CN108785657B

CN108785657B - Application of histaminin 1 polypeptide in preparation of composite material for promoting repair of large-area skin defect

Info

Publication number: CN108785657B
Application number: CN201810725789.5A
Authority: CN
Inventors: 林振; 柳毅; 王晶; 李立华; 李日旺; 吴刚
Original assignee: Hangzhou Huibo Technology Co ltd
Current assignee: Hangzhou Huibo Technology Co ltd
Priority date: 2018-07-04
Filing date: 2018-07-04
Publication date: 2022-02-11
Anticipated expiration: 2038-07-04
Also published as: CN108785657A; CN114344449B; CN114344449A

Abstract

The invention discloses application of histatin1 polypeptide in preparing a composite material for promoting repair of large-area skin defects. The invention discovers that the histamin 1(Hst1) polypeptide has the characteristics of promoting cell adhesion, vascularization and inhibiting inflammatory reaction. The Hst1 polypeptide can be loaded on various biological materials, and can be compounded with bioactive materials to prepare dressing, injection, paste, powder, hydrogel, membrane, sponge, fiber scaffold materials and the like, so that the healing of large-area skin defect can be efficiently promoted; wherein the large-area skin defect comprises skin ulcer caused by trauma, burn, tumor excision or diabetes. The Hst1 polypeptide is human polypeptide, has good biocompatibility and biological safety, and has important medical value and good industrialization prospect when being used for preparing materials for promoting large-area skin defect repair from Hst1 polypeptide.

Description

Application of histaminin 1 polypeptide in preparation of composite material for promoting repair of large-area skin defect

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to application of a histamin 1 polypeptide in preparation of a composite material for promoting repair of large-area skin defects.

Background

Large-area skin full-layer defects are commonly seen in skin ulcers and the like caused by trauma, burns, tumor resection, diabetes and the like. The large-area skin full-layer defect can not be healed by self, and autologous skin sheets and skin flaps are required to be transplanted, but the methods have the defects of limited sources, secondary operation and the like. And the allogeneic skin sheet and the skin flap have the risks of immunological rejection and the like. Thus, a number of artificial dermal implants are used for skin defect repair, however these materials do not ultimately achieve the desired therapeutic effect. Skin defects are not ideal on the one hand because of the lack of vascularity in large defect skin, failing to provide adequate oxygen and nutrients to the new tissue; in addition, excessive inflammatory responses, poor cellular chemotaxis and cell adhesion also affect healing of skin defects, resulting in delayed healing.

The wound healing speed of the oral tissue is obviously faster than that of other parts of the whole body, the repairing speed of the gingival tissue is twice of that of the skin injury, and the formation of scars can be effectively reduced, wherein one important reason is that proteins and polypeptides in the oral cavity have the effect of promoting the repair of skin defects. The histaminins are a group of histidine-rich cationic polypeptides secreted by human parotid and submandibular glands and have the length of 7-38 amino acid residues.

Histamine

1,3,5 is the most important 3 of them, accounting for 85-90% of the total content of the histamines. In recent years, histamin 1(Hst1) has been intensively studied in the biomedical field, and has various effects of promoting cell adhesion, cell extension, cell migration, vascularization, anti-inflammation and the like, but no relevant report is found on whether Hst1 can be applied to large-area skin defect repair. Histamine is currently seen in only 1 patent application: application No. 201580061786.0 entitled "salivary casein rich peptides" discloses the use of histaminins for the treatment of dental demineralization. Therefore, in order to promote the repair of large-area skin wound surfaces and solve the problems of inflammatory reaction, insufficient vascularization and the like in wound surface repair, the Hst1 polypeptide-based skin defect repair material is developed, and the important application value is realized for efficiently promoting the repair of large-area skin defects.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides the application of the histamin 1 polypeptide in preparing a composite material for promoting the repair of large-area skin defects.

