CN108440645A - A kind of biologically active polypeptide and its application - Google Patents
A kind of biologically active polypeptide and its application Download PDFInfo
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- CN108440645A CN108440645A CN201810202362.7A CN201810202362A CN108440645A CN 108440645 A CN108440645 A CN 108440645A CN 201810202362 A CN201810202362 A CN 201810202362A CN 108440645 A CN108440645 A CN 108440645A
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- biologically active
- polypeptide
- active polypeptide
- hydrogel
- mhspgad
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Abstract
The present invention relates to a kind of biologically active polypeptide and its applications, belong to polypeptide development technique field.The amino acid sequence of biologically active polypeptide provided by the invention is as shown in SEQ ID NO.1.Polypeptide of the present invention can promote migration from a variety of stem cells to damaged tissues, go back to the nest and differentiation to adult cell.It can promote angiogenesis in myocardial ischemia and lower limb ischemia disease, improve blood supply, accelerate tissue repair regeneration；It is applied in skin injury, the reparation of skin histology, the migration proliferation of induced skin stem cell and differentiation can be promoted, promote damage location angiogenesis and functional rehabilitation.
Description
Technical field
The present invention relates to polypeptide development technique fields, and in particular to a kind of biologically active polypeptide and its application.
Background technology
The morbidity and mortality of angiocardiopathy are in the various diseases umber one, according to the establishment of national cardiovascular disease center
《Chinese cardiovascular disease report 2017》It points out, cardiovascular patient number in China's is about 2.9 hundred million at present, and cardiovascular death is occupied
People's disease death constitutes 40% or more.
Blood is to organize to provide the medium of the important substances such as oxygen, nutrition with cell, due to injury of blood vessel, blood vessel blockage
Etc. amount of blood supply deficiency caused by reasons frequently can lead to tissue and cell is in hypoxemia and malnourished state, and then induction group
Knit the necrosis with cell.Since angiocardiopathy caused by tissue ischemia includes lower limb ischemia, myocardial ischemia, renal ischaemia and glycosuria
Sick skin ulcer etc..
Tissue ischemia can be effectively treated by the way that stem cell is injected directly into ischemic injury site, but its is expensive, and
Cell survival rate is relatively low, this may be in the damaging conditions of part severe (hypoxic-ischemic, lacks matrix protection at inflammation) with cell
It is related.Therefore, ideal active factors are found to promote stem cell to be controlled to ischemic migration, quickening angiogenesis ischemic disease
It treats particularly important.
Invention content
The purpose of the present invention is to provide a kind of biologically active polypeptide and its applications.Polypeptide of the present invention can promote more
Migration from kind of stem cell to damaged tissues, go back to the nest and differentiation to adult cell.In myocardial ischemia and lower limb ischemia disease its
It can promote angiogenesis, improve blood supply, promote tissue repair regeneration；It is applied in skin injury, skin histology can be promoted
Reparation, the migration proliferation of induced skin stem cell and differentiation promote damage location angiogenesis and functional rehabilitation.
The present invention provides a kind of biologically active polypeptide, the amino acid sequence such as SEQ IDNO.1 of the biologically active polypeptide
It is shown.
Preferably, the amino acid includes D types or L-type amino acid.
The present invention also provides the hydrogel for being combined with biologically active polypeptide described in above-mentioned technical proposal, the hydrogel packet
The biological active component and hydrogel carrier by physical mixed are included, the biological active component is biologically active polypeptide, described
Hydrogel carrier includes aquagel, hyaluronic acid gel, collagen hydrogel or Pluronic F127 hydrogels.
The present invention also provides the medical dressing for being combined with biologically active polypeptide described in above-mentioned technical proposal, the combination life
The medical dressing of object active peptides includes biologically active polypeptide and timbering material carrier, and the timbering material carrier includes collagen sea
Continuous or chitosan sponge.
The present invention also provides a kind of chemically modified biologically active polypeptide, the biologically active polypeptide is above-mentioned technology
Biologically active polypeptide described in scheme.
Preferably, the chemical modification includes phosphorylation modification, Azide modification or carboxylated modification.
The present invention also provides the gel for being combined with chemically modified biologically active polypeptide described in above-mentioned technical proposal, institutes
It includes the biological active component and gel carrier combined by chemical covalent to state gel, and the biological active component is chemical modification
Biologically active polypeptide afterwards, the gel carrier include chitosan gel rubber or hyaluronic acid derivatives.
The present invention also provides biology is combined described in biologically active polypeptide described in above-mentioned technical proposal or above-mentioned technical proposal
In conjunction with the medical dressing of biologically active polypeptide or above-mentioned technical proposal institute described in the hydrogel or above-mentioned technical proposal of active peptides
It states described in chemically modified biologically active polypeptide or above-mentioned technical proposal in conjunction with the solidifying of chemically modified biologically active polypeptide
Application of the glue in preparing treatment skin injury drug or dressing.
The present invention also provides one kind being coated with biologically active polypeptide or above-mentioned technical proposal institute described in above-mentioned technical proposal
State chemically modified biologically active polypeptide or above-mentioned skill in conjunction with described in the hydrogel or above-mentioned technical proposal of biologically active polypeptide
In conjunction with the hospital gauze of the gel of chemically modified biologically active polypeptide described in art scheme.
The present invention also provides biology is combined described in biologically active polypeptide described in above-mentioned technical proposal or above-mentioned technical proposal
In conjunction with the medical dressing of biologically active polypeptide or above-mentioned technical proposal institute described in the hydrogel or above-mentioned technical proposal of active peptides
It states described in chemically modified biologically active polypeptide or above-mentioned technical proposal in conjunction with the solidifying of chemically modified biologically active polypeptide
Glue is preparing the application in promoting angiogenesis, promotion tissue repair regenerating medicine or medical material.
The present invention also provides biology is combined described in biologically active polypeptide described in above-mentioned technical proposal or above-mentioned technical proposal
In conjunction with the medical dressing of biologically active polypeptide or above-mentioned technical proposal institute described in the hydrogel or above-mentioned technical proposal of active peptides
It states described in chemically modified biologically active polypeptide or above-mentioned technical proposal in conjunction with the solidifying of chemically modified biologically active polypeptide
Glue is preparing the application in preventing and/or treating ischemic disease drug or medical material.
Preferably, the ischemic disease includes myocardial infarction, lower limb ischemia and ischemic renal injury.
The present invention provides a kind of biologically active polypeptides, have amino acid sequence shown in SEQ ID NO.1.It is of the present invention
Polypeptide can promote a variety of stem cells hyperplasias and at damaged tissues migration, go back to the nest and differentiation to adult cell.In cardiac muscle
Its in ischemic and lower limb ischemia disease can promote angiogenesis, improve blood supply, reduce fibrosis；It is answered in skin injury
Used time can promote the reparation of skin histology, the migration proliferation of induced skin stem cell and differentiation, promote damage location blood vessel new
Raw and functional rehabilitation.The result shows that the product that polypeptide of the present invention is prepared can realize histology at injury tissue
Recovery on upper and function assessment；By injecting or being coated to injury tissue in ischemic disease, the new life of blood vessel can be promoted,
Promote stem cell attachment and is proliferated and is existence one suitable microenvironment of maintenance of cell；Meanwhile, it is capable to promote local skin
The reparation of tissue accelerates angiogenesis, and reduces the generation of tissue fibrosis.Such as combine the Hydrogel In Treating of polypeptide MHSPGAD
Polypeptide is conducive to raise stem cell when myocardial ischemia, enhances it and promotes cardiomyocyte proliferation, anti-apoptotic and stimulation angiogenesis
Ability improves myocardial hypertrophy and promotes the recovery of cardiac function and structure, and reduces the generation of cardiac muscular tissue's fibrosis；In conjunction with
Hemoperfusion situation is dramatically increased in the Pluronic F127 Hydrogel In Treating lower limb ischemia diseases experiment of MHSPGAD, with PBS groups
Compared to increasing about 46%, about 35% is increased compared with pure Pluronic F127 hydrogel groups, is shown in conjunction with MHSPGAD's
The recovery of effective stimulus angiogenesis and hind limb perfusion is capable of in the processing of Pluronic F127 hydrogel groups.
The product that biologically active polypeptide of the present invention is prepared has effective therapeutic effect, has no toxic side effect, damages
Hinder small, suitable prolonged application.In addition, when biologically active polypeptide of the present invention is repaired for skin injury, it is suitable for all kinds of senses
The protecting wound surface of metachromia wound, including burn, scald and the damaged surface of a wound etc., has antibiotic anti-infection, hemostasis and pain-relieving and promotion
The function of wound healing.
Description of the drawings
Fig. 1 is the mobility spectrum for a variety of ancestral cells of polypeptid induction that the embodiment of the present invention 14 provides；
Fig. 2 is under the Pluronic F127 hydrogels for the combination biologically active polypeptide that the embodiment of the present invention 17 provides improve
The hemoperfusion situation of limb ischemic.
Specific implementation mode
The present invention provides a kind of biologically active polypeptide, the amino acid sequence such as SEQ ID of the biologically active polypeptide
Shown in NO.1.In the present invention, the amino acid sequence is specially MHSPGAD.In the present invention, the amino acid includes D types
Or L-type amino acid, i.e., the amino acid sequence of the described polypeptide are more specifically (D/L types) M (D/L types) H (D/L types) S (D/L types) PG
(D/L types) A (D/L types) D.The biologically active polypeptide that amino acid sequence of the present invention is MHSPGAD is 7 transcriptional variations of HDAC
The short open reading frame (sORF) of body.HDAC 7 is specific expressed in skin in the blood vessels, helps to maintain early embryonic development mistake
Vascular integrity in journey.Histon deacetylase (HDAC) (HDACs) is the N- acetylated lysine residues removal second on histone
The enzyme family of acyl group participates in gene transcription regulation by adjusting chromatin Structure.The polypeptide of synthesis in solid state of the present invention
(MHSPGAD) can in inducing ischemic disease a variety of ancestral cells proliferation and to the migration of damaged tissues, go back to the nest and to adult
The differentiation of cell, including neural stem cell, vascular stem cell, Cardiac Stem Cells, skin progenitor cell and mescenchymal stem cell
(marrow, umbilical cord tissue, placenta tissue, adipose tissue etc.).Polypeptide (MHSPGAD) of the present invention be solve myocardial ischemia, under
Migration, proliferation and the differentiation of stem cell, the blood vessel of damage location in the diseases such as limb ischemic and acute renal ischemia and skin injury
Newborn and tissue replacement therapy provides new strategy.
