CN114344449B - Application of histamine 1 polypeptide in preparing composite material for promoting repair of large-area skin defect - Google Patents
Application of histamine 1 polypeptide in preparing composite material for promoting repair of large-area skin defect Download PDFInfo
- Publication number
- CN114344449B CN114344449B CN202210015023.4A CN202210015023A CN114344449B CN 114344449 B CN114344449 B CN 114344449B CN 202210015023 A CN202210015023 A CN 202210015023A CN 114344449 B CN114344449 B CN 114344449B
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- China
- Prior art keywords
- polypeptide
- hst1
- hydrogel
- histamine
- chitosan
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Classifications
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- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
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- A61L26/0009—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials
- A61L26/0028—Polypeptides; Proteins; Degradation products thereof
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- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
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Abstract
The invention discloses application of histamine 1 polypeptide in preparing a composite material for promoting repair of large-area skin defects. In the present invention, it was found that histamine 1 (Hst 1) polypeptides have the property of promoting cell adhesion, angiogenesis and inhibiting inflammatory reactions. The Hst1 polypeptide can be loaded on various biological materials, and can be compounded with bioactive materials to prepare dressing, injection, ointment, powder, hydrogel, membrane, sponge, fiber scaffold material and the like, so that the healing of large-area skin defects can be effectively promoted; wherein the large-area skin defect comprises skin ulcer caused by trauma, burn, tumor excision or diabetes, etc. The Hst1 polypeptide provided by the invention is a human polypeptide, has good biocompatibility and biosafety, and has important medical value and good industrialization prospect in preparing the Hst1 polypeptide to promote large-area skin defect repair materials.
Description
The application is a divisional application with the application number of 201810725789.5 and the application date of 2018, 7 and 4, and the name of the application of histamine 1 polypeptide in preparing a composite material for promoting the repair of large-area skin defects.
Technical Field
The invention belongs to the field of biomedicine, and particularly relates to application of histamine 1 polypeptide in preparing a composite material for promoting repair of large-area skin defects.
Background
Large-area full-thickness defects of skin are commonly found in trauma, burns, tumor excision, and skin ulcers caused by diabetes. The large-area skin full-layer defect can not heal by itself and needs to be transplanted by an autologous skin patch and a skin flap, but the method has the defects of limited sources, need of secondary operation and the like. And the variant skin sheets and skin flaps have the risks of immune rejection and the like. Thus, a large number of artificial dermis implants are used for skin defect repair, however these materials do not ultimately achieve the desired therapeutic effect. Skin defect treatment is undesirable on the one hand because the lack of vascular supply in large area defective skin does not provide adequate oxygen and nutrients to the new tissue; in addition, excessive inflammatory response and poor cell chemotaxis and cell adhesion can 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 healing speed of the gingival tissue is twice that of skin injury, and the scar formation can be effectively reduced, wherein one important reason is that proteins and polypeptides in the oral cavity have the effect of promoting the skin defect to repair. Histamine is a group of histidine-rich cationic polypeptides secreted by the parotid and submaxillary glands of humans and is 7 to 38 amino acid residues in length. Histamine 1,3,5 is the most important 3, accounting for 85-90% of the total content of histamine. In recent years, histamine 1 (Hst 1) has been intensively studied in the biomedical field, and has various effects of promoting cell adhesion, stretching, migration, angiogenesis, anti-inflammatory and the like, but whether Hst1 can be applied to repair of a large-area skin defect has not been reported. Histamine is only present in1 patent application: patent application No. 201580061786.0, entitled "salivary casein peptide", discloses the use of histamine in the treatment of tooth demineralization. Therefore, in order to promote the repair of the wound surface of the skin with a large area, solve the problems of inflammatory reaction, insufficient vascularization and the like of wound surface repair, the development of the skin defect repair material based on the Hst1 polypeptide has important application value for efficiently promoting the repair of the large-area defect of the skin.
Disclosure of Invention
The primary purpose of the invention is to overcome the defects and shortcomings in the prior art and provide application of histamine 1 polypeptide in preparing a composite material for promoting repair of large-area skin defects.
It is another object of the present invention to provide the use of a histamine 1 polypeptide in the manufacture of a medicament for promoting repair of a large area of skin defect.
