对光刺激的反应。
图97：在糖尿病鼠(瘦素受体缺乏模型鼠)中观察了玻璃体腔注射miR-15a-5p模拟物对糖尿病鼠视网膜双极细胞损伤的治疗作用统计结果。其中a波代表双极细胞对光刺激的反应。
图98：在糖尿病鼠(瘦素受体缺乏模型鼠)中观察了玻璃体腔注射miR-15a-5p模拟物对糖尿病鼠视网膜双极细胞损伤的治疗作用统计结果。其中op波是b波中的一组振荡电位，一般认为代表双极细胞-无长突细胞对光刺激的反应
图99：正常鼠和敲除鼠在P7时视网膜浅血管丛沿着星形胶质细胞模板生长存在差异。其中A示IB4染色的视网膜浅层血管的代表性图像。从左到右放大倍数依次为5X，10X和20X。其中B示浅层血管覆盖视网膜的面积的统计学结果。其中C示浅层血管交错产生的节点个数的统计学结果。其中D示浅层血管总长度的统计学结果。其中E示浅层血管总分支长度的统计学结果。其中F示IB4和GFAP染色的视网膜浅层血管的代表性图像。其中G示GFAP阳性区域浅层血管交错产生的节点个数的统计学结果。其中H示GFAP阳性区域管腔总长度的统计学结果。其中I区域示浅层血管与GFAP阳性区域重合程度的统计学结果。
图100：正常鼠和敲除鼠在P9时视网膜浅血管丛和深层血管丛的生长存在差异。其中A示IB4染色的视网膜浅层血管的代表性图像。从左到右放大倍数依次为5X，10X和20X。其中B示IB4染色的视网膜深层血管覆盖视网膜的代表性图像。其中C示浅层血管交错产生的节点个数的统计学结果。其中D示浅层血管网眼个数的统计学结果。其中E示浅层血管总长度的统计学结果。其中F示浅层血管总分支长度的统计学结果。其中G示申请血管覆盖面积的统计学结果。
图101：miR-15a-5p突变体对视网膜新生血管的治疗效果。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明的部分实施例，而不是全部。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例中涉及的实验方法如下：
1、miR-15a-5p对人视网膜血管内皮细胞的影响
1.1细胞培养
人视网膜血管内皮细胞(Human retinal microvascular endothelial cells，HRMEC)从angiopromie公司购买，将其在内皮细胞基本培养基(ECM)中与体积分数5％胎牛血清，体积分数1％青霉素-链霉素和内皮细胞生长补充剂(ECGS)一起培养。将细胞在37℃和体积分数5％的二氧化碳中孵育。每2天更换一次培养基。取第三至第六代细胞进行实验。
1.2 CCK-8试剂盒检测细胞增殖
使用Cell Counting Kit(CCK-8)CCK-8/WST-8试剂盒：消化细胞并制成单细胞悬浮液，以2000个/孔的密度于96孔板中接种，每组设置5个复孔，细胞贴壁后进行换液，加入目的培养基并在24小时检测细胞活性。检测前，每孔加入10μl CCK-8试剂，37℃避光孵育2h；然后将孵育后的培养基移到酶标板中，450nm处用BIO-RAD酶标仪测定的吸光度。
1.3细胞转染
在96孔板中按2000个/孔的密度接种HRMECs，细胞培养箱中培养过夜。在2个EP管中分别加入5μl Opti-MEM培养基，再向第一管中加入0.15μl的Lipofectamine 3000试剂，第二管中加入0.15μl的模拟物对照/模拟物/抑制剂对照/抑制剂，将两管液体混合，室温孵育5min，之后将10μl的混合液加入一个孔中。配液时一般需要算出所有孔加液的总量，混合后逐个孔添加，而不要每个孔单独配液避免误差。其它的模拟物对照、抑制剂对照和抑制剂也用同样的方法转染入细胞，但抑制剂对照和抑制剂需要加入比模拟物多一倍的量。
1.4细胞总RNA的提取
在细胞中加入500μl的Trizol，吹打后室温静置5min，加入100μl buffer A，剧烈震荡15s后室温静置3min，分层后4℃、15000g离心5min，吸取上层水相200μl于另一个1.5ml EP管中，加入1.5倍体积的无水乙醇，上下颠倒，加至试剂盒(EZB公司，货号EZB-RN5)提供的RNA吸附柱中，4000g离心1min，取出后倒掉收集管内的液体，将500μl的wash buffer 1液体加入离心柱然后12000g离心1min，离心直到所有液体都经过离心柱。之后向离心柱中加入500μl的wash buffer 2清洗缓冲液，12000g离心1min，取出后倒掉收集管内的液体，12000g离心1min，取出后倒掉收集管内的液体，将吸附柱换到新的1.5ml EP管中，开盖晾2min，加入20μl DEPC水，12000g离心1min，将离心液再加入吸附柱中，室温5min，12000g离心1min，收集管中液体即总RNA，用Nano Drop测浓度。
1.5逆转录
按照下述的比例在200μl的EP管中加入试剂，其中RNA的体积根据RNA浓度不同而有所不同，RNA总量500ng，之后加gDNA remover 1μl混匀室温存放5分钟。按表1加入试剂。放入PCR仪中设置程序：37℃加热15min，之后42℃加热10min，之后95℃加热3min，结束后将得到的cDNA迅速放置于冰上。
表1
1.6qRT-PCR
按照表2比例配制每孔中的反应液体，SYBR Green Master与cDNA混合，上下游引物和水混合，cDNA不足的部分用RNeasy free water补足，混匀后加入384孔板中。注意避光。加样完成后贴上封板膜，4℃2000rpm离心3分钟，然后上机。程序设置为：第一步95℃15min，第二步94℃15s——55℃30s——70℃30s并循环40次，第三步95℃15s——55℃15s——95℃15s。sEVs使用U6作为内参，血浆和玻璃体使用cel-miR-39作为外参。miRNA的表达水平用2∧-Δct来表示。各miRNA的 引物序列见表3。
表2
表3
1.7细胞划痕实验
将HRMECs消化后接种于6孔板上，细胞融合度达到75％-80％时分别转染模拟物对照、模拟物、抑制剂对照和抑制剂，6h后吸去含转染试剂的培养基并用1ml枪头在每孔细胞中央以相同的力度划一道线，之后用PBS缓冲液清洗2次洗去脱落的细胞，镜下拍照并保存，记录拍照的地方以保证之后拍照都是同一个地方，24h后再次拍照保存。图片用image J软件分析计算划痕的面积。
1.8 Transwell细胞迁移实验
Transwell细胞迁移实验用于检测miR-15a-5p转染后对细胞迁移能力的影响。预处理后，用胰蛋白酶消化HRMECs，并将8000个细胞接种入小孔上室，培养24小时后，用PBS洗涤三次。然后用PFA固定细胞并使用0.01％结晶紫缓冲液染色细胞。在显微镜下随机选择5个视野，并计数发生迁移的细胞数量。
1.9内皮细胞管腔形成实验
将Matrigel基质胶(Corning公司，货号354234)在4℃下融化12小时，在48孔细胞板的表层铺适量的基质胶，为管腔形成提供支持。然后在37℃下在固化40分钟。预处理后，将HRMECs接种到涂有Matrigel的48孔板中。3小时后，使用反相显微镜采集每个孔的五个区域。使用image-J软件计算血管管腔的总长度和管腔节点个数。
1.10免疫荧光细胞染色
预处理后，将HRMECs固定在4％多聚甲醛中15分钟，并在PBS中用0.1Triton-X100(PBST)浸泡15分钟。在室温下用5％牛血清白蛋白(BSA)封闭后，将细胞与一级抗体波形蛋白(1:300，Abcam，ab45939)、a-SMA(1:500，Abcam，ab124964)、CD31(1:150，Abcam，ab24590)在4℃下孵育过夜。用PBST洗涤三次后。然后在室温下将细胞与Alexa fluor 488缀合的山羊抗兔IgG H&L(1：1000Abcam)孵育波形蛋白，或与'Alexa fluor-647缀合的羊抗小鼠IgG H&L(1：1000，Abcam。用4，6-二氨基-2-苯基吲哚(1:500，Solarbio)标记细胞核。在共聚焦激光扫描显微镜(LSM800，蔡司德国)下观察并拍摄细胞。
2.miR-15a-5p靶基因的筛选和验证
2.1.靶基因预测
在三个数据库TargetScan,PITA,microRNAorg中分别预测筛选出的差异miRNA的靶基因。
2.2双荧光素酶报告基因实验
使用荧光素酶报告基因测定来验证Smad2是否为miR-15a-5p的靶基因。双荧光素酶报告质粒：构建了pmIRGLO-Smad2野生型和pmIRGLO-Smad2突变体，空载质粒作为对照质粒。将报告质粒与miR-15a-5p模拟物或乱序对照共转染到HEK-293细胞中。转染48小时后，根据双荧光素酶报告物测定系统(Gene Pharma公司，货号G06001)的步骤裂解细胞并收集上清液。TECAN Infinite 200(德国)用于检测萤火虫荧光素酶报告基因和雷尼拉荧光素酶报告基因的活性。萤火虫荧光素酶的比率，以Renilla荧光素酶为相对荧光素酶活性。每个实验重复3次。验证VEGF是否为miR-15a-5p的靶基因过程同上。
3.动物实验操作
3.1小鼠OIR模型的建立