Specifically, in the present invention, the biologically active polypeptide includes { L-Met } { L-His } { L-Ser } { L-Pro } G
{L-Ala}{L-Asn}、{D-Met}{L-His}{L-Ser}{L-Pro}G{D-Ala}{D-Asn}、{D-Met}{D-His}{D-
Ser } { D-Pro } G { D-Ala } { D-Asn } etc..
The present invention does not have the preparation method of the biologically active polypeptide special restriction, ripe using those skilled in the art
The polypeptide synthesis method known carries out artificial synthesized.Specifically, the solid phase synthesis process of biologically active polypeptide of the present invention
Include the following steps：
The C-terminal of (1) first amino acid is grafted on carboxy resin, and rate of carrying is 0.6mmol/g；20% piperidines is dissolved in
N, N '-dimethyl formamide (DMF) remove the Fmoc blocking groups of amino；In the present invention, the carboxy resin includes Wang Shu
Fat or 2- chlorine triphenyl chlorine resins；
(2) in condensing agent O- benzotriazole-tetramethylurea hexafluorophosphate (HBTU) and catalyst n, N- diisopropyls
Under the collective effect of ethamine (DIPEA), the carboxyl of next amino acid is combined with free amino；Repeat above step, polypeptide
Chain constantly increases, and obtains the resin for being connected with polypeptide；
(3) it is connected with the resin (each 5min, 5 times) of polypeptide described in being rinsed repeatedly with DMF, is rinsed (every time with DCM again later
1min is rinsed 3 times)；Dry resin is handled with trifluoroacetic acid (TFA), and polypeptide is detached from from resin, it is molten to be transferred into TFA
Liquid；
(4) it collects solution to be spin-dried for, obtains concentrate solution；With frost ether processing, centrifugation is precipitated；
(5) precipitation is dissolved in dimethyl sulfoxide (DMSO) (DMSO), polypeptide MHSPGAD is obtained using high-efficient liquid phase chromatogram purification.
The present invention also provides the hydrogel for being combined with biologically active polypeptide described in above-mentioned technical proposal, the hydrogel packet
The biological active component and hydrogel carrier by physical mixed are included, the biological active component is biologically active polypeptide, described
Hydrogel carrier includes aquagel, hyaluronic acid gel, collagen hydrogel or Pluronic F127 hydrogels.
In the present invention, the hydrogel of the combination biologically active polypeptide specifically include the polypeptide by physical bond biologically active polypeptide/
Aquagel, polypeptide/hyaluronic acid gel, polypeptide/collagen hydrogel and polypeptide/Pluronic F127 hydrogels.
In the present invention, the hydrogel can be used in treating the wound repair etc. after myocardial infarction, lower limb ischemia and skin injury.This hair
The bright preparation method to the hydrogel for being combined with biologically active polypeptide does not have special restriction, using those skilled in the art
The preparation method of the hydrogel of well known routine combination polypeptide.The polypeptide that the present invention combines is including being (D/L types) M (D/L
Type) H (D/L types) S (D/L types) PG (D/L types) A (D/L types) D, Azide/carboxylated polypeptide (D/L types) M (D/L types) H (D/L
Type) S (D/L types) PG (D/L types) A (D/L types) D, phosphorylated polypeptide (D/L types) M (D/L types) H { pSer } (D/L types) PG (D/L
One or more of type) A (D/L types) D.
The present invention preferably includes following steps in the preparation method for preparing the aquagel in conjunction with biologically active polypeptide：
(1) chitosan is dissolved into the acetum that mass fraction is 1%~3% with quality volume fraction 1%~5%
In, room temperature obtains CS solution overnight；
(2) dialysis 1 is carried out to the CS solution using the dialysis membrane that molecular cut off is 8kDa~10kDa in distilled water
Week remaining acetic acid is removed, solution ph is 7.2 after dialysis, obtains CS- hydrochlorides by freeze-drying later；
(3) CS- hydrochlorides described in water dissolution are distilled, MHSPGAD polypeptides are added, is uniformly mixed, obtains 1ng/mL~1mg/
Biologically active polypeptide/chitosan solution of mL；
(4) sodium β-glycerophosphate (β-GP) solution is slowly dropped to the chitosan polypeptide with the speed of 15 drops/minute
In solution, 0.5h is stirred under condition of ice bath, obtains mixed solution；Final concentration of 0.005~the 0.05mol/ of β-GP in mixed solution
L；
(5) mixed solution is transferred to 5min in 37 DEG C of insulating boxs, obtains the chitosan water for being combined with biologically active polypeptide
Gel.
The present invention observes hydrogel pattern preferably to polypeptide/aquagel of preparation using scanning electron microscope,
The performance evaluation of rheological properties test evaluation polypeptide hydrogel.
The present invention prepare in conjunction with biologically active polypeptide hyaluronic acid gel when, the preparation method preferably include with
Lower step：
(1) room temperature obtains the hyaluronic acid solution that quality volume fraction is 1~3% using distillation water dissolution hyaluronic acid；
It is 5.5 ± 0.1 to adjust pH value, and EDC/NHS is added thereto, stirs 1~2h, activates the carboxyl of hyaluronic acid, and it is saturating to obtain activation
Bright matter acid solution；
(2) mercaptoethylmaine, room temperature is added into the activation hyaluronic acid solution, it is saturating to obtain sulfydryl modification for reaction overnight
Bright matter acid；
(3) sulfydryl modification hyaluronic acid distilled water is dialysed 3 days, is freeze-dried, obtains mercapto after purification
Base modifies hyaluronic acid；
(4) sulfydryl modification hyaluronic acid after purification is dissolved in distilled water, it is 1~3% to make its quality volume fraction, to
Biologically active polypeptide is wherein added, is configured to biologically active polypeptide/hyaluronic acid solution containing 1ng/mL~1mg/mL, sulfydryl oxygen
Change forms biologically active polypeptide/hyaluronic acid gel.
The present invention preferably selects type i collagen when preparing the collagen hydrogel in conjunction with biologically active polypeptide.The present invention is to institute
The source for stating type i collagen does not have special restriction, using conventional commercial type i collagen well known to those skilled in the art.
In the present invention, the preparation method of the collagen hydrogel for being combined with biologically active polypeptide preferably includes following steps：
(1) type i collagen is added in 0.1M acetic acid solutions to the collagen solution for being made into 5~10mg/mL；
(2) sodium hydroxide is added into the collagen solution to be neutralized, obtains neutral collagen solution；
(3) biologically active polypeptide MHSPGAD is added into the neutral collagen solution, is configured to contain 1ng/mL~1mg/mL
Biologically active polypeptide/collagen solution；
(4) by polypeptide/collagen solution under conditions of pH neutrality, it is incubated 30min under the conditions of 37 DEG C, obtains combining biology living
The collagen hydrogel of property polypeptide.
For the present invention when preparing the Pluronic F127 hydrogels in conjunction with biologically active polypeptide, the preparation method is preferred
Include the following steps：
(1) Pluronic F127 powder is dissolved in the PBS buffer solution of precooling, under the conditions of 4 DEG C on magnetic stirring apparatus
It is stirred overnight, is configured to the Pluronic F127 solution that quality volume fraction is 5~20%；
(2) biologically active polypeptide is added into Pluronic F127 solution, continues to stir at 4 DEG C, is configured to contain 1ng/mL
The biologically active polypeptide of~1mg/mL/Pluronic F127 solution；
(3) it is incubated 5min or so under the conditions of 37 DEG C, obtains the PluronicF127 hydrogels in conjunction with biologically active polypeptide.
The present invention also provides the medical dressing for being combined with biologically active polypeptide described in above-mentioned technical proposal, the combination life
The medical dressing of object active peptides includes biologically active polypeptide and timbering material carrier, the timbering material carrier be include collagen
Sponge or chitosan sponge.Specifically, the medical dressing of present invention combination biologically active polypeptide includes combining biologically active polypeptide
Chitosan sponge or combine biologically active polypeptide collagen sponge.In the present invention, the medical dressing can be used in treating
The treatment use of wound repair and ischemic disease after skin injury.
For the present invention when preparing the collagen sponge in conjunction with biologically active polypeptide, the preparation method preferably includes following step
Suddenly：
(1) type i collagen is added in 0.1M acetic acid solutions to the collagen solution for being made into 5~20mg/mL；
(2) sodium hydroxide is added into the collagen solution to be neutralized, obtains neutral collagen solution；
(3) biologically active polypeptide MHSPGAD is added into the neutral collagen solution, is configured to contain 1ng/mL~1mg/mL
Biologically active polypeptide/collagen solution；
(4) collagen polypeptide solution is injected in mold, it is 0.1~10mm to make thickness, and -4 DEG C make the collagen in conjunction with polypeptide pave,
Being placed in -80 DEG C again makes polypeptide collagen solution solidify；
(5) collagen polypeptide of solidification is placed in vacuum freeze-drying machine and is freeze-dried, lyophilisation condition be -20 DEG C continue 60~
120min, -40 DEG C continue 180~240min, and 0 DEG C continues 16~19h, and 20 DEG C continue 2~3h, and vacuum degree is maintained at 0~
180pa, sub-cut are sub-packed in packing bag for medical use, the collagen sponge in conjunction with polypeptide are obtained after gamma-ray irradiation sterilizing at various specifications；
For the present invention when preparing the chitosan sponge in conjunction with biologically active polypeptide, the preparation method preferably includes following step
Suddenly：
(1) acetic acid (0.1M) dissolving chitosan (Mn=20,000,5%, w/v), room temperature is used to stay overnight；
(2) distilled water is used to dialyse to CS solution in dialysis membrane (molecular cut off is 8kDa~10kDa), 1 week,
Remaining acetic acid is removed, freeze-drying obtains chitosan hydrochloride；
(3) distillation water dissolution chitosan hydrochloride (20mg/mL), be added MHSPGAD polypeptides, be uniformly mixed, obtain polypeptide/
Chitosan (1ng/mL~1mg/mL) solution；
(4) polypeptide/chitosan solution is injected in mold, it is 0.1-10mm to make thickness, and -4 DEG C make the collagen in conjunction with polypeptide spread
It is flat, then being placed in -80 DEG C makes polypeptide/chitosan solution solidify；
(5) polypeptide/chitosan of solidification is placed in vacuum freeze-drying machine and is freeze-dried, lyophilisation condition is -20 DEG C and continues 60
~120min, -40 DEG C continue 180~240min, and 0 DEG C continues 16~19h, 20 DEG C of lasting 2-3h, and vacuum degree is maintained at 0~
180pa, sub-cut are sub-packed in packing bag for medical use at various specifications, the chitosan sea in conjunction with polypeptide are obtained after gamma-ray irradiation sterilizing
It is continuous；
The present invention also provides a kind of chemically modified biologically active polypeptide, the biologically active polypeptide is above-mentioned technology
Biologically active polypeptide described in scheme.In the present invention, the chemical modification includes phosphorylation modification, Azide modification or carboxylated
Modification.