The aim of the invention is achieved by the following technical scheme: use of a histamine 1 (Hst 1) polypeptide in the preparation of a composite material for promoting repair of a large area skin defect.
The amino acid sequence of the histamine 1 (Hst 1) polypeptide is selected from any one of the following sequences:
(1) SEQ ID NO.1 (DS P HEKRHHGYRRKFHEKHHSHREFPFYGDYGSNYLYDN) an amino acid sequence shown in seq id no;
(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, obtained by substitution, deletion and/or addition (insertion) of one or more amino acids to the amino acid sequence shown in SEQ ID NO. 1.
The dosage of the histamine 1 (Hst 1) polypeptide is 10-1000 mug/day/person, 1 time per week; the dosage and dosage range for clinical application will need to be combined with a number of factors including the mode of administration, the carrier, the physical condition of the patient, and the size of the defect.
The large-area skin defect comprises skin ulcer caused by trauma, burn, tumor excision or diabetes, etc.
The composite material is composed of histamine 1 (Hst 1) polypeptide and a carrier; the composite material can be in the form of dressing, spray, hydrogel, plaster, etc.; preferably a thiolated chitosan hydrogel loaded with an Hst1 polypeptide, a collagen sponge loaded with an Hst1 polypeptide or a polyglycolic acid porous fibrous membrane loaded with an Hst11 polypeptide; the application mode of the composite material is that dressing, spray, hydrogel, plaster and the like are directly sprayed or covered on the surface of a wound or tissue.
The carrier is a carrier with good biocompatibility and no influence on the activity of the polypeptide, and comprises degradable natural polymers, synthetic polymers and biological ceramics; the ceramic material 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 ceramic comprises hydroxyapatite, calcium hydrophosphate, tricalcium phosphate, octacalcium phosphate, calcium sulfate and the like.
The dressing comprises histamine 1 (Hst 1) polypeptide, a film or sponge and the like.
The sulfhydryl chitosan hydrogel loaded with the Hst1 polypeptide is preferably prepared by the following method:
(I) Dissolving sulfhydryl chitosan (CS-SH) into water with pH value of 8, and then adding beta-sodium glycerophosphate to adjust the pH value of 7 to obtain CS-SH hydrogel;
(II) adding the Hst1 polypeptide solution into CS-SH hydrogel, and uniformly stirring to obtain the thiol chitosan hydrogel loaded with the Hst1 polypeptide;
the sulfhydryl chitosan (CS-SH) described in the step (I) is preferably prepared by the following method:
dissolving chitosan in acetic acid aqueous solution, then adding 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDAC) and N-hydroxysuccinimide (NHS), stirring and reacting for 15min at room temperature under a dark condition, then adding cysteine (Cys), regulating pH to 5-6, stirring and reacting for 5h at room temperature under a dark condition, dialyzing, and freeze-drying to obtain the sulfhydryl chitosan (CS-SH).
The aqueous acetic acid 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 in every 100mL of acetic acid aqueous solution.
The 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDAC) was used in an amount of 50mmol/L as final concentration.
The amount of N-hydroxysuccinimide (NHS) is calculated as 50mmol/L final concentration.
The molar ratio of the chitosan to the cysteine is 1:1.
The pH regulator is NaOH solution; preferably 1mol/L NaOH solution.
The dialysis is preferably achieved by the steps of: the solution was dialyzed against light for 3 days sequentially with a HCl solution at ph=5.0, a HCl solution containing 1% (w/v) NaCl, ph=5.0, and a HCl solution at ph=5.0.
The CS-SH hydrogel in the step (I) is CS-SH hydrogel with the concentration of 5% (w/v).
The concentration of the Hst1 polypeptide solution in the step (II) is 125mg/mL.
The volume ratio of Hst1 polypeptide solution to CS-SH hydrogel in step (II) is preferably 1:9.
the collagen sponge loaded with the Hst1 polypeptide comprises collagen sponge and an Hst1 aqueous solution; wherein the collagen sponge is prepared by the following method:
(a) Dissolving type I collagen in acetic acid aqueous solution to obtain collagen solution, and freeze-drying the collagen solution to obtain a porous sponge membrane;
(b) Placing the porous sponge membrane in an ammonia gas atmosphere for 30 minutes, then washing the porous sponge membrane to be neutral, freeze-drying and sterilizing to obtain the collagen sponge.