C57BL/6J小鼠用于建立氧诱导的新生血管性视网膜病变模型。新生小鼠和哺乳期雌性小鼠在乳鼠出生后第7天(P7)至第12天(P12)暴露于75％氧气中。第12天从氧舱中取出，放置于常氧中。由于缺氧会导致新生血管生成，在出生后第17天(P17)，达到视网膜新生血管的高峰。其间伴随视网膜炎症、神经损伤及纤维化改变。
3.2小鼠玻璃体腔注药
在第12天使用34号针头(Hamilton，Reno，NV，United States)将浓度为1μg的经修饰的miR-15a-5p模拟物(Agomir)注射到小鼠玻璃体腔中。将相同数量的小鼠注射乱序的Agomir作为阴性对照组。或者，将1μl腺相关病毒注射到小鼠玻璃体腔中，滴度为1×1012。注射后使用抗生素凝胶覆盖眼表防止角膜水肿。
3.3小鼠视网膜对修饰及未修饰的miR-15a-5p的吸收差异
雄性6周龄的C57BL/6J小鼠用于该实验，麻醉后使用34号针头(Hamilton，Reno，NV，United  States)将浓度为1μg(Genepharma公司)的未经修饰的miR-15a-5p模拟物(mimics)或者经修饰的miR-15a-5p模拟物(Agomir)注射到小鼠玻璃体腔中，将相同数量的小鼠注射PBS作为阴性对照组。在注射后8小时，24小时，48小时，5天和7天取视网膜进行miR-15a-5p水平的检测。
3.4视网膜铺片
在P17处死小鼠，取出其眼球。剥离视网膜，用4％多聚甲醛固定，并切成4个放射状瓣。使用山羊血清封闭2小时，使用IB4(Thermo，货号I21411)或者抗胶质纤维酸性蛋白(GFAP)抗体(Abcam，货号ab7260)进行视网膜血管染色。清洗5小时后，使用羊抗兔IgG(Abcam 150077)作为二抗进行染色，清洗5小时后使用抗荧光衰减封片剂进行封片。使用共焦激光扫描显微镜(LSM800，德国蔡司)拍摄视网膜血管图像。使用Photoshop进行新生血管和无灌注区的量化分析。
3.5组织免疫荧光染色。
将眼球解剖并在包埋胶中快速冷冻。将视网膜切片(8μm)，并在室温下在4％多聚甲醛中固定20分钟。然后在4℃下分别与GFAP抗体，α-SMA(Abcam，货号ab1224964)，Fibronectin(Abcam，货号ab45688)，IB4(Thermo，货号I21411)孵育过夜。用PBS洗涤三次后，将视网膜切片与Alexa Fluor488缀合的IgG(Abcam，货号ab150077)孵育2小时，用PBS洗涤三次后，用4，6-二氨基-2-苯基吲哚(1:500，Solarbio)标记细胞核。用PBS洗涤三次后滴加抗荧光衰减封片剂，用盖玻片密封。然后用共焦激光扫描显微镜(LSM800，德国蔡司)对它们进行拍照。
3.6 TUNEL分析
根据制造商的说明，使用TUNEL系统(Roche，货号11684795910)对视网膜冷冻切片进行TUNEL测定。
3.7蛋白质印迹分析
经BCA定量后，取等量的蛋白样品，加入PBS定容到20μl，加入5μl蛋白上样缓冲液(5×)，95℃加热，5分钟。蛋白电泳条件为100V，90min。电转条件为100V，100min。将电转后的PVDF膜取出，封闭，过夜孵育一抗。弃去一抗，洗涤3次。加入二抗，室温摇床孵育2小时。弃去二抗，加入TBST，洗涤3次，每次10分钟。使用ECL超敏发光液进行显影。
3.8组织病理学和免疫组织化学
在P17从脱颈处死的小鼠中摘除眼睛，在P25和P42脱颈处死小鼠，取肾脏，肝脏进行固定。使用苏木精和伊红(H&E)方案对标本进行染色，并使用光学显微镜进行成像。
3.9视网膜电图(ERG)
小鼠暗适应18小时，按照说明书(Phoenix Micron VI)进行操作，并以0.01至1cd-s/m的范围发 出白光闪光。使用陷波滤波来降低60Hz的信号噪声；将低切滤波设置为0.3Hz；将高切滤波设置在500Hz。测量a波和b波的振幅。麻醉小鼠使用带有加热垫，并根据需要用一滴平衡盐水溶液湿润眼睛。
3.10 OCT
使用德国海德堡SPECTRALIS-OCT观察小鼠眼底结构改变情况。使用托吡卡胺以滴眼液对小鼠双眼进行散瞳，待小鼠进入麻醉平稳期后，在小鼠眼部涂抹玻璃酸钠凝胶，进行视网膜扫描。扫描图像以小鼠视盘为中心，进行视网膜全层厚度的扫描。
3.11激光诱导的小鼠脉络膜新生血管的模型建立
雄性6周龄的C57BL/6J小鼠用于该实验，麻醉后散大瞳孔，使用Phoenix激光发射器对准视网膜进行激光照射，破坏视网膜色素上皮层及脉络膜。7天后由脉络膜发出新生血管侵袭视网膜。
3.12小鼠视网膜下注药
雄性6周龄的C57BL/6J小鼠用于该实验，麻醉后散大瞳孔，使用34号针头(Hamilton，Reno，NV，United States)从角巩膜缘进针，挑起视网膜将1μl腺相关病毒注射到小鼠视网膜下，滴度为1×1012。注射后使用抗生素凝胶覆盖眼表防止角膜水肿。
3.13眼底照相及眼底荧光造影
取激光诱导的新生血管模型鼠，麻醉后散大瞳孔，在透明凝胶的帮助下，使用Phoenix眼底镜头对准视盘中心进行拍照。随后经腹腔注射10％荧光素钠(5mL/kg)，10分钟后，在暗场滤镜下进行眼底照相显示渗漏斑。
3.14脉络膜新生血管量化
在成模后7天脱颈处死OIR小鼠，取出其眼球。剥离脉络膜，用4％多聚甲醛固定，并切成4个放射状瓣。使用山羊血清封闭2小时，使用IB4(Thermo，货号I21411)进行脉络膜血管染色。清洗5小时后，使用抗荧光衰减封片剂进行封片。使用共焦激光扫描显微镜(LSM800，德国蔡司)拍摄脉络膜血管图像。使用Photoshop进行新生血管量化分析。
4.眼内注射miR-15a-5p对小鼠发育的安全性评估
4.1血液本的采集
眼球后采集血样，通过2000g离心15分钟，分离血清，然后在-80℃下储存。将肝脏、脾脏、大脑、肾脏解剖出来，
4.2组织病理学检测
将主要器官样品(肝脏、脾脏、肾脏)用4％中性缓冲多聚甲醛固定24小时，将样品石蜡包埋，在3μm处切割并用苏木精和伊红染色。使用奥林巴斯BX51显微镜和奥林巴斯DP71CCD相机(日本东京奥林巴斯公司)拍摄显微照片
4.3血清生化参数的测定
通过市售检测试剂盒测定肌酐(Elabscience，货号EBCK188M、尿素氮(Elabscience，货号EBCK183M)、甘油三酯(Elabscience，货号EBCK126M)、总胆固醇(Elabscience，货号EBCK109S)的血清浓度。根据制造商的说明计算每个参数的浓度。
另外，文本中使用的试剂来源：
Mimics(miR-15a-5p模拟物)购自上海吉玛制药技术有限公司，货号B02001；
Agomir(Agomir-15a-5p模拟物)购自上海吉玛制药技术有限公司，货号B06001；结构为：正义 链为SEQ ID NO：1，反义链为SEQ ID NO：6再进行5'端2个硫代骨架修饰，3'端4个硫代骨架修饰，3'端胆固醇修饰以及全链甲氧基修饰。
Inhibitor(miR-15a-5p抑制剂)购自上海吉玛制药技术有限公司，货号B03001；
Smad2-WT载体购自上海吉玛制药技术有限公司，货号C09005-36176；
Smad2-mut载体购自上海吉玛制药技术有限公司，货号C09006-36176。
VEGF-WT载体购自上海吉玛制药技术有限公司，货号C09005-56969；
VEGF-mut载体购自上海吉玛制药技术有限公司，货号C09006-56969。
腺相关病毒购自上海吉玛制药技术有限公司，货号D08001。
实施例1：向视网膜血管内皮细胞转染miR-15a-5p的模拟物或抑制剂可以分别升高和降低miR-15a-5p的表达水平
miRNA的模拟物和抑制剂可以在体外模拟其生物学作用，miR-15a-5p的模拟物是体外合成的miR-15a-5p的碱基序列，miR-15a-5p的抑制剂是体外合成的miR-15a-5p的碱基互补序列。
miR-15a-5p的模拟物：
正义链：UAGCAGCACAUAAUGGUUUGUG(SEQ ID NO：1)；
反义链：CACAAACCAUUAUGUGCUGCUA(SEQ ID NO：6)。
首先要确定使用lipofectamine 3000助转染剂转染miR-15a-5p的模拟物或抑制剂确实能够升高或降低细胞中miR-15a-5p的表达水平。qRT-PCR的结果显示与模拟物对照组(模拟物的乱序)相比，转染miR-15a-5p的模拟物可以将细胞内miR-15a-5p的含量提高约150倍(图1)，且差异有统计学意义(P<0.05)。与抑制剂对照组(抑制剂的乱序)相比，转染miR-15a-5p抑制剂的细胞内miR-15a-5p的含量下降了80％(图1)，差异有统计学意义(P<0.05)。
视网膜及脉络膜含有大量的血管，其中内皮细胞是维持血管通透性的重要细胞。因此选取视网膜内皮细胞(HRMECs)进行实验具有代表性。实施例1表明在体外向HRMECs转染miR-15a-5p的模拟物和抑制剂可以高表达或者降表达miR-15a-5p，为后续观察miR-15a-5p对细胞的作用提供研究基础。
实施例2：VEGF刺激视网膜血管内皮细胞引起病理性增殖的最佳浓度