The present invention does not have the preparation method of the biologically active polypeptide of the chemical modification special restriction, using this field
Polypeptide synthesis method known to technical staff carries out artificial synthesized.Specifically, in the present invention, the Azide/carboxyl
It includes following to change the solid phase synthesis process of polypeptide (D/L types) M (D/L types) H (D/L types) S (D/L types) PG (D/L types) A (D/L types) D
Step：
The resin for being connected with polypeptide is obtained according to Peptide systhesis scheme described in above-mentioned technical proposal；
(1) caproic acid of Azide or binary acid (succinic acid, adipic acid, decanedioic acid) are connected to peptide N-terminus respectively
On amino；
(2) it rinses the resin (each 5min is rinsed 5 times) for being connected with polypeptide repeatedly with DMF, is rinsed (every time with DCM again later
1min is rinsed 3 times)；Dry resin is handled with trifluoroacetic acid (TFA), and polypeptide is detached from from resin, it is molten to be transferred into TFA
Liquid；
(3) it collects solution to be spin-dried for, obtains concentrate solution；With frost ether processing, centrifugation is precipitated；
(4) precipitation is dissolved in dimethyl sulfoxide (DMSO) (DMSO), using high-efficient liquid phase chromatogram purification obtain end with nitrine or
Polypeptide product (D/L types) M (D/L types) H (D/L types) S (D/L types) PG (D/L types) A (D/L types) D-N3 or D/L types of carboxyl) M
(D/L types) H (D/L types) S (D/L types) PG (D/L types) A (D/L types) D-COOH.
In the present invention, phosphorylated polypeptide (D/L types) M (D/L types) H { pSer } (D/L types) PG (D/L types) A (D/L
Type) solid phase synthesis process of D includes the following steps：
The resin for being connected with polypeptide is obtained according to Peptide systhesis scheme described in above-mentioned technical proposal；
(1) after Peptide systhesis, the side chain protecting group of serine is selectively sloughed, serine uses Fmoc-Ser (trt),
It is quantitatively sloughed under the conditions of 1%TFA/DCM；Phosphorylation afterwards, using double benzyl phosphoramidites, tetrazole generates phosphoramidite tetrazolium
Reactive intermediate is connected on hydroxyl, and then oxidation generates phosphoryl under peroxy acid, completes the phosphorylation of serine；
(2) it rinses the resin (5min, 5 times) for being connected with polypeptide repeatedly with the DMF of certain volume, is rinsed again with DCM later
(1min, 3 times)；Dry resin is handled with trifluoroacetic acid (TFA), and polypeptide is detached from from resin, is transferred into TFA solution；
(3) it collects solution to be spin-dried for, obtains concentrate solution；With frost ether processing, centrifugation is precipitated；
(4) precipitation is dissolved in dimethyl sulfoxide (DMSO) (DMSO), (D/L types) M (D/L types) is obtained using high-efficient liquid phase chromatogram purification
H { pSer } (D/L types) PG (D/L types) A (D/L types) D.
The present invention also provides the gels for being combined with chemically modified biologically active polypeptide described in above-mentioned technical proposal.
In the present invention, the gel carrier includes chitosan gel rubber or hyaluronic acid derivatives.In the present invention, it specifically includes by covalent
It is grafted the polypeptide-chitosan gel rubber and polypeptide-hyaluronic acid derivatives of Azide modification.In the present invention, the gel can be used
Wound repair etc. after treatment myocardial infarction, lower limb ischemia and skin injury.
The present invention does not have special limit to the preparation method of the gel of the biologically active polypeptide for being combined with chemical modification
It is fixed, using the preparation method of the gel of routine combination polypeptide well known to those skilled in the art.The polypeptide that the present invention combines
Including being (D/L types) M (D/L types) H (D/L types) S (D/L types) PG (D/L types) A (D/L types) D, Azide/carboxylated polypeptide (D/L
Type) M (D/L types) H (D/L types) S (D/L types) PG (D/L types) A (D/L types) D, phosphorylated polypeptide (D/L types) M (D/L types) H
One or more of { pSer } (D/L types) PG (D/L types) A (D/L types) D.
The present invention is when preparing the chitosan gel rubber of the chemically modified biologically active polypeptide of covalent bond, when the chemistry
When biologically active polypeptide after modification is the biologically active polypeptide of Azide modification, it is anti-that the present invention preferably first carries out click chemistry
It answers, by covalently making MHSPGAD-N3Polypeptide is combined with chitosan.In the present invention, click chemistry reaction preferably include with
Lower step：
Chitosan is scattered in water phase, pentinoic acid is added, obtains chitosan-pentinoic acid reaction system；In 1- (3- diformazans
Aminopropyl) -3- ethyl-carbodiimide hydrochlorides catalysis under, the chitosan-pentinoic acid reaction system is condensed in ice bath
Reaction 24 hours, product obtains alkynyl chitosan through dialysis purification；
It is reacted MHSPGAD-N by click chemistry3With the alkynyl chitosan reaction：Using anhydrous cupric sulfate, resist
Bad hematic acid sodium makees catalyst, and 37 DEG C of constant temperature is stirred to react for 24 hours, and product is dialysed 3 days through water, is freeze-dried to obtain polypeptide-chitosan production
Object (CS-MHSPGAD).
In the present invention, the chitosan is preferably macromolecular chitosan (molecular weight ranges 20,000-200,000),
In the present invention, the water phase is distilled water.In the present invention, alkynyl can be grafted to chitosan molecule by the condensation reaction
In side group.In the present invention, the molar ratio of the chitosan repetitive unit and pentinoic acid is preferably 1:(0.1~0.7), more preferably
It is 1:0.5.
After obtaining alkynyl chitosan, the present invention is reacted by click chemistry by MHSPGAD-N3It is poly- with the alkynyl shell
Sugar reaction：Catalyst is done using anhydrous cupric sulfate, sodium ascorbate, 37 DEG C of constant temperature is stirred to react 24 hours, and product is through distilled water
Dialysis 3 days (dialysis retaining molecular weight is 3kDa~5kDa), -20 DEG C~-80 DEG C, 1.3~10Pa is freeze-dried to obtain polypeptide-shell
Glycan product (CS-MHSPGAD).In the present invention, the click chemistry reacts MHSPGAD-N3With anhydrous cupric sulfate, Vitamin C
The molar ratio of sour sodium is preferably (1.0-1.25):(0.1~0.5):(0.3-0.7), more preferably 1:0.2:0.4.
After obtaining polypeptide-chitosan product of covalence graft, the present invention carries out the chitosan of covalent bond MHSPGAD polypeptides
The preparation of gel, i.e.,：CS-MHSPGAD is dissolved in for 5%~20% in water phase with quality volume fraction, obtains covalent bond
Polypeptide-chitosan gel rubber of chemically modified active peptides；
The present invention is when preparing the hyaluronic acid derivatives of the chemically modified biologically active polypeptide of covalent bond, when describedization
When learning the biologically active polypeptide that the biologically active polypeptide after modification is Azide modification, it is anti-that the present invention preferably first carries out click chemistry
It answers, by covalently making MHSPGAD-N3 polypeptides be combined with hyaluronic acid.In the present invention, the click chemistry reaction preferably includes
Following steps：
Hyaluronic acid is scattered in water phase, propargylamine is added, obtains hyaluronic acid-propargylamine reaction system；In 1- (3-
Dimethylamino-propyl) -3- ethyl-carbodiimide hydrochlorides catalysis under, by the hyaluronic acid-propargylamine reaction system in ice bath
Middle condensation reaction 24 hours, product obtains ynamine hyaluronic acid through dialysis purification；
It is reacted by click chemistry and reacts MHSPGAD-N3 with the ynamine hyaluronic acid：Using anhydrous cupric sulfate,
Sodium ascorbate makees catalyst, and 37 DEG C of constant temperature is stirred to react for 24 hours, and product is dialysed 3 days through water, is freeze-dried to obtain polypeptide-hyalomitome
Acid product (HA-MHSPGAD).
In the present invention, ynamine can be grafted on hyaluronan molecule carboxyl by the condensation reaction.In the present invention,
The molar ratio of the hyaluronic acid repetitive unit and propargylamine is preferably 1:(0.1~0.7), more preferably 1:0.6.
After obtaining ynamine hyaluronic acid, the present invention reacts that MHSPGAD-N3 and the ynamineization is saturating by click chemistry
Bright matter acid reaction：Catalyst is done using anhydrous cupric sulfate, sodium ascorbate, 37 DEG C of constant temperature is stirred to react 24 hours, and product is through steaming
Distilled water is dialysed 3 days (dialysis retaining molecular weight is 3kDa~6kDa), and -20 DEG C~-80 DEG C, 1.3~10Pa is freeze-dried to obtain HA-
MHSPGAD.In the present invention, the molar ratio of click chemistry the reaction MHSPGAD-N3 and anhydrous cupric sulfate, sodium ascorbate
Preferably (1.0-1.25):(0.1~0.5)：(0.3-0.7), more preferably 1:0.2：0.4.
After obtaining polypeptide-hyaluronic acid product of covalence graft, the present invention carries out the chemically modified biology of covalent bond
The preparation of the hyaluronic acid derivatives of active peptides, i.e.,：HA-MHSPGAD is dissolved in water with quality volume fraction for 5%~20%
Xiang Zhong obtains polypeptide-hyaluronic acid derivatives of the chemically modified active peptides of covalent bond；
The present invention also provides biology is combined described in biologically active polypeptide described in above-mentioned technical proposal or above-mentioned technical proposal
In conjunction with the medical dressing of biologically active polypeptide or above-mentioned technical proposal institute described in the hydrogel or above-mentioned technical proposal of active peptides
It states described in chemically modified biologically active polypeptide or above-mentioned technical proposal in conjunction with the solidifying of chemically modified biologically active polypeptide
Application of the glue in preparing treatment skin injury drug or dressing.Biologically active polypeptide and hydrogel of the present invention can promote
The reparation of skin histology, migration, proliferation and the differentiation of induced skin stem cell promote damage location angiogenesis and function extensive
It is multiple, it can be used in the drug for preparing the good treatment skin injury of drug effect.