The collagen solution in the step (a) is a collagen solution with a concentration of 3 wt%.
The aqueous acetic acid solution in the step (a) is an aqueous acetic acid solution with a concentration of 1% (v/v).
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 mug/mL.
The porous polyglycolic acid fiber membrane loaded with the Hst11 polypeptide comprises a porous polyglycolic acid (PGA) fiber membrane and an aqueous Hst1 solution; among them, polyglycolic acid (PGA) fiber porous membrane is preferably prepared by electrospinning.
The concentration of the Hst1 aqueous solution is preferably 40 mug/mL.
Application of histamine 1 (Hst 1) polypeptide in preparing medicament for promoting repair of large-area skin defect.
The medicine for promoting the repair of the large-area skin defect is administrated in a mode of 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, excipient, filler, binder, wetting agent, disintegrating agent, absorption enhancer, adsorption carrier, surfactant or the like.
The medicine for promoting the repair of the large-area skin defect can be prepared into various dosage forms including powder, injection and the like by adopting a conventional method in the field.
Compared with the prior art, the invention has the following advantages and effects:
(1) The invention discovers that histamine 1 (Hst 1) polypeptide has the characteristics of promoting cell adhesion, angiogenesis and inhibiting inflammatory reaction. In vitro experiments prove that the Hst1 polypeptide can promote the adhesion, the extension, the vascularization, the migration, the inhibition of inflammatory reaction, the up-regulation of the vascularization factor of the stem cells and the activation of the mesenchymal stem cells-endothelial cells to form vascular-like tissues, and the vascularization promoting capacity is improved by 2.8 times compared with that of a blank control group, so that the Hst1 polypeptide can effectively promote the repair of skin defects. Animal experiments prove that after the Hst1 polypeptide is subcutaneously injected, the healing speed of the skin defect of the rat can be accelerated, and the healing speed of the skin defect of the rat is faster than that of a control group. In vivo experiments prove that Hst1 polypeptide and materials such as hydrogel, membrane, ointment, sponge and the like of the composite group Hst1 can promote the healing speed of rat skin defects: the Hst1 polypeptide loaded chitosan hydrogel can efficiently promote the healing of the skin defect of the rat, and the skin defect is repaired by 82% in 7 days, while the control group is only repaired by 51%.
(2) The Hst1 polypeptide applied by the invention is a human-derived polypeptide, has good biocompatibility and biosafety, does not find that animals have acute toxic reaction, has no side effects such as immune rejection and the like, and has no obvious tumorigenicity in the observation of tumorigenesis.
(3) The difficulty in repairing large-area skin defects is mainly caused by difficult cell adhesion, insufficient vascularization and wound repair inflammatory reaction, and the Hst1 polypeptide applied by the invention has anti-inflammatory and antibacterial effects and can effectively 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 bioactive materials to prepare dressing, injection, ointment, powder, hydrogel, membrane, sponge, fiber scaffold material and the like, which can efficiently promote the healing of large-area skin defects, and has important medical value; and the Hst1 polypeptide composite artificial material is simple to prepare and low in price. Therefore, the application of the Hst1 polypeptide to prepare the material for promoting the repair of the large-area skin defect has good industrialization prospect.
Drawings
FIG. 1 is a graph showing the results of research on the effect of Hst1 on the adhesion and expansion of mesenchymal stem cells; wherein, figure A is the effect of Hst1 on mesenchymal stem cell adhesion; panel B shows the effect of Hst1 on mesenchymal stem cell expansion.
FIG. 2 is a schematic representation of the cellular activity of the thiolated chitosan injectable hydrogels of example 1.
FIG. 3 is a microscopic image of HUVECs cells seeded on thiolated chitosan injectable hydrogels of example 1; wherein, figure a is a hydrogel; panel B is an Hst1 loaded hydrogel.
FIG. 4 is a graph showing the results of the repair of Hst1 polypeptide in example 2 on 7 days after repair of rat skin defects; wherein, figure a skin repair map; figure B is a statistical plot of skin repair proportions.
FIG. 5 is a graph showing the repair result of the thiol chitosan injectable hydrogel of example 3 to repair rat skin defects; wherein, figure a skin repair map; figure B is a statistical plot of skin repair proportions.