VEGF是已知的诱导体内体外血管新生的强效因子，在早产儿视网膜病变，糖尿病视网膜病变，脉络膜新生血管中具有较强的致病作用。在体外实验中，VEGF作用于HRMECs可以建立病理性增殖的模型。因此需要确定VEGF刺激的浓度。选择了0、10、20ng/mL的VEGF分别刺激HRMECs，CCK-8检测结果显示10ng/mL以上浓度均可以引起HRMECs显著增殖(图2)，同时该浓度不会引起HRMECs中miR-15a-5p表达量发生变化(图3)。
实施例3：miR-15a-5p在体外对视网膜血管内皮细胞病理性增殖的治疗作用
在确定10ng/mL的VEGF可引起HRMECs病理性增殖且miR-15a-5p表达水平不变后，继续研究miR-15a-5p在生理及病理条件(10ng/mL的VEGF)下对HRMECs的作用。CCK-8实验是对细胞增殖分裂活力进行测定的常用方法，结果显示，miR-15a-5p模拟物可以降低VEGF引起的细胞异常增殖(图4)。细胞划痕实验结果显示，miR-15a-5p模拟物可以减弱VEGF诱导的细胞增殖(图5-图6)。Transwell实验目的是观察细胞的迁移能力，结果显示miR-15a-5p模拟物可以减弱VEGF诱导的细胞异常迁移(图7-图8)。管腔形成实验模拟的是细胞在体外形成管腔的能力，结果显示miR-15a-5p模拟物可以减弱VEGF诱导的管腔增多(图9-图11)。以上差异均具有统计学意义(P<0.05)。
VEGF可以在体内引起内皮细胞增殖，进而诱导新生血管的发育，新生血管不同于生理性血管，新生血管壁缺少紧密连接，其内物质通过管壁渗漏到视网膜及玻璃体中，造成眼底渗出及出血，极大的影响了正常的视功能。在体外向HRMECs转染miR-15a-5p模拟物后，可以减少VEGF诱导的细胞异常增殖、迁移及管腔形成。这表明miR-15a-5p模拟物在体外对HRMECs的病理性增殖具有显著的治疗作用。
实施例4：小鼠视网膜对修饰后的miR-15a-5p模拟物的吸收更好
常见的miRNA的模拟物为mimic，其是由未经修饰的碱基组成，在体外，细胞对其有良好的吸收效果。在体内实验中，未经修饰的碱基容易被体内无处不在的核酸酶降解，因此，本实施例采用修饰后的miRNA，例如添加胆固醇等修饰。为对比视网膜对修饰及未修饰的模拟物的吸收效果，本实施例对比了小鼠在不同时间吸收同样量miR-15a-5p模拟物的效率。其中，经修饰的模拟物为Agomir。结果如图12所示，注射模拟物24小时后，出现明显差异，Agomir具有良好的组织相容性，视网膜对Agomir的吸收效果为mimic的2.5倍，并且高水平持续至注射后第7天。因此在后续动物实验中，选取Agomir(Agomir-15a-5p)作为miR-15a-5p的模拟物进行体内注射。
实施例5：miR-15a-5p对高氧诱导的小鼠视网膜新生血管的治疗作用
为探究miR-15a-5p对新生血管的作用，选择了氧诱导视网膜病变(OIR)模型，模型构建流程图见图13，该模型是最常用的视网膜新生血管模型，模拟视网膜新生血管(RNV)形成的病理过程。在此过程中，选取生长发育的时间点，检测视网膜中miR-15a-5p的表达水平，结果如图14所示，在新生血管的高峰期，出生后14天和17天，miR-15a-5p的表达水平升高，差异有统计学意义。随后为探索miR-15a-5p对体内新生血管的作用，首先设置Agomir-15a-5p模拟物的浓度梯度，在出生后第12天注射不同剂量的模拟物和模拟物对照，在出生后第17天经IB4染色视网膜新生血管分析。结果表明Agomir-15a-5p对新生血管的抑制作用具有剂量依赖性(图15-图16)，0.5μg-1.5μg的Agomir-15a-5p模拟物可显著抑制视网膜新生血管(图15-图16)。
同时选取VEGF单抗作为阳性对照，结果表明2μg的VEGF单抗药物对视网膜血管有最佳的抑制效果，随后选取2μg作为治疗浓度进行进一步验证(图17)。为了观察玻璃体腔注射后agomir在视网膜中的分布即持续时间，将CY3标记的agomir-15a-5p注射到小鼠的玻璃体内。其在注射后8小时便在视网膜中弥散分布，并且荧光强度在注射后24小时达到峰值(图18中的A，B)。此外，使用聚合酶链式反应检测视网膜中agomir的升高倍数。Agomir-15a-5p在注射后24小时增加约2-3倍，在注射后48小时达到约3-5倍的峰值浓度，其高表达持续5天(图18中的C)。
随后选取1μg作为治疗浓度进行进一步验证，同时选取VEGF单抗作为阳性对照，结果表明Agomir-15a-5p模拟物可以降低视网膜新生血管至对照组的65％(图19-图20)。相比于VEGF单抗，Agomir-15a-5p模拟物还可以促进视网膜无灌注区的恢复，视网膜无灌注区的面积减少至对照组的73％(图19和图21)。
腺相关病毒是高效的基因递送工具，使用腺相关病毒可以感染视网膜细胞，使其高表达miR-15a-5p。其中，腺相关病毒携带的核酸序列为TAGCAGCACATAATGGTTTGTG(SEQ ID NO：9)，其可以在体内转录为miR-15a-5p后发挥治疗作用。
在氧诱导小鼠出生后第五天，进行球内注射包含miR-15a-5p的腺相关病毒(1×1012)以及对照病毒(1×1012)。病毒结构如图22所示，在第12天取材视网膜，通过眼球冰冻切片观察腺相关病毒感染的部位，图23结果显示包含miR-15a-5p的腺相关病毒感染视网膜，视网膜出现弥散的荧光。随后通过PCR检测各组视网膜miR-15a-5p含量，结果如图24所示，注射了包含miR-15a-5p的腺相关病毒组视网膜的miR-15a-5p是对照组的13倍，说明该病毒可以成功过表达miR-15a-5p。在出生后第17天对氧诱导小鼠的视网膜新生血管进行定量，结果显示注射了包含miR-15a-5p的腺相关病毒组视网膜新生血管和无灌注区明显减少(图25-图27)。以上结果表明包含miR-15a-5p的腺相关病毒可以将miR-15a-5p成功递送到小鼠视网膜细胞中，并且对高氧诱导的小鼠视网膜新生血管具有治疗作用，可以促进无灌注区恢复。
实施例5采用了两种方法递送miR-15a-5p，一种是经修饰的miR-15a-5p，另一种是腺相关病毒。通过玻璃体腔注射两种物质分别达到了使视网膜中miR-15a-5p含量升高的目的。采取的氧诱导视网膜病变模型，是经典的眼底疾病模型，可以模拟多种眼底疾病，例如糖尿病视网膜病变和早产儿视网膜病变等疾病的新生血管表型。本实施例观察了miR-15a-5p对该模型的新生血管和无灌注区的治疗作用，发现miR-15a-5p对视网膜新生血管具有治疗作用，并且可以促进无灌注区恢复。
实施例6：miR-15a-5p促进了高氧诱导的小鼠视网膜无灌注区的恢复
内皮顶端细胞和星形胶质细胞的附着对无灌注区域的血管重塑至关重要。为了解释miR-15a-5p在 促进无灌注区血管重建中的作用，我们观察了第17天小鼠视网膜中星形胶质细胞和血管芽之间的关系。OIR小鼠视网膜血管闭塞区域的星形胶质细胞退化，星形胶质细胞的缺失伴随着Müller细胞的GFAP反应性增加，表现为浅血管丛中Müler细胞末端的斑点染色(图28A第二列)。具体来看，图28A最后一行显示常氧组视网膜星形胶质细胞呈现星形舒展形态，高氧对照治疗组，缺乏舒展的星形细胞形态。取而代之的是斑点状不规则细胞形态，是活化的Müller细胞的足板，代表Müller细胞的炎症反应性增加。然而，与OIR未治疗组视网膜相比，经过Agomir处理后，小鼠的视网膜无灌注区域中的星形胶质细胞形成了更好的网络，并保留了其正常的星形/树突形态和密度(图28A第三列)。图28B显示无灌注区域边缘的血管尖端内皮发出的丝状伪足，表明退化的血管的重建。我们的实验表明，与OIR对照组相比，经过Agomir治疗后，无灌注区的附近的内皮顶端细胞发出的丝状伪足增加了5倍(图28C)，而无灌注区的星型胶质细胞为丝状伪足从尖端内皮细胞延伸到闭塞视网膜中提供了支架和模板(图28D)。具体来看，图28D的第二行，常氧组血管与星形胶质细胞无缝隙攀附贴合，而高氧未治疗组血管无处攀附生长，反而有很多斑点状的细胞，经过Agomir治疗后，血管攀附星形胶质细胞有所恢复，最终促进了无灌注区恢复。VEGF单抗对上述血管和星形胶质细胞的攀附关系无修复效果。因此，miR-15a-5p介导的视网膜无灌注区的血管挽救与血管闭塞区内源性星形胶质细胞的保护有关。
实施例7：miR-15a-5p缺乏对高氧诱导的小鼠视网膜新生血管和无灌注区的影响
MiR-15a-5p对新生血管的抑制作用在miR-15a-5p敲除鼠中进行验证。选择了氧诱导视网膜病变(OIR)模型，进一步验证miR-15a-5p在视网膜血管生成中的作用。分别将正常鼠和敲除鼠置于高氧环境中以诱导视网膜病理性新生血管形成(图29)。在出生后第17天经IB4染色分析视网膜新生血管簇和血管闭塞面积。结果表明miR-15a-5p缺乏会明显增加OIR视网膜无灌注区及新生血管面积(图30-图31)。这表明miR-15a-5p的缺失影响了小鼠OIR模型中血管的形成。接下来，我们研究了miR-15a-5p模拟物(Agomir-15a-5p)对正常鼠和敲除鼠氧诱导新生血管模型的影响，以进一步确定miR-15a-5p是否调节视网膜异常血管生成(图32)。结果表明，玻璃体腔注射Agomir-15a-5p后，不仅可以降低正常鼠视网膜无灌注区和新生血管面积，也可以逆转敲除鼠中miR-15a-5p缺乏导致的视网膜无灌注区和病理性新生血管面积增加(图32-图34)。