The present invention also provides one kind being coated with biologically active polypeptide or above-mentioned technical proposal institute described in above-mentioned technical proposal
State chemically modified biologically active polypeptide or above-mentioned skill in conjunction with described in the hydrogel or above-mentioned technical proposal of biologically active polypeptide
In conjunction with the hospital gauze of the gel of chemically modified biologically active polypeptide described in art scheme.Biologically active polypeptide of the present invention
It is coated on hospital gauze with hydrogel, a kind of gauze for treating all kinds of infected wounds, the infected wound can be obtained
Including burn, scald and damaged surface of a wound etc., the gauze can the anti-sense of antibacterial it is right, hemostasis and pain-relieving, wound healing etc..
The present invention also provides biology is combined described in biologically active polypeptide described in above-mentioned technical proposal or above-mentioned technical proposal
In conjunction with the medical dressing of biologically active polypeptide or above-mentioned technical proposal institute described in the hydrogel or above-mentioned technical proposal of active peptides
It states described in chemically modified biologically active polypeptide or above-mentioned technical proposal in conjunction with the solidifying of chemically modified biologically active polypeptide
Glue is preparing the application in promoting angiogenesis, promotion tissue repair regenerating medicine or medical material.
The present invention also provides biology is combined described in biologically active polypeptide described in above-mentioned technical proposal or above-mentioned technical proposal
In conjunction with the medical dressing of biologically active polypeptide or above-mentioned technical proposal institute described in the hydrogel or above-mentioned technical proposal of active peptides
It states described in chemically modified biologically active polypeptide or above-mentioned technical proposal in conjunction with the solidifying of chemically modified biologically active polypeptide
Glue is preparing the application in preventing and/or treating ischemic disease drug or medical material.In the present invention, the ischemic disease
Disease includes myocardial infarction, lower limb ischemia and ischemic renal injury.Polypeptide and hydrogel of the present invention can promote blood vessel new
It is raw, improve blood supply, reduces the generation of fibrosis, realize the treatment of ischemic disease.
A kind of biologically active polypeptide of the present invention and its application are done further in detail with reference to specific embodiment
Introduction, technical scheme of the present invention includes but not limited to following embodiment.
Embodiment 1
The synthesis in solid state of { L-Met } { L-His } { L-Ser } { L-Pro } G { L-Ala } { L-Asn } polypeptide：
Polypeptide is by ripe synthesis in solid state (SPPS) step, using 2- chlorine trityl chloride resin and side chain by tert-
Buty radical protections and amino is by fmoc-protected Amino acid synthesis.Step is：
The C-terminal of (1) first amino acid is grafted on 2- chlorine trityl chloride resins, and rate of carrying is 0.6mmol/g.20%
Piperidines be dissolved in N, N '-dimethyl formamide (DMF) removes the Fmoc blocking groups of amino；
(2) in condensing agent O- benzotriazole-tetramethylurea hexafluorophosphate (HBTU) and catalyst n, N- diisopropyls
Under the collective effect of ethamine (DIPEA), the carboxyl of next amino acid is combined with free amino；Repeat above step, polypeptide
Chain can constantly increase；
(3) it rinses the resin (5min, 5 times) for being connected with polypeptide repeatedly with the DMF of certain volume, is rinsed again with DCM later
(1min, 3 times)；Dry resin is handled with trifluoroacetic acid (TFA), and polypeptide is detached from from resin, is transferred into TFA solution；
(4) it collects solution to be spin-dried for, obtains concentrate solution；With frost ether processing, centrifugation is precipitated；
(5) precipitation is dissolved in dimethyl sulfoxide (DMSO) (DMSO), polypeptide { L-Met } { L- is obtained using high-efficient liquid phase chromatogram purification
His}{L-Ser}{L-Pro}G{L-Ala}{L-Asn}。
Embodiment 2
The synthesis in solid state of { D-Met } { L-His } { L-Ser } { L-Pro } G { D-Ala } { D-Asn } polypeptide：
Polypeptide is by ripe synthesis in solid state (SPPS) step, using 2- chlorine trityl chloride resin and side chain by tert-
Buty radical protections and amino is by fmoc-protected Amino acid synthesis.Step is：
The C-terminal of (1) first amino acid is grafted on 2- chlorine trityl chloride resins, and rate of carrying is 0.6mmol/g.20%
Piperidines be dissolved in N, N '-dimethyl formamide (DMF) is used to remove the Fmoc blocking groups of amino；
(2) in condensing agent O- benzotriazole-tetramethylurea hexafluorophosphate (HBTU) and catalyst n, N- diisopropyls
Under the collective effect of ethamine (DIPEA), the carboxyl of next amino acid is combined with free amino；Repeat above step, polypeptide
Chain can constantly increase；
(3) it rinses the resin (5min, 5 times) for being connected with polypeptide repeatedly with the DMF of certain volume, is rinsed again with DCM later
(1min, 3 times)；Dry resin is handled with trifluoroacetic acid (TFA), and polypeptide is detached from from resin, is transferred into TFA solution；
(4) it collects solution to be spin-dried for, obtains concentrate solution；With frost ether processing, centrifugation is precipitated；
(5) precipitation is dissolved in dimethyl sulfoxide (DMSO) (DMSO), polypeptide { D-Met } { L- is obtained using high-efficient liquid phase chromatogram purification
His}{L-Ser}{L-Pro}G{D-Ala}{D-Asn}。
Embodiment 3
The synthesis in solid state of { D-Met } { D-His } { D-Ser } { D-Pro } G { D-Ala } { D-Asn } polypeptide：
Polypeptide is by ripe synthesis in solid state (SPPS) step, using 2- chlorine trityl chloride resin and side chain by tert-
Buty radical protections and amino is by fmoc-protected Amino acid synthesis.Step is：
The C-terminal of (1) first amino acid is grafted on 2- chlorine trityl chloride resins, and rate of carrying is 0.6mmol/g.20%
Piperidines be dissolved in N, N '-dimethyl formamide (DMF) is used to remove the Fmoc blocking groups of amino；
(2) in condensing agent O- benzotriazole-tetramethylurea hexafluorophosphate (HBTU) and catalyst n, N- diisopropyls
Under the collective effect of ethamine (DIPEA), the carboxyl of next amino acid is combined with free amino；Repeat above step, polypeptide
Chain can constantly increase；
(3) it rinses the resin (5min, 5 times) for being connected with polypeptide repeatedly with the DMF of certain volume, is rinsed again with DCM later
(1min, 3 times)；Dry resin is handled with trifluoroacetic acid (TFA), and polypeptide is detached from from resin, is transferred into TFA solution；
(4) it collects solution to be spin-dried for, obtains concentrate solution；With frost ether processing, centrifugation is precipitated；
(5) precipitation is dissolved in dimethyl sulfoxide (DMSO) (DMSO), polypeptide { D-Met } { D- is obtained using high-efficient liquid phase chromatogram purification
His}{D-Ser}{D-Pro}G{D-Ala}{D-Asn}。
Embodiment 4
The synthesis in solid state of Azide polypeptide { L-Met } { L-His } { L-Ser } { L-Pro } G { L-Ala } { L-Asn }-N3：
Polypeptide is by ripe synthesis in solid state (SPPS) step, using 2- chlorine trityl chloride resin and side chain by tert-
Buty radical protections and amino is by fmoc-protected Amino acid synthesis.Step is：
1. the C-terminal of first amino acid is grafted on 2- chlorine trityl chloride resins, rate of carrying is 0.6mmol/g.20%
Piperidines be dissolved in N, N '-dimethyl formamide (DMF) is used to remove the Fmoc blocking groups of amino；
2. under the collective effect of condensing agent HBTU and catalyst DIPEA, the carboxyl of next amino acid and free ammonia
Base junction is closed；Above step is repeated, polypeptide chain can constantly increase；
3. the synthesis of Azide caproic acid：6- bromocaproic acids ethyl ester is reacted with sodium azide in DMF, later at sodium hydroxide
Reason, obtains the caproic acid of Azide；
4. finally the Azide caproic acid of previous step is connected to same step on the N- terminal amino groups of polypeptide；
5. rinsing the resin (5min, 5 times) for being connected with polypeptide repeatedly with the DMF of certain volume, rinsed again with DCM later
(1min, 3 times)；
6. handling dry resin with TFA, polypeptide is detached from from resin, is transferred into TFA solution；
7. collecting solution to be spin-dried for, concentrate solution is obtained；With frost ether processing, centrifugation is precipitated；
8. precipitation is dissolved in DMSO, polypeptide product { L-Met } { L- with nitrine is obtained using high-efficient liquid phase chromatogram purification
His}{L-Ser}{L-Pro}G{L-Ala}{L-Asn}-N3。
Embodiment 5
The synthesis in solid state of phosphorylated polypeptide MH { pSer } PGAD：
Polypeptide is by ripe synthesis in solid state (SPPS) step, using 2- chlorine trityl chloride resin and side chain by tert-
Buty radical protections and amino is by fmoc-protected Amino acid synthesis.Step is：
The C-terminal of (1) first amino acids methionine is grafted on 2- chlorine trityl chloride resins, and the rate of carrying is
0.6mmol/g.20% piperidines is dissolved in N, and N '-dimethyl formamide (DMF) is used to remove the Fmoc blocking groups of amino；
(2) in condensing agent O- benzotriazole-tetramethylurea hexafluorophosphate (HBTU) and catalyst n, N- diisopropyls
Under the collective effect of ethamine (DIPEA), the carboxyl of next amino acid is combined with free amino；Repeat above step, polypeptide
Chain can constantly increase；
(3) after Peptide systhesis, the side chain protecting group of serine is selectively sloughed, serine uses Fmoc-Ser (trt),
What can be quantified under the conditions of 1%TFA/DCM sloughs.Phosphorylation afterwards, using double benzyl phosphoramidites, tetrazole generates phosphorous acyl
Amine tetrazolium reactive intermediate, is connected on hydroxyl, and then oxidation generates phosphoryl under peroxy acid, completes the phosphoric acid of serine
Change；
(4) it rinses the resin (5min, 5 times) for being connected with polypeptide repeatedly with the DMF of certain volume, is rinsed again with DCM later
(1min, 3 times)；Dry resin is handled with trifluoroacetic acid (TFA), and polypeptide is detached from from resin, is transferred into TFA solution；
(5) it collects solution to be spin-dried for, obtains concentrate solution；With frost ether processing, centrifugation is precipitated；
(6) precipitation is dissolved in dimethyl sulfoxide (DMSO) (DMSO), MH { pSer } PGAD is obtained using high-efficient liquid phase chromatogram purification.