FIG. 6 is a map of masson staining of repair of rat skin defects by collagen sponges loaded with Hst1 polypeptides.
FIG. 7 is a general diagram of the application of Hst1 polypeptide-loaded porous fibrous membrane of polyglycolic acid in example 5 to promote repair of rat skin defects.
Detailed Description
The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto. The raw materials and reagents used in the following examples were commercially available unless otherwise specified.
The examples relate to Hst1 polypeptide powder (synthesized by Shanghai TaoPu biosystems) which has the amino acid sequence: DS (DS) P HEKRHHGYRRKFHEKHHSHREFPFYGDYGSNYLYDN (serine 2 is phosphorylated).
Example 1: research on promoting cell adhesion, stretching and vascularization by Hst1 polypeptide
(1) Mesenchymal stem cells (purchased from Cyagen Biosciences Inc (USA), lot 170221I 31) were seeded in 96-well plates at a density of 5X 10 per well 3 Each mesenchymal stem cell (BMSCs) was stained with 50. Mu.l of serum-free medium (a-MEM, gibco) containing Hst1 polypeptide at a final concentration of 10. Mu.M, and cultured in a single serum-free medium (a-MEM, gibco) for 1.5 hours, and after 3 hours, the cells were stained with phalloidin to observe cell adhesion and expansion.
(2) BMSCs were seeded in 96-well plates at a density of 5X 10 per well 3 Mu.l of serum-free medium containing Hst1 polypeptide at a final concentration of 10. Mu.M was added to each cell for 1d (day), 3d and 5d, then 90. Mu.l of culture broth (a-MEM, gibco) and 10. Mu.l of CCK-8 solution (Japanese Kogyo Co., ltd.) were added to each well at each time point, and incubated again at 37℃for 2h. 100 μl of the reaction solution was removed from each well of the 24-well 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 in 96-well plate with concentration of 125mg/mL at 40 ul/well, inoculating BMSCs in 96-well plate with density of 5×10/well 3 The control group contains no Hst1 polypeptide, and after 3 days of culture, the adhesion and stretching of the cells are observed by a microscope. The preparation method of the chitosan hydrogel containing the Hst1 polypeptide (namely the sulfhydryl chitosan hydrogel loaded with the Hst1 polypeptide) comprises the following steps:
(1) the preparation method of the sulfhydryl chitosan injectable hydrogel comprises the following steps:
1g of chitosan is weighed and dissolved in 100mL of 0.5% (v/v) acetic acid aqueous solution, 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDAC) and N-hydroxysuccinimide (NHS) with the final concentration of 50mmol/L are respectively added, stirring reaction is carried out at room temperature and in a dark place for 15min, cys is added into the reaction system (the mol ratio of chitosan to cysteine is 1:1), the pH is regulated to 5-6 by using 1mol/L NaOH solution, and stirring reaction is carried out at room temperature and in a dark place for 5 h; sequentially dialyzing with pH=5.0 HCl solution, pH=5.0 HCl solution containing 1% (w/v) NaCl and pH=5.0 HCl solution for 3 days in dark, and freeze-drying to obtain sulfhydryl chitosan (CS-SH) sample, and storing at 4deg.C.
(2) Adding CS-SH into deionized water solution with pH=8, fully dissolving, finally preparing into CS-SH solution with 5% (w/v), and then adding beta-sodium glycerophosphate to adjust the final pH=7 at room temperature to obtain CS-SH hydrogel;
(3) adding and stirring uniformly according to the volume ratio of CS-SH hydrogel to Hst1 polypeptide solution (the concentration of Hst1 polypeptide solution is 125 mg/mL) of 9:1, and obtaining gel precursor solution (the thiol chitosan hydrogel loaded with Hst1 polypeptide).
(4) Results
Cell adhesion, stretching and vascularization results show that Hist1 obviously promotes BMSCs adhesion, and the cell adhesion quantity of the BMSCs is 2.4 times and 1.8 times of that of a control group (Con) in 1.5h and 3h respectively; hist1 significantly promoted BMSCs expansion with cell expansion ratios of 1.5h and 3h 2-fold and 1.5-fold, respectively, compared to the control group (see fig. 1).