之后我们在第17天的正常鼠和敲除鼠视网膜中观察星形胶质细胞和血管芽之间的关系。OIR小鼠视网膜血管闭塞区的星形胶质细胞退化，星形胶质细胞的缺失伴随着Müller细胞的GFAP反应性增加。并且相对于正常鼠，敲除鼠视网膜的GFAP染色相对稀疏。图中所示为浅血管丛中Müler细胞末端的斑点染色(图35)。然而，无论是正常鼠还是敲除鼠，Agomir处理后的小鼠的中央视网膜中星形 胶质细胞形成了更好的网络，并保留了其正常的星形/树突形态(图35)。之后，我们观察在无灌注区域边缘的血管芽发出的丝状伪足来观察退化血管的重建。结果表明，无论在正常鼠还是敲除鼠视网膜中，Agomir治疗增加了内皮顶端细胞的密度(图36)，而星型胶质细胞为尖端内皮细胞的丝状伪足延伸到闭塞视网膜中提供了支架和模版(图36)。
实施例8：miR-15a-5p对激光诱导的小鼠脉络膜新生血管的治疗作用
MiR-15a-5p对新生血管的抑制作用在脉络膜新生血管模型中也进行了验证。首先用激光诱导脉络膜新生血管模型，模型构建流程如图37所示，并向小鼠玻璃体腔注射Agomir-15a-5p的模拟物、模拟物对照和VEGF单抗。一周后，通过眼底荧光造影观察到与对照组相比，Agomir-15a-5p的模拟物及VEGF单抗组荧光渗漏面积明显减少，提示二者可抑制脉络膜新生血管的形成(图38-图39)。取脉络膜新生血管小鼠的脉络膜铺片行IB4染色以定量新生血管簇的面积(图40)，经统计，Agomir-15a-5p的模拟物及VEGF单抗组两组可明显抑制脉络膜新生血管簇的形成(图41)。
在脉络膜新生血管模型中同样采取如图22所示的病毒进行miR-15a-5p的递送。其中，腺相关病毒携带的核酸序列为TAGCAGCACATAATGGTTTGTG(SEQ ID NO：9)，其可以在体内转录为miR-15a-5p后发挥治疗作用。
在小鼠进行造模前7天，进行视网膜下注射包含miR-15a-5p的腺相关病毒(1×1012量)以及对照病毒(1×1012)。在注射后的第7天取材视网膜，通过眼球冰冻切片观察腺相关病毒感染的部位，图42结果显示包含miR-15a-5p的腺相关病毒感染视网膜和脉络膜，视网膜色素上皮层和脉络膜出现弥散的荧光。随后通过PCR检测各组视网膜脉络膜miR-15a-5p含量，结果如图43所示，注射了包含miR-15a-5p的腺相关病毒组视网膜脉络膜的miR-15a-5p是对照组的7倍，说明该病毒可以成功感染视网膜和脉络膜并过表达miR-15a-5p。在激光诱导后的第7天进行小鼠视网膜新生血管定量，结果显示注射了包含miR-15a-5p的腺相关病毒组视网膜新生血管明显减少(图44-图45)。以上结果表明包含miR-15a-5p的腺相关病毒可以将miR-15a-5p成功递送到小鼠视网膜脉络膜细胞中，并且对激光诱导的小鼠视网膜新生血管具有治疗作用。
实施例8同样采用了两种方法递送miR-15a-5p，一种是经修饰的miR-15a-5p，另一种是腺相关病毒。通过眼内注射两种物质分别达到了使视网膜脉络膜中miR-15a-5p含量升高的目的。采取的激光诱导脉络膜新生血管模型，可以模拟脉络膜新生血管。本实施例观察了miR-15a-5p对该模型的新生血管的治疗作用，发现miR-15a-5p对脉络膜新生血管具有治疗作用。
实施例9：miR-15a-5p对氧诱导的小鼠视网膜结构和功能损伤的治疗作用
实施例5表明miR-15a-5p可恢复OIR视网膜的早期血流灌注，这对视网膜神经细胞的营养及发 育至关重要。本实施例继续从视网膜厚度及视神经功能两个方面进行miR-15a-5p对视网膜保护作用评估。首先采用H&E染色可以直观的观察到视网膜的结构改变，图46显示Agomir-15a-5p模拟物治疗组小鼠视网膜结构完整，外丛状层(星型标志所示层)厚度正常，而VEGF单抗组小鼠外丛状层明显变薄。因此Agomir-15a-5p模拟物可明显改善氧诱导小鼠视网膜结构和功能的损伤。之后，通过光学相干断层成像OCT来量化视网膜各层厚度(图47-图48)，结果显示，Agomir-15a-5p模拟物可明显改善氧诱导小鼠的视网膜变薄，而VEGF单抗组视网膜厚度与未处理小鼠无明显差异，无法改善氧诱导小鼠的视网膜变薄(图49)。视网膜电生理检测(ERG)广泛用于视网膜功能的评估，分别在早期(小鼠出生后25天)及晚期(小鼠出生后42天)观察了Agomir-15a-5p模拟物对氧诱导小鼠视网膜功能的治疗作用(图50)。与未处理的常氧组相比，氧诱导各组小鼠视网膜的a波及b波振幅均降低。但氧诱导组视网膜经Agomir-15a-5p模拟物治疗后均明显增加了早期及晚期a波及b波振幅。而VEGF单抗对氧诱导小鼠视网膜的神经功能损伤无治疗作用(图51)。
视网膜是神经组织的一部分，其主要功能为将光信号转化为电信号并传导至大脑。视网膜的结构是正常发挥视功能的基础，视功能直接决定了患者的视力。各种眼底病变均伴随视网膜结构和功能的损耗。本实施例采取的氧诱导的视网膜病变模型，在发病后出现明显的视网膜变薄及电生理功能减弱，而眼内给药miR-15a-5p后，视网膜结构和神经功能均有显著改善，并且优于VEGF单抗组。
实施例10：miR-15a-5p对激光诱导的小鼠脉络膜视网膜结构和功能损伤的治疗作用
使用H&E染色评估视网膜层间结构、细胞形态和脉络膜新生血管的病灶。结果如图52所示，在对照组中，脉络膜发出新生血管长入视网膜层下，视网膜局部隆起。Agomir-15a-5p模拟物治疗组隆起较少，病灶基本消失，VEGF单抗组有少许病灶残留。使用ERG评估脉络膜新生血管小鼠视网膜电生理功能，结果如图53-图54显示，Agomir-15a-5p模拟物治疗组大幅改善脉络膜新生血管小鼠视网膜功能损伤，VEGF单抗组对视网膜功能损伤无显著治疗作用。另外，在氧化应激下，视网膜会出现神经胶质增生，GFAP是神经胶质细胞活化的指标，代表视网膜炎症水平的增加。Agomir-15a-5p治疗后，OIR视网膜中GFAP的激活显著降低(图55中A、B、C、D)，表明miR-15a-5p会抑制OIR视网膜的神经胶质细胞增殖。然而，与对照组相比。VEGF单抗处理并无抑制GFAP活化的趋势，且无统计学意义。
本实施例采取的激光诱导的脉络膜视网膜损伤模型，在发病后也出现明显的视网膜变薄及电生理功能减弱，而眼内给药miR-15a-5p后，视网膜结构和神经功能均有显著改善，并且优于VEGF单抗组。
实施例11：miR-15a-5p对高氧诱导和激光诱导的小鼠视网膜炎症的治疗作用
氧诱导的小鼠视网膜病变伴随炎症水平的增加，为探究miR-15a-5p对小鼠视网膜炎症的治疗作用，在P17、P25、P42取视网膜检测TNFα表达水平，图56结果显示，注射Agomir-15a-5p模拟物组小鼠在P17时TNFα蛋白表达水平降低，VEGF单抗组对视网膜TNFα水平升高无治疗作用。在P25和P42两个时间点各组间无统计学差异。细胞间粘附分子1(ICAM-1)通常表达于内皮细胞和免疫细胞，对白细胞迁移和活化具有关键作用。氧诱导鼠视网膜ICAM-1表达水平升高，注射Agomir-15a-5p模拟物组小鼠ICAM-1表达水平降低(图57-图58)。激光诱导的小鼠眼底出现脉络膜新生血管，该病变伴随视网膜脉络膜的炎症水平的增加。图59结果显示，注射Agomir-15a-5p模拟物可以降低激光诱导的各组小鼠视网膜炎症因子水平。而VEGF单抗组对激光诱导的各组小鼠视网膜炎症无治疗作用。
眼底疾病常伴随炎症因子的表达水平升高，导致视网膜脉络膜疾病加重并迁延不愈。TNFα和IL-1β常是经典的致炎因子，募集炎症细胞聚集。ICAM-1是内皮细胞黏附分子，可以募集白细胞黏附到内皮细胞表明，增加血管通透性。本实施例检测了不同治疗节点视网膜的炎症因子水平，结果表明眼内给药miR-15a-5p可以明显减少视网膜脉络膜的炎症因子的表达水平。VEGF单抗组并未显示明显的改善视网膜炎症的效果。
实施例12：miR-15a-5p靶向调节VEGF