Embodiment 6
The preparation of the chitosan gel rubber of covalent bond MHSPGAD polypeptides is reacted by click chemistry：
The synthesis of the chitosan of alkynyl substituted
1. dissolving the chitosan in distilled water, pentinoic acid is added thereto, utilizes 1M hydrochloric acid (HCL) and 1M sodium hydroxides
(NaOH) solution, which adjusts pH value, makes chitosan be completely dissolved；
2. by 1- ethyls-(3- dimethylaminopropyls) carbodiimide hydrochloride (EDC, according to EDC:Pentinoic acid=3:1
Ratio) it is added in CS solution and (adds 1 time, totally 3 times per half an hour), during which ice bath；
3. reacting at room temperature for 24 hours；
4. a pair product dialysed (5mM HCl and 1wt%NaCl, 2 days；3mM HCl, 1 day；1mM HCl, 2 days；Distillation
Water, 3 days, 4 DEG C)；
5. freeze-drying obtains final product.
The synthesis of CS-MHSPGAD
Using click chemistry (" click " reacts) synthesis CS-MHSPGAD, steps are as follows：
1. under the conditions of nitrogen protection, alkynyl chitosan is dissolved with distilled water (10mL).
2. click-reaction condition：MHSPGAD-N3:CuSO4:The raw materials components mole ratio of sodium ascorbate is 1:0.2:0.4.
3. under the conditions of 37 DEG C, reaction is for 24 hours；
4. being dialysed 3 days using distilled water；
5. obtaining CS-MHSPGAD materials by freeze-drying；
CS-MHSPGAD is by MHSPGAD-N3Nitrine and alkynyl chitosan alkynes carry out click-reaction and
Synthesis.First, the sequence (MHSPGAD) of heptapeptide is synthesized using solid phase Fmoc- chemistry of amino acids methods.Then, anti-using condensation
The ends N- that 6- azido caproic acids should be connected to heptapeptide obtain MHSPGAD-N3.Then, pass through the condensation reaction of CS and 4- pentinoic acids
Synthesize alkynyl chitosan.Finally, CS-MHSPGAD compounds are synthesized using click-reaction.CS-MHSPGAD compounds are with quality
Volume fraction is dissolved in formation polypeptide-chitosan gel rubber in distilled water for 20%.
Embodiment 7
The preparation of the hyaluronic acid derivatives of covalent bond MHSPGAD polypeptides is reacted by click chemistry：
The synthesis of the hyaluronic acid of ynamine substitution
1. hyaluronic acid (HA) is dissolved in distilled water, propargylamine is added thereto；
2. by 1- ethyls-(3- dimethylaminopropyls) carbodiimide hydrochloride (EDC, according to EDC:Pentinoic acid=3:1
Ratio) it is added in hyaluronic acid solution and (adds 1 time, totally 3 times per half an hour), during which ice bath；
3. reacting at room temperature for 24 hours；
4. a pair product is dialysed (distilled water, 3 days)；
5. freeze-drying obtains the hyaluronic acid of final product ynamine.
The synthesis of HA-MHSPGAD
Using click chemistry (" click " reacts) synthesis HA-MHSPGAD, steps are as follows：
1. under the conditions of nitrogen protection, ynamine hyaluronic acid is dissolved with distilled water (10mL).
2. click-reaction condition：MHSPGAD-N3:CuSO4:The raw materials components mole ratio of sodium ascorbate is 1:0.2:0.4.
3. under the conditions of 37 DEG C, reaction is for 24 hours；
4. being dialysed 3 days using distilled water；
5. obtaining HA-MHSPGAD materials by freeze-drying；
HA-MHSPGAD is to carry out click-reaction by the alkynes of nitrine and ynamine hyaluronic acid to MHSPGAD-N3
And synthesize.First, the sequence (MHSPGAD) of heptapeptide is synthesized using solid phase Fmoc- chemistry of amino acids methods.Then, condensation is utilized
The ends N- that 6- azido caproic acids are connected to heptapeptide by reaction obtain MHSPGAD-N3.Then, pass through the contracting of hyaluronic acid and propargylamine
Close reaction synthesis ynamine hyaluronic acid.Finally, HA-MHSPGAD products are synthesized using click-reaction.HA-MHSPGAD products with
Quality volume fraction, which is dissolved in for 10% in distilled water, prepares polypeptide-hyaluronic acid derivatives.
Embodiment 8
Preparation in conjunction with the aquagel of MHSPGAD and performance evaluation：
In conjunction with the preparation of the aquagel of MHSPGAD
(1) acetic acid (0.1M) dissolving chitosan (1.5%, w/v), room temperature is used to stay overnight；
(2) distilled water is used to dialyse to CS solution in dialysis membrane (molecular cut off is 8kDa -10kDa), 1 week,
Remaining acetic acid is removed, CS- hydrochlorides are obtained；
(3) MHSPGAD polypeptides are added in distillation water dissolution CS- hydrochlorides (20mg/mL), are uniformly mixed, obtain polypeptide/shell
Glycan (10ng/mL) solution；
(4) sodium β-glycerophosphate (β-GP) solution is added drop-wise in chitosan solution, stirs 0.5h under condition of ice bath, obtains
Mixed solution；
(5) the final concentration of 0.023mol/L of β-GP in mixed solution are 7.2 or so through dialysis solution pH value；
(5) mixed solution is transferred in 37 DEG C of insulating boxs, and 5min can form the aquagel in conjunction with MHSPGAD.
Scanning electron microscope observes hydrogel pattern
After hydrogel is lyophilized, be put into drying machine it is dry for 24 hours, using the micro- sem observation CS of Field Emission Scanning Electron and
In conjunction with configuration of surface and porous structure (accelerating potential 10kV, the material surface spray before observing of the aquagel of MHSPGAD
Gold processing).
Hydrogel rheological properties are tested
(1) rheometer test is completed using AR 2000ex (TAinstrument) rheometer, is justified using 25mm stainless steel flat plates
Plate clamp, 400 μm of fixture gap, temperature is set as 25 DEG C.Test needs 1ml solution to each sample every time.
(2) by CS solution (containing 0.023M β-GP, pH value 7.2) and CS-MHSPGAD solution (containing 0.023M β-GP,
PH value is 7.12) to be added in rheometer, and the rheological properties of sample are measured within the temperature range of 10 DEG C -50 DEG C.
(3) it tests and records hydrogel elastic modulus (storage modulus) (G ') and viscous modulus (loss modulus) (G ") with temperature
Spend the change situation of variation, frequency 1rad/s.
(4) using delayed phase (δ) come gelling temperature when determining that G ' and G " is equal.Constant heating rate (1 DEG C/
Min change temperature in the case of).
The morphological feature of freeze-drying hydrogel is observed using SEM, it is found that two kinds of hydrogels all have the porous web of homogeneous intercommunication
Shape structure, pore size are 50-100 μm.Compare the rheological property that two kinds of biomaterials vary with temperature.As temperature is from 10
50 DEG C DEG C are increased to, CS/MHSPGAD/ β-GP and CS/ β-GP all show dramatically increasing for storage modulus (G '), prompt from solution
Phase transition process of the state to gel state.The gelling temperature of CS/MHSPGAD/ β-GP is 35 DEG C -36 DEG C, with CS/ β-GP phases
Seemingly, it was demonstrated that CS/MHSPGAD/ β-GP are also suitable under the conditions of normal body temperature, in vivo gel in-situ.
Embodiment 9
In conjunction with the preparation of the hyaluronic acid gel of MHSPGAD：
(1) using distillation water dissolution hyaluronic acid (1.5%, w/v), room temperature；It is 5.5 or so to adjust pH value, is added thereto
Enter EDC/NHS, activated carboxyl reacts 1-2h；
(2) add a certain amount of mercaptoethylmaine, room temperature, reaction overnight into the hyaluronic acid solution after activation；
(3) distilled water is dialysed 3 days, and the hyaluronic acid after sulfydryl modification is obtained by freeze-drying；
(4) sulfydryl modification hyaluronic acid after purification is dissolved in distilled water (1.5%, w/v), it is living that biology is added thereto
Property polypeptide, prepare biologically active polypeptide/hyaluronic acid solution of 20ng/mL, sulfhydryl oxidase, which is formed, combines biologically active polypeptide/thoroughly
Bright matter acid hydrogel.
Embodiment 10
In conjunction with the preparation of the collagen hydrogel of MHSPGAD：
(1) 1mg type i collagens are added in 0.13mL 0.1M acetic acid solutions and are made into 7.69mg/ml concentration collagen solutions；
(2) 0.159mL PBS are added in above-mentioned 0.13mL collagen solutions, and 100uL 0.1M sodium hydroxides are added and neutralize；
(3) polypeptide MHSPGAD is added into collagen solution, prepares polypeptide/collagen solution (MHSPGAD/ collagens, 10ng/
mL)；
(4) polypeptide/collagen solution is incubated 30min under conditions of pH neutrality, under the conditions of 37 DEG C and can be formed in conjunction with biological living
The collagen hydrogel of property polypeptide.
Embodiment 11
In conjunction with the preparation of the Pluronic F127 hydrogels of MHSPGAD：
(1) the Pluronic F127 powder for weighing 0.1g is dissolved in the PBS buffer solution of precooling, in magnetic force under the conditions of 4 DEG C
The Pluronic F127 solution that the quality volume fraction being configured to is 10% is stirred overnight on blender.
(2) polypeptide is added into Pluronic F127 solution, continues to stir at 4 DEG C, is configured to MHSPGAD/Pluronic
F127 (10ng/mL) solution.
(3) MHSPGAD/Pluronic F127 solution is incubated 5min or so under the conditions of 37 DEG C, you can is formed and is combined
The Pluronic F127 hydrogels of MHSPGAD.