Another objective of the invention is to provide an application of the histamin 1 polypeptide in preparing a medicament for promoting repair of large-area skin defects.

The purpose of the invention is realized by the following technical scheme: application of histatin 1(Hst1) polypeptide in preparing composite material for promoting repair of large-area skin defect.

The amino acid sequence of the histatin 1(Hst1) polypeptide is selected from any one of the following sequences:

(1) as shown in SEQ ID NO.1 (DS)^PHEKRHHGYRRKFHEKHHSHREFPFYGDYGSNYLYDN);

(2) a polypeptide comprising part or all of the amino acid sequence of SEQ ID No. 1;

(3) an amino acid sequence having at least 80% homology with the amino acid sequence shown in SEQ ID No.1, which is obtained by substituting, deleting and/or adding (inserting) one or more amino acids to the amino acid sequence shown in SEQ ID No. 1.

The dosage of the histatin 1(Hst1) polypeptide is 10-1000 mug/day/person, 1 time per week; the dosage and dosage range to be used clinically will need to be combined with a number of factors including the mode of administration, the carrier, the condition of the patient, the size of the defect, etc.

The large-area skin defect comprises skin ulcer caused by trauma, burn, tumor excision or diabetes, and the like.

The composite material is composed of histatin 1(Hst1) polypeptide and a carrier; the composite material can be in the form of dressing, spray, hydrogel, plaster and the like; preferably sulfhydrylation chitosan hydrogel loaded with Hst1 polypeptide, collagen sponge loaded with Hst1 polypeptide or polyglycolic acid porous fiber membrane loaded with Hst11 polypeptide; the use mode of the composite material is that the dressing, the spraying agent, the hydrogel, the plaster and the like are directly sprayed or covered on the surface of the wound or the tissue.

The carrier is good in biocompatibility and does not influence the activity of the polypeptide, and comprises degradable natural macromolecules, synthetic macromolecules and biological ceramics; the biological ceramic material specifically comprises more than one of natural materials and derivatives thereof, synthetic polymer materials and derivatives thereof, and biological ceramics.

The natural materials comprise chitosan, hyaluronate, sodium alginate, cellulose, starch, lignin, collagen, gelatin, carrageenan and the like.

The synthetic polymer material comprises polylactic acid, polyglycolic acid, polycaprolactone, polyhydroxyalkanoate, polysiloxane, polyurethane and the like.

The biological ceramics comprise hydroxyapatite, calcium hydrophosphate, tricalcium phosphate, octacalcium phosphate, calcium sulfate and the like.

The dressing comprises histamin 1(Hst1) polypeptide, a film or a sponge and the like.

The SH-chitose hydrogel loaded with Hst1 polypeptide is preferably prepared by the following method:

(I) dissolving thiolated chitosan (CS-SH) into water with pH of 8, and then adding beta-sodium glycerophosphate to adjust the pH of 7 to obtain CS-SH hydrogel;

(II) adding an Hst1 polypeptide solution into the CS-SH hydrogel, and uniformly stirring to obtain a sulfhydrylation chitosan hydrogel loaded with Hst1 polypeptide;

the thiolated chitosan (CS-SH) described in step (I) is preferably prepared by the following method:

dissolving chitosan in acetic acid aqueous solution, adding 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDAC) and N-hydroxysuccinimide (NHS), stirring and reacting for 15min at room temperature in the dark, adding cysteine (Cys), adjusting the pH value to 5-6, stirring and reacting for 5 hours at room temperature in the dark, dialyzing, and freeze-drying to obtain the thiolated chitosan (CS-SH).

The acetic acid aqueous solution is 0.5% (v/v) acetic acid aqueous solution.

The dosage of the chitosan is calculated according to the proportion of 1g of chitosan per 100mL of acetic acid aqueous solution.

The dosage of the 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDAC) is calculated according to the final concentration of 50 mmol/L.

The dosage of the N-hydroxysuccinimide (NHS) is calculated according to the final concentration of 50 mmol/L.