miR-15a-5p是一段碱基序列，其调控下游基因的方式为碱基互补配对结合在目标基因的RNA上，阻碍目标基因翻译为蛋白质的过程。本实施例通过体内体外实验，观察miR-15a-5p对VEGF的靶向调节作用。体外实验使用未经修饰的miR-15a-5p模拟物(mimics)，体内实验使用经修饰的miR-15a-5p模拟物(Agomir)。通过碱基互补配对发现miR-15a-5p可以与VEGF的mRNA特异性结合，因此后续实施例分别在细胞和动物中进行验证。视网膜色素上皮(RPE)细胞是眼内VEGF的来源之一，已知TGF-β1可以在体外引起RPE细胞分泌的VEGF水平升高，因此向RPE细胞转染miR-15a-5p模拟物，并使用TGF-β1诱导VEGF分泌，结果显示，miR-15a-5p可以减少TGF-β1诱导的VEGF表达增加，图60示VEGF的mRNA水平，图61-图62示VEGF蛋白水平，图63示RPE细胞上清中的VEGF蛋白水平。图64示双荧光素酶报告实验，野生的VEGF mRNA可以与miR-15a-5p结合，突变型的VEGF无法与miR-15a-5p结合，因此荧光仅在结合了的组中发生淬灭，提示miR-15a-5p可以特异性调控VEGF转录。图65-图66示玻璃体腔注射Agomir-15a-5p模拟物可以逆转高氧诱导的小鼠视网膜VEGF表达水平增加。图67示玻璃体腔注射Agomir-15a-5p模拟物可以逆转激光诱导的小鼠视网膜VEGF的mRNA表达水平增加。
实施例13：miR-15a-5p相较抗-VEGF可以更长时间抑制视网膜ERK磷酸化信号激活
miR-15a-5p特异性结合VEGF的mRNA抑制VEGF转录。VEGF单抗拮抗VEGF蛋白，二者原 理不同。因此在对比二者疗效时，可以选择VEGF下游信号通路的激活情况进行观察。VEGF与其受体VEGFR2特异性结合后，通过磷酸化ERK等信号通路，激活内皮细胞增殖。因此选择ERK磷酸化信号通路作为观察指标。
注射及取材示意图见图68中A图。结果显示，高氧诱导组小鼠在注射当天即P12，视网膜出现磷酸化ERK信号增强(图68中B图和图68中I图)；在注射后第一天，即P13，VEGF单抗组小鼠视网膜磷酸化ERK信号首先出现降低趋势(图68中C图和图68中J图)；在注射后第二天，即P14，VEGF单抗和Agomir-15a-5p模拟物组均出现磷酸化ERK信号降低趋势(图68中D图和图68中K图)；在注射后的第四天，即P15，VEGF单抗组ERK磷酸化信号恢复高水平，而Agomir-15a-5p模拟物组保持磷酸化ERK信号降低趋势，且二者之间存在统计学差异(图68中E图和图68中L图)；新生血管的高峰期，P17同P15，Agomir-15a-5p模拟物组保持磷酸化ERK信号降低趋势，VEGF单抗组无抑制作用，且二者之间存在统计学差异(图68中F图和图68中M图)；新生血管恢复期即P20和P25，各组视网膜之间磷酸化ERK信号无显著差异(图68中G图、图68中H图、图68中N图和图68中O图)。在上述过程中ERK的总蛋白水平无明显差异，因此将各时间段磷酸化ERK信号的相对变化做折线图。图68中P图可以明显看出，未治疗组小鼠视网膜ERK的磷酸化信号自P13起升高，到P17逐渐降低，一直处于最高水平，VEGF单抗组在P13相较于Agomir-15a-5p模拟物抑制ERK磷酸化程度更高，但自P14起，Agomir-15a-5p模拟物抑制ERK磷酸化程度高于VEGF单抗，并在P15和P17和VEGF单抗组比较，差异有统计学意义。二者在P17时差距达到高峰，在P20和P25对ERK磷酸化均无抑制效果。这表明miR-15a-5p通过结合VEGF的mRNA能够更持久的抑制ERK信号通路，从而减少病理性新生血管的生成。
实施例14：miR-15a-5p靶向调节Smad2减少视网膜内皮细胞间充质转化
通过碱基互补配对发现miR-15a-5p可以与Smad2的mRNA特异性结合，因此后续实施例分别在细胞和动物中进行验证。体外实验使用未经修饰的miR-15a-5p模拟物(mimic)，体内实验使用经修饰的miR-15a-5p模拟物(Agomir)。首先在视网膜内皮细胞中转染了miR-15a-5p的模拟物和抑制剂，经PCR(图69)、Western Blot(图70-图71)实验表明，miR-15a-5p可抑制Smad2的RNA和蛋白表达水平，而miR-15a-5p的抑制剂可促进Smad2的RNA和蛋白表达水平。这提示miR-15a-5p可调节Smad2的表达。而为了确定miR-15a-5p与Smad2的直接结合，通过双荧光素酶报告进行验证(图72)。结果表明，与对照组相比，miR-15a-5p模拟物显著降低了Smad2野生型载体的荧光素酶活性，对突变型Smad2的荧光素酶活性没有任何影响。因此，miR-15a-5p通过结合Smad2的mRNA抑制其转录及表达，即Smad2是miR-15a-5p的直接调控靶点。
在眼底疾病中，例如糖尿病视网膜病变，新生血管性视网膜疾病，脉络膜新生血管，增殖性玻璃体视网膜病变等疾病都伴随着视网膜纤维化的病理改变。而Smad2作为TGF-β信号通路中的一环，是典型的促纤维化通路蛋白。因此为了验证miR-15a-5p能否通过抑制Smad2抑制视网膜纤维化的发生，使用TGF-β1刺激视网膜内皮细胞诱导内皮-间充质转化(EndoMT)模型模拟视网膜纤维化改变，并向细胞内转染miR-15a-5p模拟物，观察miR-15a-5p模拟物对EndoMT的抑制作用。
通过细胞免疫荧光染色(图73-图74)和Western Blot(图75-图76)实验可以直观的观察到，TGF-β1刺激内皮细胞后，纤维化的标志物波形蛋白和α-SMA表达明显增高，而内皮细胞标志物CD31表达降低。这表明TGF-β1诱导内皮细胞向纤维细胞转化，也就是发生了EndoMT。而转染miR-15a-5p模拟物逆转了TGF-β1诱导后波形蛋白、α-SMA表达增高以及CD31表达降低。因此，miR-15a-5p可以通过抑制Smad2的磷酸化及总Smad2的表达而逆转TGF-β1所诱导的EndoMT(图77-图78)。
实施例15：miR-15a-5p靶向调节Smad2减少视网膜Müller细胞纤维化
Müller细胞是贯穿整个视网膜的一种特化的神经胶质细胞，具有维持视网膜的正常结构和功能的作用，还参与多种病理过程，尤其是眼底增殖性病变，如视网膜新生血管、糖尿病视网膜病变等，体外培养Müller细胞是研究这些增殖性眼底病变的途径之一。TGF-β2可以在体外刺激Müller细胞出现纤维化的表型。图79和图80结果显示1ng/mL的TGF-β2可以引起Müller细胞活化伴随纤维化相关蛋白表达水平升高。因此本实施例采取1ng/mL的TGF-β2进行造模。向Müller细胞转染了miR-15a-5p的模拟物及模拟物对照，随后加入1ng/mL的TGF-β2进行共培养，结果显示转染了miR-15a-5p的模拟物的Müller细胞GFAP表达水平降低，纤维化相关蛋白表达水平降低(图81和图82)。进一步也采取了细胞免疫荧光进行观察，首先用Müller细胞标志物GS以鉴定Müller细胞(图83)，模拟物转染后可以逆转Müller细胞的活化和纤维化(图84)。此外，转染了miR-15a-5p的模拟物后，Müller细胞中炎症因子包括TNF-α和MCP1的表达降低(图85和图86)。
由于在视网膜内皮细胞中证实miR-15a-5p可以靶向调节Smad2减少TGFβ信号通路活化，因此在Müller细胞中进行验证，图87显示转染了miR-15a-5p的模拟物的Müller细胞总Smad2表达水平降低，磷酸化信号减弱(图88)。因此miR-15a-5p可以靶向调节Smad2表达减少视网膜Müller细胞纤维化。
实施例16：miR-15a-5p靶向调节Smad2减少高氧诱导小鼠视网膜纤维化趋势
在高氧诱导小鼠玻璃体腔中注射Agomir-15a-5p模拟物，模拟物对照以及VEGF单抗。在P17取小鼠的眼球进行冰冻切片，并提其蛋白进行Western Blot检测。图89示α-SMA和视网膜血管在视网膜上的表达共定位情况，可见高氧小鼠对照组α-SMA表达水平更高，与血管共定位，而玻璃体腔中 注射Agomir-15a-5p模拟物组α-SMA表达水平更低，视网膜共定位少。图90示纤维连接蛋白和视网膜血管的表达定位，高氧小鼠对照组纤维连接蛋白表达水平更高，与血管共定位，而玻璃体腔中注射Agomir-15a-5p模拟物组纤维连接蛋白表达水平更低，视网膜共定位少，VEGF单抗组纤维连接蛋白表达水平更高。图91示α-SMA和视网膜活化的Müller细胞表达定位，其中GFAP示活化的Müller细胞。结果显示高氧小鼠对照组和VEGF单抗组的α-SMA和视网膜活化的Müller细胞共定位更显著。这表明Agomir-15a-5p模拟物可以抑制视网膜的纤维化趋势，而VEGF单抗会加重视网膜的纤维化趋势。Western Blot检测结果见图92，进一步证实miR-15a-5p可以减少视网膜纤维化蛋白的表达水平，减少视网膜纤维化改变。而VEGF单抗治疗对视网膜纤维化病变无治疗作用。
实施例17：miR-15a-5p对氧诱导小鼠发育的安全性评估