Embodiment 12
In conjunction with the preparation of the porous collagen sponge of MHSPGAD：
(1) type i collagen is added in 0.1M acetic acid solutions to the collagen solution for being made into 10mg/mL；
(2) sodium hydroxide is added into the collagen solution to be neutralized, obtains neutral collagen solution；
(3) biologically active polypeptide MHSPGAD is added into the neutral collagen solution, is configured to the biology containing 20ng/mL
Active peptides/collagen solution；
(4) polypeptide/collagen solution is injected in mold, it is 0.1-10mm to make thickness, and -4 DEG C make the collagen in conjunction with polypeptide pave,
Being placed in -80 DEG C again makes polypeptide collagen solution solidify；
(5) collagen polypeptide of solidification is placed in vacuum freeze-drying machine and is freeze-dried, lyophilisation condition, which is -20 DEG C, to be continued
60min, -40 DEG C of lasting 240min, 0 DEG C of lasting 19h, 20 DEG C of lasting 3h, vacuum degree are maintained at 0-180pa, and sub-cut is at various rule
Lattice are sub-packed in packing bag for medical use, the collagen sponge in conjunction with polypeptide are obtained after gamma-ray irradiation sterilizing；
Embodiment 13
In conjunction with the preparation of the porous chitosan sponge of MHSPGAD：
(1) acetic acid (0.1M) dissolving chitosan (Mn=20,000,5%, w/v), room temperature is used to stay overnight；
(2) distilled water is used to dialyse to CS solution in dialysis membrane (molecular cut off is 8kDa -10kDa), 1 week,
Remaining acetic acid is removed, freeze-drying obtains chitosan hydrochloride；
(3) distillation water dissolution chitosan hydrochloride (20mg/mL), be added MHSPGAD polypeptides, be uniformly mixed, obtain polypeptide/
Chitosan (50ng/mL) solution；
(4) polypeptide/chitosan solution is injected in mold, it is 0.1-10mm to make thickness, and -4 DEG C make the collagen in conjunction with polypeptide spread
It is flat, then being placed in -80 DEG C makes polypeptide/chitosan solution solidify；
(5) polypeptide/chitosan of solidification is placed in vacuum freeze-drying machine and is freeze-dried, lyophilisation condition, which is -20 DEG C, to be continued
60min, -40 DEG C of lasting 180min, 0 DEG C of lasting 19h, 20 DEG C of lasting 3h, vacuum degree are maintained at 0-180pa, and sub-cut is at various rule
Lattice are sub-packed in packing bag for medical use, the chitosan sponge in conjunction with polypeptide are obtained after gamma-ray irradiation sterilizing；
Embodiment 14
The migration of the external evoked cells of polypeptide MHSPGAD：
Migration experiment uses 24-transwell cell culture cell (the PET filter membranes containing 8 μm of apertures, Millipore
Corporation,Billerica,MA).By cell Nature enemy 12h, with 105μ L RPMI1640 (10% tires of a cell/100
Cow's serum) density by cell seeding in the small interiors transwell.The blood serum medium that lower layer's orifice plate is not added to peptide factor is made
For control group.After co-culturing 4-6h, the cell that transwell films upper surface does not migrate is wiped, transwell films lower surface
Cell fix 5min with 4%PFA, then with 0.5% violet staining 30min.Pass through ordinary optical microscope (Nikon
EclipseTE2000-U Kanagawa, Japan) count migrating cell number.
Specific stem cell migration experimental procedure：
(1) film pre-processes
By 48.5mLH2+ 150 μ L collagens of O+1.5mL acetic acid are placed in the centrifuge tube of 50mL.It is made of pencil in the rough surface of film
Label is put into above-mentioned solution 4 and spends night.
It is taken the film out before experiment, is put into the culture dish equipped with PBS and rinses one time, then washed one time with starvation media, inhaled
New starvation media is added after removing culture medium, is put into 37 degree of incubators.
(2) cell is handled
Mesenchymal stem cell, 1 positive cells of Sca, CD34 positive cells, NG positive cells, CD 44 is positive thin
Born of the same parents distinguish Nature enemy 12h, vitellophag, degree of thickening 1 × 105A/mL blood serum mediums, cell migration apparatus lower layer are added
166 μ L 10ng/mL contain the blood serum medium of polypeptide factor, at the same at transwell room to be not added with the blank of polypeptide factor
Blood serum medium does control group.Film (asperities is downward) carefully is completed, installs plastic mattress, fixed higher device.Upper layer is separately added into
100 μ L each group stem cell suspensions migrate 4-6h.
(3) fixed
Carefully film is taken off, hangs the cell of shiny surface, the asperities court on new filter paper on the filter paper got wet with PBS
Upper placement, thoroughly dries.Then asperities fixes 5min upward in methanol again, thoroughly dries.
(4) it dyes
It is thick to be placed face down on 30min in crystal violet dye liquor, it takes out film and is rinsed one time with distilled water, asperities is placed on load glass upward
On piece counts cell.
Migrating cell is counted by ordinary optical microscope (Nikon Eclipse TE2000-U Kanagawa, Japan)
Number, compare blank serum culture medium, and for experimental group under the action of polypeptide factor, the mobility of mesenchymal stem cell is empty
3.2 times of white control group, the mobility of 1 positive cells of Sca is 4.3 times of blank serum control group, the migration of CD34 positive cells
Rate is 3 times of blank serum control group, and the mobility of NG positive cells is 4.6 times of blank serum control group, and CD 44 is positive thin
The mobility of born of the same parents is 4 times of blank serum control group.Experimental result such as Fig. 1 shows, can under the chemotaxis of polypeptide factor
It induces some ancestral cells to the directional migration at damage or ischemic tissue, accelerates the reparation of ischemic or injury tissue.
Embodiment 15
In conjunction with reparation of the aquagel after myocardial infarction ischemic of MHSPGAD
Coronary artery left anterior descending branch permanently ligatures foundation and polypeptide hydrogel transplanting：
(1) mouse anesthesia：With the pre- numb mouse of 5% isoflurane gas, four limbs and the head of mouse are fixed with medical adhesive tape
In on surgical plate, cutting off pars cervicalis tracheae, anesthesia respirator positive airway pressure is connected after trachea cannula, the content for adjusting isoflurane is about
1%-1.5%, respiratory rate 120/min；
(2) chest preserved skin is carried out disinfection with medicinal alcohol and Iodophor；
(3) row thoracotomy opens chest and exposure heart at the 4th intercostal, carefully tears the pericardium exposure left heart of mouse
Room antetheca；
(4) lower section of arteria coroaria sinistra initiating terminal is at left auricle of heart about 1mm, with the suture following coronary artery occlusion of 7-0
Left anterior descending branch (is whitened or is occurred atropurpureus and electrocardiogram with a lighter for ligation point lower section cardiac muscle
Myocardium ST sections of the electricity of (electrocardiogram, ECG) detection display is raised to ligature successfully mark；
(5) 10 μ l physiological saline, chitosan water are injected after ligaturing successfully respectively at Ligation lower-left, each 1mm in bottom right
Gel or the aquagel for combining MHSPGAD；
(6) chest is closed, muscle is sutured and skin, wound is sterilized again with Iodophor, properly raise.
(7) sham-operation (sham) control group：Chest is opened, threading does not connect bundle and do not transplant any solution, and the rear chest that closes sutures muscle.
Myocardial Regeneration situation is evaluated using means such as immunostaining, cardiac ultrasonics.It is commented using echocardiogram within postoperative 28 days
Estimate cardiac function, compared with sham-operation group, all infarct mouse show the reduction of left ventricle anterior wall thickness and cardiac function.Its
Aquagel treatment group and aquagel (LVAWd of the middle injection in conjunction with MHSPGAD：0.88±0.04mm/LVAWs:
0.38 ± 0.03mm) (LVAWd is compared with physiological saline group：0.68±0.09mm/LVAWs:0.238 ± 0.03mm), significant increasing
Add left ventricle antetheca end-diastolicthickness (LVAWd：1.22 ± 0.08mm) and LV antetheca end-systole thickness (LVAWs：0.72±
0.02mm), with sham-operation group close to (LVAWd：1.44±0.06mm/LVAWs:1.00±0.03mm).In conjunction with the shell of MHSPGAD
Glycan Hydrogel In Treating group also reduces left ventricular end diastolic diameter (LVEDd：6.7 ± 0.6mm) and LV myocardial contraction latter stages it is straight
Diameter (LVEDs：4.6 ± 0.2mm), with sham-operation group close to (LVEDd：5.9±0.6mm/LVEDs:3.8±0.03mm).According to
Ejection fraction (EF) and the definition for shortening score (FS), aquagel treatment group significant improvement of the injection in conjunction with MHSPGAD
LV contractile functions.
Using combine MHSPGAD aquagel group be conducive to raise stem cell, enhance its promote cardiomyocyte proliferation,
Anti-apoptotic and stimulation angiogenesis ability, improve myocardial hypertrophy and promote the recovery of cardiac function and structure, and reduce cardiac muscle
The generation of tissue fibrosis.
Embodiment 16
In conjunction with reparation of the hyaluronic acid gel after myocardial infarction ischemic of MHSPGAD
Coronary artery left anterior descending branch permanently ligatures foundation and polypeptide hydrogel transplanting：
(1) mouse anesthesia：With the pre- numb mouse of 5% isoflurane gas, four limbs and the head of mouse are fixed with medical adhesive tape
In on surgical plate, cutting off pars cervicalis tracheae, anesthesia respirator positive airway pressure is connected after trachea cannula, the content for adjusting isoflurane is about
1%-1.5%, respiratory rate 120/min；
(2) chest preserved skin is carried out disinfection with medicinal alcohol and Iodophor；
(3) row thoracotomy opens chest and exposure heart at the 4th intercostal, carefully tears the pericardium exposure left heart of mouse
Room antetheca；
(4) lower section of arteria coroaria sinistra initiating terminal is at left auricle of heart about 1mm, with the suture following coronary artery occlusion of 7-0
Left anterior descending branch (is whitened or is occurred atropurpureus and electrocardiogram with a lighter for ligation point lower section cardiac muscle
Myocardium ST sections of the electricity of (electrocardiogram, ECG) detection display is raised to ligature successfully mark；
(5) 10 μ l physiological saline, hyaluronic acid are injected after ligaturing successfully respectively at Ligation lower-left, each 1mm in bottom right
Hydrogel or the MHSPGAD/ hyaluronic acid gels for combining 20ng/mL；
(6) chest is closed, muscle is sutured and skin, wound is sterilized again with Iodophor, properly raise.