The molar ratio of the chitosan to the cysteine is 1: 1.

The pH regulator is NaOH solution; preferably a 1mol/L NaOH solution.

The dialysis is preferably achieved by: the mixture was dialyzed against 1% (w/v) NaCl, HCl solution at pH 5.0, and HCl solution at pH 5.0 in this order for 3 days.

The CS-SH hydrogel in the step (I) is the CS-SH hydrogel with the concentration of 5% (w/v).

The concentration of the Hst1 polypeptide solution in the step (II) is 125 mg/mL.

The volume ratio of the Hst1 polypeptide solution to the CS-SH hydrogel in the step (II) is preferably 1: 9.

the collagen sponge loaded with the Hst1 polypeptide comprises collagen sponge and Hst1 aqueous solution; the collagen sponge is prepared by the following method:

(a) dissolving the I-type collagen in an acetic acid aqueous solution to obtain a collagen solution, and then freeze-drying the collagen solution to obtain a porous sponge membrane;

(b) and (3) placing the porous sponge membrane in an ammonia atmosphere for 30 minutes, then washing the porous sponge membrane to be neutral, and performing freeze drying and sterilization to obtain the collagen sponge.

The collagen solution in the step (a) is a collagen solution with a concentration of 3 wt%.

The acetic acid aqueous solution in the step (a) is 1% (v/v) acetic acid aqueous solution.

The porous sponge membrane in the step (a) is a porous sponge membrane with the diameter of 5mm, the height of 2mm and the porosity of more than 90 percent.

The concentration of the Hst1 aqueous solution is preferably 40 mu g/mL.

The polyglycolic acid porous fiber membrane loaded with the Hst11 polypeptide comprises a polyglycolic acid (PGA) fiber porous membrane and an Hst1 aqueous solution; among them, polyglycolic acid (PGA) fiber porous membrane is preferably produced by electrospinning.

The concentration of the Hst1 aqueous solution is preferably 40 mu g/mL.

Application of histatin 1(Hst1) polypeptide in preparing medicine for promoting repair of large-area skin defect is disclosed.

The administration mode of the medicament for promoting the repair of the large-area skin defect is local administration, subcutaneous injection, intradermal injection, intramuscular injection or intravenous injection.

The medicament for promoting the repair of the large-area skin defect can also contain one or at least two pharmaceutically acceptable carriers.

The carrier is preferably a sustained-release agent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption enhancer, an adsorption carrier, a surfactant or the like.

The medicine for promoting the repair of the large-area skin defect can be prepared into various dosage forms by adopting the conventional method in the field, including powder, injection and the like.

Compared with the prior art, the invention has the following advantages and effects:

(1) the invention discovers that the histatin 1(histatin1, Hst1) polypeptide has the characteristics of promoting cell adhesion, vascularization and inhibiting inflammatory reaction. In vitro experiments prove that the Hst1 polypeptide can promote the adhesion, extension, vascularization, migration and inflammation reaction of mesenchymal stem cells, up-regulate vascularization factors of the stem cells and activate mesenchymal stem cells-endothelial cells to form vascular tissues, the vascularization promoting capacity of the polypeptide is improved by 2.8 times compared with that of a blank control group, and the Hst1 polypeptide can effectively promote the repair of skin defects. Animal experiments prove that the Hst1 polypeptide can accelerate the healing speed of the skin defect of a rat after subcutaneous injection, and the healing speed of the skin defect of the rat is faster than that of a control group. In vivo experiments prove that materials such as Hst1 polypeptide and hydrogel, film, paste and sponge of the composite group Hst1 can promote the healing speed of rat skin defect: hst1 polypeptide-loaded chitosan hydrogel can effectively promote the healing of rat skin defect, and at 7 days, the skin defect is repaired by 82%, while the control group is only repaired by 51%.