实施例5和实施例9确定了玻璃体腔注射Agomir-15a-5p模拟物对氧诱导小鼠的新生血管的治疗作用后，应对其药物安全性进行评估，以确定这种治疗方案对小鼠的生长发育、代谢及重要脏器有无不良影响。图93中A图显示自给药当天P12(出生后第12天)至小鼠基本成年P42(出生后42天)，小鼠的体重变化。结果表明，从出生后15天开始常氧组小鼠的体重增长率明显高于高氧的三组小鼠，但从给药至成年，高氧的三组小鼠体重增长率无明显差异(图93中A图)，因此玻璃体腔注射Agomir-15a-5p模拟物不会对高氧组小鼠的生长发育产生不良影响。为进一步观察玻璃体腔注射Agomir-15a-5p模拟物的安全性，分别在P17、P25、P42三个时间点收集小鼠的血清，检测总胆固醇、甘油三酯、肌酐及尿素氮等指标，评估各组小鼠的肝肾功能。如图93中B图显示，VEGF单抗治疗组小鼠血浆颜色与其余各组颜色不同。说明VEGF单抗治疗可能对小鼠脂代谢产生影响。如图93中C图显示，与常氧组相比，在P17和P25两个时间点显示，高氧的三组小鼠血清肌酐水平明显降低，但各组间无明显差异，可能与体重较低有关。图93中D图表明，P17时，与其他组相比，VEGF单抗治疗组的小鼠血清尿素水平明显升高。P25时，高氧对照组小鼠血清尿素水平明显升高，Agomir-15a-5p模拟物治疗组与常氧组小鼠血清尿素含量无明显差异。图93中E图显示血浆甘油三酯结果，除P17时，VEGF单抗治疗组的小鼠血清甘油三酯水平明显升高，其余组间无明显差异。图93中F图显示总胆固醇的变化趋势，在P17时，高氧组的总胆固醇水平表达均高于常氧组，但高氧组各组间无明显差异。此外，使用H&E染色观察了P17、P25及P42小鼠的肝脏及肾脏结构(图93中G图和图93中H图)。以上结果说明玻璃体腔注射Agomir-15a-5p模拟物不会对高氧组小鼠的生长发育、代谢及脏器产生不良影响。而VEGF单抗眼内注射会导致模型组小鼠血脂代谢异常。
实施例18：miR-15a-5p对正常小鼠发育及视网膜的安全性评估
按照上述治疗方案对未造模的小鼠进行玻璃体腔注射。对各组小鼠进行体重检测，发现从P21开 始，VEGF单抗组的体重呈下降趋势，P24时，体重下降显著，与其余三组差异有统计学意义，并持续至P42。而Agomir-15a-5p模拟物不会对正常小鼠的体重造成影响(图94中A图)。同样分别在P17、P25、P42三个时间点收集小鼠的血清，观察肌酐(图94中B图)、尿素氮(图94中C图)、甘油三酯(图94中D图)和总胆固醇(图94中E图)等指标，评估各组小鼠脂质代谢的肝肾功能。发现除VEGF单抗组在P17时甘油三酯水平较常氧组升高外，其余均无差异，这表明VEGF单抗影响小鼠的甘油三酯代谢。玻璃体腔注射荧光分子(CY3)标记的Agomir-15a-5p模拟物后，取小鼠肝肾进行检测，结果显示肝肾无明显荧光，表明Agomir-15a-5p模拟物未在肝肾残留(图94中F图)。取小鼠肾(图94中G图)肝(图94中H图)的组织切片进行H&E染色，结果显示无明显异常。给未造模小鼠行玻璃体腔注药，观察玻璃体腔注射Agomir-15a-5p模拟物对正常小鼠的眼部及生长发育是否有副作用。OCT结果显示Agomir-15a-5p模拟物对视网膜厚度无影响，但是VEGF单抗组小鼠视网膜变薄(图94中I图、图94中J图、图94中K图)。经GFAP染色显示，玻璃体腔注射Agomir-15a-5p模拟物不会引起胶质细胞的激活(图94中L图、图94中M图)。这表明玻璃体腔注射VEGF单抗会导致小鼠发育迟缓，血脂代谢异常，视网膜变薄。
实施例19：miR-15a-5p对糖尿病鼠视网膜神经损伤的保护作用
糖尿病患者血糖水平较高，对神经系统产生不可逆损伤。糖尿病患者的视功能下降明显，本实施例选取自发的糖尿病模型鼠(瘦素敲除鼠)作为视网膜神经变性的模型进行研究，观察miR-15a-5p眼内注射对神经变性的保护作用。给12周龄的小鼠行玻璃体腔注射Agomir-15a-5p模拟物，给同样数量的小鼠玻璃体腔注射模拟物对照。在16周使用视网膜电生理检测糖尿病小鼠的视网膜功能。结果表明，Agomir-15a-5p模拟物相较于其对照可以明显改善糖尿病鼠视网膜光感受器细胞(图95、图96和图98)和双极细胞(图95和图97)的振幅强度，即Agomir-15a-5p模拟物对糖尿病鼠视网膜神经损伤具有保护作用。
实施例20：miR-15a-5p缺失导致小鼠血管发育异常
小鼠的视网膜血管是在出生后以视盘为中心发出，在视网膜内表面呈放射状蔓延生长，逐渐形成浅层血管网覆盖整个视网膜，随后向深层发育，形成完整的视网膜血管系统。在此期间，P7时浅层血管已经分化出血管主干和分支，并且覆盖80％的视网膜区域，并开始向深层发育。P9时浅层血管完全覆盖视网膜，深层血管逐渐覆盖50％的视网膜。与此同时，星形胶质细胞与视网膜血管网的发育密切相关。星形胶质细胞同样由视盘发出，在视网膜内面浅层由视盘逐渐向周边蔓延，其细胞个体呈现星状结构，总体呈现类似血管网的网状结构，并且发育早于血管，为视网膜血管的发育提供了模板。在本实施例中，我们使用了miR-15a-5p敲除鼠，观察了P7和P9的视网膜血管及星形胶质细胞的发育情 况。图99结果表明，出生后7天，miR-15a-5p敲除鼠浅层血管发育迟缓，其覆盖视网膜的程度低于正常小鼠，差异有统计学意义(图99中A图和图99中B图)，这说明缺失miR-15a-5p会减缓小鼠视网膜浅层血管的发育。并且，miR-15a-5p敲除鼠已经形成的浅层血管网的总长度，分支长度，和交叉的血管形成的节点个数均少于正常鼠(图99中C图，图99中D图和图99中E图)，这表明缺失miR-15a-5p会导致血管网的规则程度下降。图99中F图是血管和星形胶质细胞的共同染色的代表性图像，正常鼠的周边区尽管没有血管灌注，但是仍然存在大量星形胶质细胞排列而成的网状结构，但是敲除鼠的周边区缺少星形胶质细胞。经统计，敲除鼠视网膜GFAP阳性的星形胶质细胞构成的网状结构的节点个数和总长度数少于正常鼠(图99中G图和H图)。敲除鼠的血管网与星形胶质细胞构成的网状结构重合度低于正常鼠(图99中I图)。
小鼠出生后9天，浅层血管网基本覆盖视网膜，但是敲除鼠的浅层血管网规则程度较差(图100中A图)，浅层血管节点个数，浅层血管网眼个数，浅层血管总长度和浅层血管总分支长度均低于正常鼠(图100中C图，图100中D图，图100中E图和图100中F图)。深层血管发育情况如图100中B图所示，敲除鼠的深层血管网覆盖视网膜程度低于正常鼠(图100中G图)。因此，缺失miR-15a-5p会导致视网膜浅层及深层血管发育迟缓，星形胶质细胞发育异常，进而导致血管网与星形胶质细胞网之间的连接缺失。即miR-15a-5p对血管发育具有重要的调控作用。
上述20个实施例中，实施例1和实施例4表明，未经修饰的miR-15a-5p或修饰的miR-15a-5p具有生物活性，可在体外被细胞吸收，或者在体内被视网膜细胞吸收，并迅速发挥生物学作用。实施例2和实施例3表明，在体外，miR-15a-5p可以抑制病理性新生血管生成。实施例5-11表明，在体内，通过眼内给药的方式，miR-15a-5p可以抑制病理性新生血管、促进无灌注区恢复、减少视网膜炎症因子表达并促进神经损伤修复。VEGF是经典的促新生血管生成因子，参与多种眼底疾病的进展。其中实施例6和7表明miR-15a-5p可以拯救星形胶质细胞，为血管顶端细胞提供模板，相较于VEGF单抗可以明显促进无灌注区恢复，血管的灌注对于神经细胞的存活至关重要。实施例12表明miR-15a-5p可以直接靶向调节VEGF的mRNA表达水平，实施例13进一步对比了miR-15a-5p与VEGF单抗在抑制视网膜新生血管信号通路的持久度，结果表明miR-15a-5p可以更持久的发挥抑制作用。在眼底疾病中，例如糖尿病视网膜病变，新生血管性视网膜疾病，脉络膜新生血管，增殖性玻璃体视网膜病变等疾病都伴随着视网膜纤维化的病理改变。而Smad2作为TGF-β信号通路中的一环，是典型的促纤维化通路蛋白。实施例14-16表明miR-15a-5p通过抑制Smad2抑制视网膜纤维化的发生。实施例17-18评估了眼内注射miR-15a-5p和VEGF单抗对氧诱导的小鼠发育及正常小鼠发育的影响，结果表明VEGF单抗可导致正常小鼠体重降低，血脂异常，而miR-15a-5p对小鼠发育无不良影响。实施例19表 明miR-15a-5p可以缓解糖尿病鼠视网膜神经变性。实施例20表明，缺失miR-15a-5p会导致小鼠视网膜浅层及深层血管发育迟缓，星形胶质细胞发育异常，进而导致血管网与星形胶质细胞网之间的连接缺失，影响视网膜发育，即miR-15a-5p对血管发育具有重要的调控作用。综上表明，miR-15a-5p或修饰的miR-15a-5p在治疗眼底疾病方面具有明确效果，在生物医药领域中具有非常好的应用和研究价值。
实施例21：miR-15a-5p突变体对视网膜新生血管的治疗效果
实施例1-20证明了miR-15a-5p或修饰的miR-15a-5p在治疗眼底疾病方面具有明确效果。本实施例进一步在miR-15a-5p基础上进行突变，确定miR-15a-5p突变体在治疗眼底疾病中的作用。突变位点如表4所示，其中加粗序列为种子序列(治疗疾病必须包含的序列)，下划线序列为突变序列，分别突变1，2，3，5个位点。随后使用突变序列对视网膜新生血管模型进行治疗，结果如表4和图101所示，野生型序列可以抑制新生血管达74.8％，非种子区突变序列仍然可以达到类似的抑制新生血管的效果。因此本实施例证实miR-15a-5p种子序列对二者存在调控作用。
表4

以上详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这些简单变型均属于本发明的保护范围。