(7) sham-operation (sham) control group：Chest is opened, threading does not connect bundle and do not transplant any solution, and the rear chest that closes sutures muscle.
Myocardial Regeneration situation is evaluated using means such as immunostaining, cardiac ultrasonics.After treatment 30 days, separation each group animal
Heart does 7 μm of paraffin sections.Histotomy carries out histologic analysis after HE and sirius red dyeing.With echocardiogram knot
Fruit is consistent, and the left ventricle antetheca of physiological saline group and hyaluronic acid gel group is significantly thinning, and in conjunction with the saturating of MHSPGAD
The anterior wall thickness of bright matter acid hydrogel group dramatically increases.By sirius red coloration result as it can be seen that sham-operation group animal cardiac muscle only
It can be seen that fragmentary collagen distribution, and there is a large amount of collagen deposition in the infarcted region of each group myocardial infarction animal.Statistical result showed, respectively
The infarction size and infarcted region collagen content of group myocardial infarction animal are without significant difference.Compared with sham-operation group, physiological saline
Collagen I/III ratios of group and simple hyaluronic acid gel group significantly increase about 10 times and 8 times.But in conjunction with
Collagen I/III odds ratio physiological saline groups of the hyaluronic acid gel group injection group of MHSPGAD reduce>75%.Experiment knot
Fruit display uses the hyaluronic acid gel in conjunction with MHSPGAD to be conducive to raise stem cell, enhances it and promotes cardiomyocyte proliferation, resists
Apoptosis and stimulation angiogenesis ability, improve myocardial hypertrophy and promote the recovery of cardiac function and structure, and reduce myocardium group
The generation of textured fiber.
Embodiment 17
In conjunction with MHSPGAD Pluronic F127 hydrogels lower limb ischemia reparation in application
Lower limb femoral artery ligation detachment establishes lower limb ischemia model and polypeptide hydrogel transplanting
(1) mouse anesthesia：With the pre- numb mouse of 5% isoflurane gas, mouse is fixed on surgical plate with medical adhesive tape.It is real
Test three groups of animal point, respectively PBS groups, aquagel group and the aquagel group for combining MHSPGAD.
(2) by the way that mouse lower limb femoral artery ligation detachment is manufactured lower limb ischemia model.By certain density solution (PBS
Group, Pluronic F127 hydrogels groups and combination 10ng/mL polypeptide MHSPGAD/PluronicF127 hydrogels group) it is ligaturing
Locate Surrounding muscles multi-point injection, total amount is 50 microlitres/.
It is postoperative that angiogenesis situation is observed by angiography.Pass through Doppler scanning within the 7th day and the 14th day after surgery
Instrument measures pedal blood perfusion, and the Pluronic F127 hydrogels group ischemic seriousness and degree of necrosis in conjunction with MHSPGAD are notable
It is lighter than other two groups, this polypeptide that may be benefited from the Pluronic F127 hydrogels in conjunction with MHSPGAD can promote ischemic
New Sca1 in tissue+The formation of cell ecological position.
It drew materials respectively at 7,14 days, by the Doppler scanning imaging such as further analyses of Fig. 2-1 by not treating and merely
The restoration of blood flow situation of Pluronic F127 Hydrogel In Treatings and polypeptide therapeutic rear left lower limb ischemia.Quantitative result such as Fig. 2-2
Left ischemic limb is shown compared with the blood flow of right healthy lower limb, between each group after treatment 14 days, in conjunction with MHSPGAD's
Pluronic F127 hydrogel group hemoperfusion situations, increase about 46%, with Pluronic F127 water-settings compared with PBS groups
Glue group shows that the Pluronic F127 hydrogel groups processing in conjunction with MHSPGAD being capable of effective stimulus blood compared to increasing about 35%
Pipe generates and the recovery of hind limb perfusion.Therefore, MHSPGAD polypeptides are the principal elements of hind leg angiogenesis and recovery, it was demonstrated that more
Peptide has effectively facilitated angiogenesis, and vessel density is promoted to significantly improve.
Embodiment 18
In conjunction with MHSPGAD aquagel lower limb ischemia reparation in application
Lower limb femoral artery ligation detachment establishes lower limb ischemia model and polypeptide hydrogel transplanting
(1) mouse anesthesia：With the pre- numb mouse of 5% isoflurane gas, mouse is fixed on surgical plate with medical adhesive tape.It is real
Test three groups of animal point, respectively PBS groups, chitosan alone hydrogel group and the aquagel group for combining MHSPGAD.
(2) by the way that mouse lower limb femoral artery ligation detachment is manufactured lower limb ischemia model.By certain density solution (PBS
Group, aquagel group and polypeptide/aquagel group) the Surrounding muscles multi-point injection at ligation, total amount be 50 microlitres/
Only.
Postoperative to observe angiogenesis situation by angiography, the aquagel group ischemic in conjunction with MHSPGAD is serious
Property and degree of necrosis are significantly lighter than other two groups.Thin vessels in immuning fluorescent dyeing analysis musculature and capillary
Distribution, quantitative result is shown, in conjunction with the significant increase (p of MHSPGAD/ aquagel group capillary densities of 10ng/mL<
0.05), aquagel treatment is shown in terms of reducing ischemic severity and increasing capillary density (about 16%)
To stimulating the moderate effect of angiogenesis, but result is not notable compared with the aquagel group for combining MHSPGAD, shows to tie
The recovery of effective stimulus angiogenesis and hind limb perfusion is capable of in the aquagel group processing for closing MHSPGAD.Prove that polypeptide has
Effect promotes angiogenesis, and vessel density significantly improves.
Embodiment 19
The foundation of skin corium full thickness skin damage model and the collagen hydrogel transplanting for combining MHSPGAD：
(1) 4% chloraldurate of injection anaesthetizes sb. generally in nude mice abdominal cavity.
(2) after anaesthetic effect satisfaction, 75% alcohol disinfecting nude mice skin of back 3 times.
(3) holostrome nude mice skin and sarcolemma are wiped out with eye scissors, exposure muscle layer, l.0cm it is to prepare area2Skin
Defect area.
(4) the sterile waterproof wound dressing flap coverages of IV3000, pad pasting edge invest nude mice with Shang mouthfuls of Nian He Ji Dot and carry on the back
Portion's skin.
(5) postoperative nude mice single cage raising, Wound dressing is replaced from 3d every 2d.
(6) 200ul physiological saline (not treatment group), collagen hydrogel, knot is added dropwise in every nude mice wound of each experimental group
Close the polypeptide MHSPGAD/ collagen hydrogels of 10ng/mL.
(7) postoperative substantially observation and healing curve measure, and observe and record the state of mind, the feed drink of each group nude mice daily
Regimen condition, energy, stool and urine situation.
(8) the postoperative row living imaging first after anesthesia in 2 days detects, and then removes wound dressing, carefully removes the surface of a wound
Exudate, crust, whether there is or not infection, wound healing situations for the observation surface of a wound, and carry out the surface of a wound and take pictures while retouching print wound with transparent membrane
Face.
(9) after taking pictures, the surface of a wound uses the sterile waterproof wound dressing pad pasting coverings of IV3000, pad pasting edge viscous with wound again
He Ji Dot invest nude mice skin of back.It is postoperative 3rd using Image J softwares analysis each group, 6,9, l2d remain surface of a wound area, and
Draw healing curve.
In addition, evaluating skin tissue regeneration situation using immunostaining.
Mouse skin defect model is established, the collagen hydrogel local injection in conjunction with MHSPGAD to defect of skin portion is used
Position.Further determine that wound closure, experimental result display combine based on the measurement of wound area in 14 days time courses
The healing rate of the collagen hydrogel group of MHSPGAD at every point of time is all faster than other two groups.At the 7th day, with other group of phase
Than showing the function of accelerating wound healing in conjunction with the collagen hydrogel treatment group of MHSPGAD, skin corium is complete within the 14th day after surgery
Layer skin injury is almost healed, make rate 95.31%, hence it is evident that is higher than collagen hydrogel group and physiological saline group.Due to blood vessel
Oxygen and nutriment can be taken to wound to, and then angiogenesis is most important in wound healing and repair process.Pass through
CD31 immunofluorescence dyeings assess the angiogenesis of wound location after various treatments, compared with control group physiological saline group, in conjunction with
The collagen hydrogel group of MHSPGAD showed higher microvessel density at the 7th day, and nascent blood vessel density is up to 56%.With control group
Physiological saline is compared with collagen hydrogel group, visible in the granulation tissue in conjunction with the collagen hydrogel treatment group new life of MHSPGAD
More new capillary vessels, this is directly related with healing improvement.It is all these statistics indicate that, in conjunction with the collagen water of MHSPGAD
Polypeptide in gel promotes the reparation of local skin tissue, accelerates angiogenesis, accelerate skin corium full thickness skin injury repair into
Journey.
Embodiment 20
The foundation of skin corium full thickness skin damage model and the aquagel transplanting for combining MHSPGAD：
(1) 4% chloraldurate of injection anaesthetizes sb. generally in nude mice abdominal cavity.
(2) after anaesthetic effect satisfaction, 75% alcohol disinfecting nude mice skin of back 3 times.
(3) holostrome nude mice skin and sarcolemma are wiped out with eye scissors, exposure muscle layer, l.0cm it is to prepare area2Skin
Defect area.
(4) the sterile waterproof wound dressing flap coverages of IV3000, pad pasting edge invest nude mice with Shang mouthfuls of Nian He Ji Dot and carry on the back
Portion's skin.
(5) postoperative nude mice single cage raising, Wound dressing is replaced from 3d every 2d.
(6) every nude mice wound of each experimental group be added dropwise 200ul physiological saline (not treatment group), aquagel,
In conjunction with the MHSPGAD/ aquagels of 10ng/mL.
(7) postoperative substantially observation and healing curve measure, and observe and record the state of mind, the feed drink of each group nude mice daily
Regimen condition, energy, stool and urine situation.
(8) the postoperative row living imaging first after anesthesia in 2 days detects, and then removes wound dressing, carefully removes the surface of a wound
Exudate, crust, whether there is or not infection, wound healing situations for the observation surface of a wound, and carry out the surface of a wound and take pictures while retouching print wound with transparent membrane
Face.