(2) The Hst1 polypeptide applied by the invention is human polypeptide, has good biocompatibility and biosafety, does not show that animals have acute toxic reaction, does not have side effects such as immunological rejection and the like, and simultaneously has no obvious tumorigenicity in the observation of tumorigenicity of the Hst11 polypeptide.

(3) The large-area skin defect repair difficulty is mainly caused by cell adhesion difficulty, insufficient vascularization and wound repair inflammatory reaction, and the Hst1 polypeptide applied by the invention has the anti-inflammatory and antibacterial effects and can efficiently promote wound healing.

(4) The Hst1 polypeptide applied by the invention can be loaded on various biological materials, has wide application prospect, for example, the Hst1 polypeptide is compounded with a biological active material to prepare dressing, injection, paste, powder, hydrogel, membrane, sponge, fiber scaffold material and the like, can efficiently promote the healing of large-area skin defect, and has important medical value; and the Hst1 polypeptide composite artificial material is simple to prepare and low in price. Therefore, the Hst1 polypeptide is applied to prepare the material for promoting the repair of large-area skin defects, and has good industrialization prospect.

Drawings

FIG. 1 is a graph showing the results of the study of the effect of Hst1 on the adhesion and expansion of mesenchymal stem cells; wherein, panel a is the effect of Hst1 on mesenchymal stem cell adhesion; panel B is the effect of Hst1 on mesenchymal stem cell expansion.

Fig. 2 is a schematic diagram of the cell activity of the thiolated chitosan injectable hydrogel of example 1.

FIG. 3 is a microscope photograph of HUVECs cells seeded on thiolated chitosan injectable hydrogel in example 1; wherein, panel A is a hydrogel; and the picture B is Hst1 loaded hydrogel.

FIG. 4 is a graph showing the results of 7 days after the Hst1 polypeptide of example 2 repaired a rat skin defect; wherein panel a is a skin repair panel; and the B is a statistical chart of the skin repair ratio.

FIG. 5 is a graph showing the results of repairing rat skin defects with thiolated chitosan injectable hydrogel in example 3; wherein panel a is a skin repair panel; and the B is a statistical chart of the skin repair ratio.

Figure 6 is a massson staining pattern of Hst1 polypeptide loaded collagen sponges for repair of rat skin defects.

FIG. 7 is a general view of the application of the Hst1 polypeptide-loaded polyglycolic acid porous fiber membrane in example 5 to the promotion of rat skin defect repair.

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. The raw materials and reagents used in the following examples are commercially available unless otherwise specified.

The example relates to Hst1 polypeptide powder (synthesized by Shanghai Tanpu Bio Inc.) which is Hst1 polypeptide powder and has the amino acid sequence: DS (direct sequence)^PHEKRHHGYRRKFHEKHHSHREFPFYGDYGSNYLYDN (serine 2 is phosphorylated).

Example 1: study on Hst1 polypeptide promoting cell adhesion, cell expansion and cell vascularization

(1) Bone marrow mesenchymal stem cells (purchased from Cyagen Biosciences Inc (USA), batch No. 170221I31) were seeded in a 96-well plate at a density of 5X 10 per well³Bone marrow mesenchymal stem cells (BMSCs) were cultured in 50. mu.l of serum-free medium (a-MEM, Gibco) containing Hst1 polypeptide at a final concentration of 10. mu.M, and the control group was serum-free medium alone (a-MEM, Gibco), and after 1.5 hours and 3 hours of culture, Coprin staining was performed to observe cell adhesion and spreading.

(2) BMSCs were seeded in 96-well plates at a density of 5X 10 per well³Cells were cultured for 1d (day), 3d and 5d by adding 50. mu.l of serum-free medium containing Hst1 polypeptide at a final concentration of 10. mu.M, then 90. mu.l of culture solution (a-MEM, Gibco) and 10. mu.l of CCK-8 solution (Homophilus Japan) were added to each well at each time point, and incubated again for 2h at 37 ℃. 100. mu.l of the reaction solution was taken out from each well of the 24-well culture plate and transferred to a clean 96-well plate, and the absorbance of the solution at 450nm was measured using a spectrophotometric microplate reader.