CCK-8 experiments prove that the Hst1 polypeptide has good cell compatibility, and the cell activities of the Hst1 polypeptide are 1,3 and 5d, and the cell activities of the Hst1 polypeptide and the control group have no statistical difference (see figure 2).
The results show that the Hst1 polypeptide-loaded hydrogel can significantly promote the expansion of BMSCs cells (see FIG. 3).
Example 2: research of promoting repair of rat skin defect by Hst1 polypeptide
(1) Construction of SD rat Large area skin Defect model
SD rats (weight 250-300 g, total 5, purchased from animal experiment center of southern medical university) were anesthetized with 3% (w/v) pentobarbital sodium intraperitoneal injection, prone on an operating table, fixed limbs, shaved on the back, and 1% (w/v) iodine disinfection drape. The skin puncher is used for carrying out circular full-layer skin defect with the diameter of 5mm on the back side, gentamicin (the injection amount is 4 mg/kg) is injected every day to prevent infection within 3d after operation, and the injection of the kaibuprofen (the injection amount is 12.5 mg/kg) is used for easing pain and carrying out normal feeding in separate cages.
(2) Repair of rat skin defects with Hst1 polypeptides
Using SD rats as a control, with one side being the experimental group, prepared powders of Hst1 polypeptide were implanted into skin defects (covered on the wound surface), each defect was filled with about 100. Mu.g of Hst1 polypeptide powder, and covered with gauze. The other side is a control group, which is not specially treated. The dressing is not required to be changed in the whole experimental process, and the animal is observed every day to avoid the dressing from falling off.
The wound condition was recorded with a digital camera at postoperative day 0d (postoperative day) and 7 d. The experimental results are shown in FIG. 4. The Image analysis software (Image J) determined the area of the skin defect suggesting that application of Hst1 significantly accelerates the rate of skin defect repair, with histamine 1 repairing approximately 75% of the skin area at 7 days, whereas the control (Con) repaired only 51%. Wherein, the wound healing area is calculated by the following formula:
P＝[(I-R)/I]×100％；
wherein: i is the initial skin defect area, and R is the skin defect area at a specific time point.
Example 3: hst1 polypeptide-loaded sulfhydrylation chitosan hydrogel for promoting repair of rat skin defect
(1) The preparation method of the thiolated chitosan hydrogel loaded with the Hst1 polypeptide is the same as in example 1.
(2) Repair of rat skin defect by Hst1 polypeptide-loaded sulfhydrylation chitosan hydrogel
Injecting Hst1 polypeptide-loaded thiolated chitosan hydrogel into an SD rat (model construction method is the same as that of example 1), and injecting Hst1 polypeptide-loaded thiolated chitosan hydrogel (40 ul hydrogel is injected into each skin defect) into the skin defect (phi=5mm) of the SD rat; the other side is a control group; wherein the control group (Con) was not specially treated. The results are shown in fig. 5, which shows that the thiol-modified chitosan loaded with the Hst1 polypeptide obviously promotes skin repair.
Example 4: application of collagen sponge loaded with Hst1 polypeptide to promotion of repair of rat skin defects
(1) Preparation of collagen sponge loaded with Hst1 polypeptide
(1) Preparation of a porous sponge film: dissolving type I collagen (Sichuan Ming Biotechnology Co., ltd.) in 1% (v/v) acetic acid water solution, preparing 3% (wt%) collagen solution, lyophilizing to obtain porous sponge membrane with diameter of 5mm and height of 2mm, and porosity of above 90%; then placing for 30 minutes in an ammonia atmosphere, and washing with water to be neutral; finally, freeze-drying, sterilizing and sealing and preserving at low temperature;
(2) preparation of Hst1 solution: 250 μg of Hst1 polypeptide powder was dispersed and dissolved with 2mL of sterilized deionized water.
(2) Repair of rat skin defects by collagen sponges loaded with Hst1 polypeptides
SD rats (model construction method is the same as that of example 1) are adopted for self control, one side is an experimental group, a pre-prepared porous sponge film is implanted (covered on the surface of a wound) at a skin defect (phi=5 mm), and then Hst1 solution (40 mu l of Hst1 solution is loaded on the porous sponge film with the diameter of 5 mm) is added; the other side is pure collagen (pure porous sponge film) 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 of the experimental group was denser and more similar to the normal skin tissue than the control group (Con) (see fig. 6).