(9) after taking pictures, the surface of a wound uses the sterile waterproof wound dressing pad pasting coverings of IV3000, pad pasting edge viscous with wound again
He Ji Dot invest nude mice skin of back.It is postoperative 3rd using Image J softwares analysis each group, 6,9, l2d remain surface of a wound area, and
Draw healing curve.
In addition, evaluating skin tissue regeneration situation using histological stain.
Mouse skin defect model is established, the aquagel local injection in conjunction with MHSPGAD to defect of skin is used
Position.The healing of skin corium full-thickness cutaneous wound is one and is related to keratinocyte, and fibroblast, endothelial cell and macrophage are thin
The many cells of born of the same parents and the process of interaction, migration, proliferation and differentiation lead to new organize the formation of and final wound closure.For
The regeneration situation in wound is given farther insight into, carries out HE dyeing to disclose skin in wound
The metamorphosis of layer.At the 7th day, obviously added with the wound epithelium regeneration epithelium for combining the aquagel of MHSPGAD to handle
Speed.In addition, in conjunction with MHSPGAD aquagel group compared with other groups, wound location tissue is thicker, tissue preferably
Granulation tissue is formed, this is of great significance to tissue repair and regeneration.At the 14th day, in conjunction with the chitosan water-setting of MHSPGAD
Glue group and aquagel group show relatively complete skin regeneration, but examining to the microstructure of regenerating tissues
Significant difference.Wound that best epidermal-dermal engages epithelium again completely is shown in conjunction with the aquagel group of MHSPGAD
Change.Experimental result shows that the reparation for promoting local skin tissue in conjunction with the aquagel of MHSPGAD, quickening form granulation group
It knits and the engagement of best epidermal-dermal.
Embodiment 21
The foundation of skin corium full thickness skin damage model and the collagen sponge transplanting for combining MHSPGAD：
(1) 4% chloraldurate of injection anaesthetizes sb. generally in nude mice abdominal cavity.
(2) after anaesthetic effect satisfaction, 75% alcohol disinfecting nude mice skin of back 3 times.
(3) holostrome nude mice skin and sarcolemma are wiped out with eye scissors, exposure muscle layer, l.0cm it is to prepare area2Skin
Defect area.
(4) the sterile waterproof wound dressing flap coverages of IV3000, pad pasting edge invest nude mice with Shang mouthfuls of Nian He Ji Dot and carry on the back
Portion's skin.
(5) postoperative nude mice single cage raising, collagen sponge dressing is replaced from 3d every 2d.
(6) l.0cm every nude mice wound of each experimental group sticks2The collagen sponge of size, in conjunction with polypeptide
MHSPGAD/ collagen sponges, while blank control is set, Ji Bu treatment groups.
(7) postoperative substantially observation and healing curve measure, and observe and record the state of mind, the feed drink of each group nude mice daily
Regimen condition, energy, stool and urine situation.
(8) the postoperative row living imaging first after anesthesia in 2 days detects, and then removes wound dressing, carefully removes the surface of a wound
Exudate, crust, whether there is or not infection, wound healing situations for the observation surface of a wound, and carry out the surface of a wound and take pictures while retouching print wound with transparent membrane
Face.
(9) after taking pictures, the surface of a wound uses the sterile waterproof wound dressing pad pasting coverings of IV3000, pad pasting edge viscous with wound again
He Ji Dot invest nude mice skin of back.It is postoperative 3rd using Image J softwares analysis each group, 6,9, l2d remain surface of a wound area, and
Draw healing curve.
In addition, evaluating skin tissue regeneration situation using histological stain and immunofluorescence dyeing.Compared with comparative example, knot
The rush ability of cell proliferation for closing the MHSPGAD/ collagen sponges of polypeptide is remarkably reinforced, the addition of polypeptide MHSPGAD so that
MHSPGAD/ collagen sponges have the extracellular microenvironment (extracellular matrix) of high cell affinity and appropriate load ability, are
Proliferation, migration and the metabolism of cell provide indispensable biological place.
Embodiment 22
Application in coating the preparation of the hospital gauze of polypeptide and repairing after surgery：
To the wound of traumatic bleeding frequently in being applied on wound, western hemostatic, then use dressing and gauze bandage to wrap up
Method, to stop blooding, relieve pain and prevent wound infection.The method that this drug is detached with gauze bandage makes when rescuing the wounded,
Formality increases, and anthemorrhagic speed is slow, can not relieve pain in time, increases the pain of the sick and wounded.
Present embodiments provide a kind of novel polypeptide hospital gauze easy to carry of the rapid hemostasis and pain-relieving of energy.Polypeptide is medical
Gauze is mainly used for all kinds of infected wounds, including burn, scald, for after skin and dermatoplasty, Urology Surgery, oral cavity extraction,
The surface of a wound in ulcer and the damaged surface of a wound etc. and various surgeries, gynemetrics, Department of B urn, all kinds of operative incisions of cosmetology and plastic department is protected
Shield has antibiotic anti-infection, and hemostasis and pain-relieving, wound healing, the preparation process for coating the hospital gauze of polypeptide is as follows：
(1) polypeptide MHSPGAD is dissolved in certain mass volume fraction in sterile water, by medical absorbent cotton/gauze or medical
Non-woven cloth is immersed in aforementioned polypeptides aqueous solution, and is dried, and dryness polypeptide hospital gauze is obtained；
(2) hydrogel of MHSPGAD polypeptides will be combined uniformly to be applied on the inside of medical absorbent cotton/gauze or medical non-woven cloth,
It is sealed, obtains moist polypeptide hospital gauze；
(3) hospital gauze for coating polypeptide is obtained into polypeptide hospital gauze after 60 irradiation sterilizations of Co.
To in the selection of hospital gauze, the case where according to wound area, the large mesh polypeptide hospital gauze of meduim yarn, needle are chosen
For the more surface of a wound of those exudates, and the polypeptide hospital gauze of spun yarn fine mesh is used for the less surface of a wound of exudate.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Sequence table
<110>Xu Qingbo
<120>A kind of biologically active polypeptide and its application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 7
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 1
Met His Ser Pro Gly Ala Asp
1 5
Claims (12)
1. a kind of biologically active polypeptide, which is characterized in that the amino acid sequence of the biologically active polypeptide such as SEQ ID NO.1 institutes
Show.
2. biologically active polypeptide according to claim 1, which is characterized in that the amino acid includes D types or L-type amino
Acid.
3. being combined with the hydrogel of biologically active polypeptide described in claims 1 or 2, which is characterized in that the hydrogel includes logical
The biological active component and hydrogel carrier of physical mixed are crossed, the biological active component is biologically active polypeptide, the water-setting
Glue carrier includes aquagel, hyaluronic acid gel, collagen hydrogel or Pluronic F127 hydrogels.
4. being combined with the medical dressing of biologically active polypeptide described in claims 1 or 2, which is characterized in that the combination biology is lived
The medical dressing of property polypeptide includes biologically active polypeptide and timbering material carrier, the timbering material carrier include collagen sponge,
Chitosan sponge.
5. a kind of chemically modified biologically active polypeptide, which is characterized in that the biologically active polypeptide is claims 1 or 2
The biologically active polypeptide.
6. chemically modified biologically active polypeptide according to claim 5, which is characterized in that the chemical modification includes
Phosphorylation modification, Azide modification or carboxylated modification.
7. being combined with the gel of the chemically modified biologically active polypeptide of claim 5 or 6, which is characterized in that described solidifying
Glue includes the biological active component and gel carrier combined by chemical covalent, and the biological active component is chemically modified
Biologically active polypeptide, the gel carrier include chitosan gel rubber or hyaluronic acid derivatives.
8. a kind of be coated with described in biologically active polypeptide or claim 3 described in claims 1 or 2 combines biologically active polypeptide
After chemical modification being combined described in hydrogel or the chemically modified biologically active polypeptide of claim 5 or 6 or claim 7
Biologically active polypeptide gel hospital gauze.
9. combining the hydrogel or power of biologically active polypeptide described in biologically active polypeptide or claim 3 described in claims 1 or 2
Profit requires the medical dressing of the 4 combination biologically active polypeptides or the chemically modified bioactivity of claim 5 or 6 more
Painting overgrowth described in peptide or claim 7 in conjunction with described in the gel of chemically modified biologically active polypeptide or claims 8
Application of the hospital gauze of object active peptides in preparing treatment skin injury drug or dressing.
10. described in biologically active polypeptide or claim 3 described in claims 1 or 2 in conjunction with biologically active polypeptide hydrogel or
In conjunction with the medical dressing of biologically active polypeptide or claim 5 or the 6 chemically modified bioactivity described in claim 4
It preparing promotion angiogenesis in conjunction with the gel of chemically modified biologically active polypeptide described in polypeptide or claim 7, promoting
Application in tissue repair regenerating medicine or medical material.
11. described in biologically active polypeptide or claim 3 described in claims 1 or 2 in conjunction with biologically active polypeptide hydrogel or
In conjunction with the medical dressing of biologically active polypeptide or claim 5 or the 6 chemically modified bioactivity described in claim 4
Described in polypeptide or claim 7 prevention and/or treatment ischemic are being prepared in conjunction with the gel of chemically modified biologically active polypeptide
Application in property disease medicament or medical material.
12. application according to claim 9, which is characterized in that the ischemic disease includes myocardial infarction, lower limb ischemia
And ischemic renal injury.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810202362.7A CN108440645A (en) | 2018-03-13 | 2018-03-13 | A kind of biologically active polypeptide and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810202362.7A CN108440645A (en) | 2018-03-13 | 2018-03-13 | A kind of biologically active polypeptide and its application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108440645A true CN108440645A (en) | 2018-08-24 |
Family
ID=63194648
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| Application Number | Title | Priority Date | Filing Date |
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| Country | Link |
|---|---|
| CN (1) | CN108440645A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SE1951495A1 (en) * | 2019-12-18 | 2021-06-19 | Amferia Ab | Improved wound care device |
-
2018
- 2018-03-13 CN CN201810202362.7A patent/CN108440645A/en not_active Withdrawn
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SE1951495A1 (en) * | 2019-12-18 | 2021-06-19 | Amferia Ab | Improved wound care device |
| SE544460C2 (en) * | 2019-12-18 | 2022-06-07 | Amferia Ab | Improved wound care device |
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Application publication date: 20180824 |