(3) Spreading chitosan hydrogel containing Hst1 polypeptide on 96-well plate with concentration of 125mg/mL and 40ul per well, inoculating BMSCs in 96-well plate with density of 5 × 10 per well³The cells and the control group do not contain Hst1 polypeptide water, and after 3 days of culture, the adhesion and the extension of the cells are observed by a microscope. The preparation method of the chitosan hydrogel containing Hst1 polypeptide (namely sulfhydrylation chitosan hydrogel loaded with Hst1 polypeptide) is as follows:

the preparation method of the sulfhydryl chitosan injectable hydrogel comprises the following steps:

weighing 1g of chitosan, dissolving in 100mL of 0.5% (v/v) acetic acid aqueous solution, respectively adding 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDAC) and N-hydroxysuccinimide (NHS) with the final concentration of 50mmol/L, stirring at room temperature and in the dark for reaction for 15min, then adding Cys into a reaction system (the molar ratio of chitosan to cysteine is 1:1), adjusting the pH to 5-6 by using 1mol/L NaOH solution, and stirring at room temperature and in the dark for reaction for 5 hours; after dialysis with a pH 5.0HCl solution, a pH 5.0HCl solution containing 1% (w/v) NaCl, and a pH 5.0HCl solution in the dark for 3 days, the samples were lyophilized to obtain thiolated chitosan (CS-SH) samples, which were stored at 4 ℃.

Adding CS-SH into deionized water solution with pH of 8, dissolving fully, preparing 5% (w/v) CS-SH solution, adding beta-sodium glycerophosphate, and adjusting the final pH to 7 at room temperature to obtain CS-SH hydrogel;

and thirdly, adding the CS-SH hydrogel and the Hst1 polypeptide solution (the concentration of the Hst1 polypeptide solution is 125mg/mL) according to the volume ratio of 9:1, and uniformly stirring to obtain a gel precursor solution (the sulfhydrylation chitosan hydrogel loaded with the Hst1 polypeptide).

(4) Results

The results of cell adhesion, stretching and vascularization show that Hist1 obviously promotes the adhesion of BMSCs, and the cell adhesion quantity is 2.4 times and 1.8 times of that of a control group (Con) at 1.5h and 3h respectively; hist1 significantly promoted the expansion of BMSCs at 2-fold and 1.5-fold higher cell expansion ratios at 1.5h and 3h, respectively, than the control group (see fig. 1).

CCK-8 experiment proves that Hst1 polypeptide has good cell compatibility, the cell activity is 1,3 and 5d, and no statistical difference is generated between the Hst1 polypeptide and the control group (see figure 2).

The results show that the hydrogel loaded with the Hst1 polypeptide can obviously promote the expansion of the BMSCs cells (see figure 3).

Example 2: research on promotion of Hst1 polypeptide on rat skin defect repair

(1) Construction of large-area skin defect model of SD rat

SD rats (250-300 g in weight, 5 in total, purchased from southern medical university animal experiment center) were anesthetized by intraperitoneal injection with 3% (w/v) sodium pentobarbital, and then were prone on an operating table, the limbs were fixed, the back was shaved, and a 1% (w/v) iodine sterilized drape was applied. A circular full-layer skin defect with the diameter of 5mm is made on the back side of the skin puncher, gentamicin (the injection amount is 4mg/kg) is injected every day to prevent infection within 3 days after operation, carprofen (the injection amount is 12.5mg/kg) is injected to relieve pain, and the animals are fed normally in cages.