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本发明对各种可能的组合方式不再另行说明。

Claims (32)

  1. miRNA和/或修饰的miRNA和/或包含转录为miRNA的核酸的载体在制备治疗和/或预防眼底疾病的产品中的应用，所述的miRNA的核苷酸序列包括ACGACGAU(SEQ ID NO：10)。
  2. 根据权利要求1所述的应用，其特征在于，所述的miRNA的核苷酸序列包括SEQ ID NO：10，并且与SEQ ID NO：1的核苷酸序列具有60％以上同一性或包含一个或多个核苷酸的取代、缺失或插入的核苷酸序列。
  3. 根据权利要求2所述的应用，其特征在于，所述的miRNA的核苷酸序列与SEQ ID NO：1的核苷酸序列具有65％、70％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、95％或99％以上同一性或包含不超过十个核苷酸的取代、缺失或插入的核苷酸序列。
  4. 根据权利要求1所述的应用，其特征在于，所述的miRNA的核苷酸序列为SEQ ID NO：1。
  5. 根据权利要求1所述的应用，其特征在于，所述转录为miRNA的核酸包含TAGCAGCA(SEQ ID NO：11)。
  6. 根据权利要求1所述的应用，其特征在于，所述转录为miRNA的核酸包含SEQ ID NO：11，且与SEQ ID NO：9的核苷酸序列具有60％以上同一性或包含一个或多个核苷酸的取代、缺失或插入的核苷酸序列。
  7. 根据权利要求1所述的应用，其特征在于，所述转录为miRNA的核酸为SEQ ID NO：9。
  8. 根据权利要求1所述的应用，其特征在于，所述的眼底疾病选自玻璃体病变、视网膜病变、视神经病变或脉络膜病变中的一种或两种以上。
  9. 根据权利要求1所述的应用，其特征在于，所述的眼底疾病选自早产儿视网膜疾病、视网膜新生血管疾病、脉络膜新生血管疾病或糖尿病视网膜病变中的一种或两种以上。
  10. 根据权利要求1所述的应用，其特征在于，所述miRNA的序列包含修饰，例如在碱基上进行的修饰。
  11. 根据权利要求1-10任一所述的应用，其特征在于，所述的载体为病毒载体或非病毒载体。
  12. 根据权利要求11所述的应用，其特征在于，所述的病毒载体包括慢病毒载体、逆转录病毒载体、腺病毒载体、腺相关病毒载体、痘病毒载体或疱疹病毒载体中的一种或两种以上。
  13. 根据权利要求11所述的应用，其特征在于，所述的非病毒载体包括脂质体、脂质纳米颗粒、聚合物、多肽、抗体、适配体或N-乙酰半乳糖胺中的一种或两种以上。
  14. 根据权利要求1所述的应用，其特征在于，修饰的miRNA包括在碱基上进行的修饰。
  15. 根据权利要求10或14所述的应用，其特征在于，所述碱基上进行的修饰包括3'端进行胆固醇修饰、5'端两个硫代骨架修饰、3'端四个硫代骨架修饰或全链甲氧基修饰中的一种或两种以上。
  16. 根据权利要求1所述的应用，其特征在于，所述的产品包含正义链和反义链，所述的正义链包括SEQ ID NO：1或与SEQ ID NO：1的核苷酸序列具有60％以上同源性或包含一个或多个核苷酸的取代、缺失或插入的核苷酸序列，所述的反义链包括SEQ ID NO：6或与SEQ ID NO：6的核苷酸序列具有60％以上同源性或包含一个或多个核苷酸的取代、缺失或插入的核苷酸序列；
    优选的，所述的反义链上包含碱基的修饰。
  17. 一种包含转录为miRNA的核酸的载体，其特征在于，所述的miRNA的核苷酸序列包括SEQ ID NO：10。
  18. 根据权利要求17所述的载体，其特征在于，所述的miRNA的核苷酸序列包括SEQ ID NO：10，并且与SEQ ID NO：1的核苷酸序列具有60％以上同一性或包含一个或多个核苷酸的取代、缺失或插入的核苷酸序列；
    优选的，所述的miRNA的序列为SEQ ID NO：1。
  19. 根据权利要求17所述的载体，其特征在于，所述转录为miRNA的核酸包含TAGCAGCA(SEQ ID NO：11)。
  20. 根据权利要求17所述的载体，其特征在于，所述转录为miRNA的核酸包含SEQ ID NO：11，且与SEQ ID NO：9的核苷酸序列具有60％以上同一性或包含一个或多个核苷酸的取代、缺失或插入的核苷酸序列。
  21. 根据权利要求17所述的载体，其特征在于，所述转录为miRNA的核酸为SEQ ID NO：9。
  22. 根据权利要求17所述的载体，其特征在于，所述的载体为病毒载体或非病毒载体；
    优选的，所述病毒载体包括慢病毒载体、逆转录病毒载体、腺病毒载体、腺相关病毒载体、痘病毒载体或疱疹病毒载体中的一种或两种以上；
    优选的，所述的非病毒载体包括脂质体、脂质纳米颗粒、聚合物、多肽、抗体、适配体或N-乙酰半乳糖胺中的一种或两种以上。
  23. 一种药物或药物组合物，其特征在于，所述的药物或药物组合物包括miRNA或其模拟物、修饰的miRNA或者权利要求17-22任一所述的载体，以及药学上可接受的辅料；
    所述的miRNA的核苷酸序列包括ACGACGAU(SEQ ID NO：10)。
  24. 根据权利要求23所述的药物或药物组合物，其特征在于，所述的miRNA的核苷酸序列包括SEQ ID NO：10，并且与SEQ ID NO：1的核苷酸序列具有60％以上同一性或包含一个或多个核苷酸的取代、缺失或插入的核苷酸序列；
    优选的，所述的miRNA的核苷酸序列为SEQ ID NO：1。
  25. 根据权利要求23所述的药物或药物组合物，其特征在于，修饰的miRNA包括在碱基上进行的修饰；优选的，所述碱基上进行的修饰包括3'端进行胆固醇修饰、5'端两个硫代骨架修饰、3'端四个硫代骨架修饰或全链甲氧基修饰中的一种或两种以上。
  26. 一种治疗和/或预防眼底疾病的方法，其特征在于，所述的方法包括向有需要的受试者施用有效量的miRNA、miRNA模拟物、修饰的miRNA、权利要求17-22任一所述的载体或权利要求23-25任一所述的药物或药物组合物；
    其中，所述的miRNA的核苷酸序列包括ACGACGAU(SEQ ID NO：10)。
  27. 根据权利要求26所述的方法，其特征在于，所述的眼底疾病选自玻璃体病变、视网膜病变、视神经病变或脉络膜病变中的一种或两种以上。
  28. 根据权利要求26所述的方法，其特征在于，所述的眼底疾病选自早产儿视网膜疾病、视 网膜新生血管疾病、脉络膜新生血管疾病或糖尿病视网膜病变中的一种或两种以上。
  29. 根据权利要求26所述的方法，其特征在于，所述的miRNA的核苷酸序列包括SEQ ID NO：10，并且与SEQ ID NO：1的核苷酸序列具有60％以上同一性或包含一个或多个核苷酸的取代、缺失或插入的核苷酸序列；
    优选的，所述的miRNA的核苷酸序列为SEQ ID NO：1。
  30. 根据权利要求26所述的方法，其特征在于，所述的miRNA或其模拟物包含正义链和反义链，所述的正义链包含SEQ ID NO：1或与SEQ ID NO：1的核苷酸序列具有60％以上同源性或包含一个或多个核苷酸的取代、缺失或插入的核苷酸序列，所述的反义链包括SEQ ID NO：6或与SEQ ID NO：6的核苷酸序列具有60％以上同源性或包含一个或多个核苷酸的取代、缺失或插入的核苷酸序列。
  31. 根据权利要求26所述的方法，其特征在于，修饰的miRNA包括在碱基上进行的修饰；
    优选的，所述碱基的修饰包括3'端进行胆固醇修饰、5'端两个硫代骨架修饰、3'端四个硫代骨架修饰或全链甲氧基修饰中的一种或两种以上。
  32. 根据权利要求26所述的方法，其特征在于，所述施用的部位可以为受试者的眼内空间或腔；例如前房中的房水、悬吊韧带、睫状体、睫状体内和肌肉、晶状体或虹膜、玻璃体、视网膜、脉络膜或视神经等中的一种或两种以上。
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116785312B (zh) * 2023-06-21 2023-11-14 天津医科大学眼科医院 miR-15a-5p在治疗眼底疾病中的应用
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Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101585794B1 (ko) * 2014-10-07 2016-01-18 가톨릭대학교 산학협력단 miRNA를 이용한 안과 질환의 예방 또는 치료
CN107683341A (zh) * 2015-05-08 2018-02-09 新加坡科技研究局 用于慢性心力衰竭的诊断和预后的方法
CN109414459A (zh) * 2016-03-24 2019-03-01 斯坦姆实验室 源自脐带血的外泌体用于组织修复的用途