(2) Repair of rat skin defect by Hst1 polypeptide

Using SD rats self-control, one side being experimental, a previously prepared Hst1 polypeptide powder was implanted into skin defects (covering the wound surface), each defect was filled with approximately 100 μ g Hst1 polypeptide powder, and bandaged with gauze. The other side is a control group without special treatment. Dressing change is not needed in the whole experiment process, and the activity of the animal is observed every day so as to avoid dressing falling off.

Wound surface condition was recorded with a digital camera at 0d (day after surgery) and 7d post-operatively. The results of the experiment are shown in FIG. 4. Image analysis software (Image J) measuring the area of skin defects suggested that the application of Hst1 significantly accelerated the rate of skin defect repair, with histamin 1 repairing approximately 75% of the skin area at 7 days, while the control (Con) repaired only 51%. Wherein, the wound healing area is calculated by the following formula:

P＝[(I-R)/I]×100％；

in the formula: i is the initial skin defect area and R is the skin defect area at a particular time point.

Example 3: hst1 polypeptide-loaded thiolated chitosan hydrogel for promoting rat skin defect repair

(1) The preparation method of the SH chitosan hydrogel loaded with Hst1 polypeptide is the same as that of example 1.

(2) Repair of rat skin defect by sulfhydrylation chitosan hydrogel loaded with Hst1 polypeptide

Adopting an SD rat (the model construction method is the same as that of the embodiment 1) for self control, injecting SH 1-loaded polypeptide thiolated chitosan hydrogel into one side of an experimental group, and injecting the Hst 1-loaded polypeptide thiolated chitosan hydrogel (40 ul hydrogel is injected into each skin defect) into the skin defect (phi is 5mm) of the SD rat; the other side is a control group; the control group (Con) was not treated specifically. The result is shown in figure 5, which shows that the thiolated chitosan loaded with Hst1 polypeptide obviously promotes skin repair.

Example 4: the collagen sponge loaded with Hst1 polypeptide is applied to promote the repair of rat skin defects.

(1) Preparation of Hst1 polypeptide-loaded collagen sponge

Preparing a porous sponge membrane: dissolving type I collagen (Szechwan Ming Biotech Co., Ltd.) in 1% (v/v) acetic acid water solution, preparing 3% (wt%) collagen solution, and freeze-drying to obtain porous sponge membrane with diameter of 5mm and height of 2mm, and porosity of above 90%; then placing the mixture in an ammonia atmosphere for 30 minutes, and washing the mixture to be neutral; finally, freeze-drying, sterilizing, and storing at low temperature in a sealing way;

preparing an Hst1 solution: 250 μ g of Hst1 polypeptide powder was dissolved in 2mL of sterile deionized water.

(2) Repair of rat skin defect by Hst1 polypeptide-loaded collagen sponge

An SD rat (a model construction method is the same as that of example 1) is adopted for self control, one side is an experimental group, a prepared porous sponge membrane is implanted (covered on the surface of a wound) at a skin defect position (phi is 5mm), and then Hst1 solution (the porous sponge membrane with the diameter of 5mm is loaded with 40 mul Hst1 solution) is added; the other side is a pure collagen (pure porous sponge membrane) control group.

As a result, it was found that the material was partially degraded after 5 days, and the new skin covered the entire defect area, and the new skin was denser and closer to the normal skin tissue in the experimental group than in the control group (Con) (see fig. 6).

Example 5: the polyglycolic acid porous fiber membrane loaded with Hst1 polypeptide is applied to promoting the repair of rat skin defects.

Polyglycolic acid (PGA) fiber porous membranes (fiber diameter about 100nm, membrane porosity 95%; reference is made to Min Suk Lee, Taufiq Ahmad, Jinkyu Lee, Hassa n K.Awada, Yadong Wang, Kyobum Kim, Heungsoo Shin and Hee Seok Yan g, Dual delivery of growth factors with co-owned modified poly (lactic-co-glycolic acid) nanofiber impropressions neviralization in a mouse skin model, biometricals, 10.1016/j. biomaterials.2017.01.036,124, (65-77), (2017).) moist heat sterilization.