US20190136320A1 (en) * 2016-04-25 2019-05-09 Instytut Biologii Doswiadczalnej Im. Marcelego Nenckiego Polska Akademia Nauk Microrna biomarkers in blood for diagnosis of alzheimer's disease
WO2019191109A1 (en) * 2018-03-28 2019-10-03 The Board Of Trustees Of The Leland Stanford Junior University Microrna inhibitors and their use in treating epiretinal membranes
CN111184734A (zh) * 2020-03-05 2020-05-22 北京市心肺血管疾病研究所 miRNA在治疗心肌肥厚中的应用
CN111394447A (zh) * 2020-02-17 2020-07-10 天津医科大学眼科医院 一种血浆小细胞外囊泡miR-431-5p的应用
CN113481237A (zh) * 2021-06-29 2021-10-08 厦门朔望医药科技有限公司 一种预防和治疗新生血管眼部疾病的基因药物
CN115948392A (zh) * 2022-08-03 2023-04-11 中山大学中山眼科中心 miR-1910-5p拮抗剂在治疗病理性新生血管中的应用
CN116785312A (zh) * 2023-06-21 2023-09-22 天津医科大学眼科医院 miR-15a-5p在治疗眼底疾病中的应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080227704A1 (en) * 2006-12-21 2008-09-18 Kamens Joanne S CXCL13 binding proteins
AR112600A1 (es) * 2017-05-30 2019-11-20 Teijin Pharma Ltd Anticuerpo anti-receptor de igf-i
IL270835B2 (en) * 2017-06-12 2025-01-01 Sinai Health Sys Allograft tolerance without the need for systemic immunosuppression

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101585794B1 (ko) * 2014-10-07 2016-01-18 가톨릭대학교 산학협력단 miRNA를 이용한 안과 질환의 예방 또는 치료
CN107683341A (zh) * 2015-05-08 2018-02-09 新加坡科技研究局 用于慢性心力衰竭的诊断和预后的方法
CN109414459A (zh) * 2016-03-24 2019-03-01 斯坦姆实验室 源自脐带血的外泌体用于组织修复的用途
US20190136320A1 (en) * 2016-04-25 2019-05-09 Instytut Biologii Doswiadczalnej Im. Marcelego Nenckiego Polska Akademia Nauk Microrna biomarkers in blood for diagnosis of alzheimer's disease
WO2019191109A1 (en) * 2018-03-28 2019-10-03 The Board Of Trustees Of The Leland Stanford Junior University Microrna inhibitors and their use in treating epiretinal membranes
CN111394447A (zh) * 2020-02-17 2020-07-10 天津医科大学眼科医院 一种血浆小细胞外囊泡miR-431-5p的应用
CN111184734A (zh) * 2020-03-05 2020-05-22 北京市心肺血管疾病研究所 miRNA在治疗心肌肥厚中的应用
CN113481237A (zh) * 2021-06-29 2021-10-08 厦门朔望医药科技有限公司 一种预防和治疗新生血管眼部疾病的基因药物
CN115948392A (zh) * 2022-08-03 2023-04-11 中山大学中山眼科中心 miR-1910-5p拮抗剂在治疗病理性新生血管中的应用
CN116785312A (zh) * 2023-06-21 2023-09-22 天津医科大学眼科医院 miR-15a-5p在治疗眼底疾病中的应用

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
"China Master Thesis", 31 December 2020, CHENGDE MEDICAL COLLEGE, CN, article HUANG YANJIE: "The Study of MiR-15a-5p Regulating Multidrug Resistance through the Wnt/β-catenin Pathway in Colorectal Cancer", pages: 1 - 57, XP093251612 *
HE DAN, RUAN ZHONG-BAO, SONG GUI-XIAN, CHEN GE-CAI, WANG FEI, WANG MEI-XIANG, YUAN MAO-KUN, ZHU LI: "miR-15a-5p regulates myocardial fibrosis in atrial fibrillation by targeting Smad7", PEERJ, vol. 9, pages e12686, XP093251616, ISSN: 2167-8359, DOI: 10.7717/peerj.12686 *
JIN LU, LI YIFAN, HE TAO, HU JIA, LIU JIAJU, CHEN MINGWEI, ZHANG ZENG, GUI YAOTING, MAO XIANGMING, YANG SHANGQI, LAI YONGQING: "miR-15a-5p acts as an oncogene in renal cell carcinoma", MOLECULAR MEDICINE REPORTS, SPANDIDOS PUBLICATIONS, GR, vol. 15, no. 3, 1 March 2017 (2017-03-01), GR , pages 1379 - 1386, XP093251611, ISSN: 1791-2997, DOI: 10.3892/mmr.2017.6121 *
LI YULONG, ZONG WEI; LIU HAILING; WANG PING;: "Effect of miR-15a-5p on the proliferation of gastric cancer cells", JOURNAL OF SHANXI MEDICAL UNIVERSITY, CN, vol. 50, no. 5, 31 May 2019 (2019-05-31), CN , pages 566 - 569, XP093251610, ISSN: 1007-6611 *
QIAN JIANSHAENG, LI YU; DOU JIAN-WEI: "miR-15a-5p inhibits proliferation and migration of human hepatocellular carcinoma SMMC-7721 cells", CHINESE JOURNAL OF PATHOPHYSIOLOGY, ZHONGGUO BINGLI SHENGLI XUEHUI, BEIJING, CN, vol. 33, no. 2, 27 February 2017 (2017-02-27), CN , pages 344 - 348 +351, XP093251608, ISSN: 1000-4718 *
何倩欣 (HE, QIANXIN): "miR-15a-5p在腹膜透析相关的腹膜纤维化中的作用及分子机制 (Effect of miR-15a-5p on Peritoneal Fibrosis Caused by Peritoneal Dialysis)", 万方数据库 (WANFANG DATA), 2 September 2020 (2020-09-02), XP009559529 *

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