SD rats (the model construction method is the same as that in example 1) with the weight of 250-300 g and 5 animals in total are purchased from the southern university of medical science animal experiment center. Taking the self as a control and one side as an experimental group, implanting a PGA fiber porous membrane, implanting the PGA fiber porous membrane into a skin defect part (phi is 5mm) on one side of a rat, dispersing and dissolving 250 mu g of Hst1 polypeptide powder by using 2mL of sterilized deionized water, and then adding 40 mu L of Hst1 solution (containing 5 mu g of Hst1) into each part; blank control on the other side.

As a result, the skin was repaired by 78% in the experimental group after 7 days, while that in the blank control group was only 50% (see fig. 7).

In conclusion, the Hst1 polypeptide can obviously promote the repair of large-area skin defects, and the hydrogel, the film, the paste, the sponge and the like compounded with various biological materials can achieve similar effects of promoting the repair of the skin defects. Hst1 polypeptide may be natural polypeptide or synthetic polypeptide, or polypeptide with variation of whole amino acid sequence or individual, multiple amino acid substitution, deletion and insertion, and its homologue, because partial amino acid changes do not affect its essential function. In addition to the above materials, other composite materials such as degradable natural polymers, synthetic polymers and bioceramics can also promote the repair of rat skin defects, for example, natural materials and derivatives thereof such as chitosan, hyaluronate, sodium alginate, cellulose, starch, lignin, collagen, gelatin, carrageenan, synthetic polymer materials and derivatives thereof such as polylactic acid, polyglycolic acid, polycaprolactone, polyhydroxyalkanoate, polysiloxane, polyurethane, and derivatives thereof, bioceramics such as hydroxyapatite, calcium hydrogen phosphate, tricalcium phosphate, octacalcium phosphate, calcium sulfate, and composite materials composed of the above materials, and the like.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> Hangzhou coma science and technology Co., Ltd

Application of <120> histamin 1 polypeptide in preparation of composite material for promoting repair of large-area skin defect

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 38

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Hst1 polypeptide

<400> 1

Asp Ser His Glu Lys Arg His His Gly Tyr Arg Arg Lys Phe His Glu

1 5 10 15

Lys His His Ser His Arg Glu Phe Pro Phe Tyr Gly Asp Tyr Gly Ser

20 25 30

Asn Tyr Leu Tyr Asp Asn

35

Claims

1. The application of the histatin1 polypeptide in preparing the composite material for promoting the adhesion and extension of the bone marrow mesenchymal stem cells is characterized in that the histatin1 polypeptide is humanized histatin1 polypeptide, and the amino acid sequence is as follows: DS (direct sequence)^PHEKRHHGYRRKFHEKHHSHREFPFYGDYGSNYLYDN, respectively; the composite material is sulfhydrylation chitosan hydrogel loaded with Hst1 polypeptide;

the SH-chitose hydrogel loaded with Hst1 polypeptide is prepared by the following method:

(I) dissolving thiolated chitosan into water with pH =8, and then adding beta-sodium glycerophosphate to adjust the pH =7 to obtain CS-SH hydrogel;

the CS-SH hydrogel in the step (I) is a CS-SH hydrogel with the concentration of 5% (w/v);

the concentration of the Hst1 polypeptide solution in the step (II) is 125 mg/mL;

the volume ratio of the Hst1 polypeptide solution to the CS-SH hydrogel in the step (II) is 1: 9.

2. the use according to claim 1, wherein the thiolated chitosan of step (I) is prepared by:

dissolving chitosan in an acetic acid aqueous solution, adding 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride and N-hydroxysuccinimide, stirring and reacting for 15min at room temperature in a dark place, then adding cysteine, adjusting the pH value to 5-6, stirring and reacting for 5 hours at room temperature in a dark place, dialyzing, and freeze-drying to obtain the thiolated chitosan.