Example 5: application of polyglycolic acid porous fiber membrane loaded with Hst1 polypeptide in promoting repair of rat skin defect
Polyglycolic acid (PGA) fiber porous membranes prepared by electrospinning (fiber diameter about 100nm, membrane porosity 95%; synthetic methods were referenced by Min Suk Lee, taufiq Ahmad, jinkyu Lee, hassan K.Awada, yadong Wang, kyobium Kim, heungsoo Shin and Hee Seok Yang, dual delivery of growth factors with coacervate-coated poly (lactic-co-glycolic acid) nanofiber improves neovascularization in amouse skin flap model, biomaterials,10.1016/j. Biomaterials.2017.01.036,124, (65-77), (2017)) were sterilized by damp-heat.
SD rats (model construction method same as in example 1) with a weight of 250-300 g were used, 5 animals were purchased from the university of south medical science animal experiment center. Taking the PGA fiber porous membrane as a control, implanting the PGA fiber porous membrane into a skin defect part (phi=5mm) on one side of a rat, dispersing and dissolving 250 mu g of Hst1 polypeptide powder with 2mL of sterilized deionized water, and then adding 40 mu L of Hst1 solution (containing 5 mu g of Hst 1) into each part; the other side was blank.
As a result, it was found that the skin of the experimental group was repaired 78% after 7 days, whereas the blank group was only 50% (see fig. 7).
In conclusion, the Hst1 polypeptide can obviously promote the repair of large-area skin defects, and hydrogel, a membrane, ointment, sponge and the like which are compounded with various biological materials can achieve similar effects of promoting the repair of skin defects. Hst1 polypeptides may be natural or synthetic polypeptides, or may be polypeptides and homologs of all amino acid sequences or individual, multiple amino acid substitutions, deletions and insertions of variants, as changes in part of the amino acids do not affect their actual function. Besides the above materials, other composite materials such as degradable natural polymers, synthetic polymers and bioceramics can also promote repair of rat skin defects, such as natural materials including chitosan, hyaluronate, sodium alginate, cellulose, starch, lignin, collagen, gelatin, carrageenan and the like, and derivatives thereof, synthetic polymer materials including polylactic acid, polyglycolic acid, polycaprolactone, polyhydroxyalkanoate, polysiloxane, polyurethane and the like, and derivatives thereof, bioceramics including hydroxyapatite, calcium hydrophosphate, tricalcium phosphate, octacalcium phosphate, calcium sulfate and the like, and composite materials formed by the above materials, and the like.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (4)
1. Use of a histamine 1 polypeptide for the preparation of a composite material for promoting mesenchymal stem cells to adhere and stretch on chitosan hydrogel to promote repair of a large area skin defect, characterized in that the amino acid sequence of the histamine 1 polypeptide is: DS (DS) P HEKRHHGYRRKFHEKHHSHREFPFYGDYGSNYLYDN; the composite material is a sulfhydrylation chitosan hydrogel loaded with histamine 1 polypeptide, and the sulfhydrylation chitosan hydrogel loaded with histamine 1 polypeptide is prepared by the following method:
(I) Dissolving sulfhydryl chitosan into water with pH value of 8, and then adding beta-sodium glycerophosphate to adjust the pH value of 7 to obtain CS-SH hydrogel;
(II) adding the Hst1 polypeptide solution into CS-SH hydrogel, and uniformly stirring to obtain the thiol chitosan hydrogel loaded with the Hst1 polypeptide;
the CS-SH hydrogel in the step (I) is CS-SH hydrogel with the concentration of 5% (w/v);
the concentration of the Hst1 polypeptide solution in the step (II) is 125mg/mL;
the volume ratio of the Hst1 polypeptide solution to the CS-SH hydrogel in the step (II) is 1:9.
2. use according to claim 1, wherein the thiolated chitosan of step (I) is prepared by the following method:
dissolving chitosan in acetic acid aqueous solution, then 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 pH to 5-6, stirring and reacting for 5h at room temperature in a dark place, dialyzing, and freeze-drying to obtain the sulfhydryl chitosan.
3. The use according to claim 2, wherein the aqueous acetic acid solution is an aqueous acetic acid solution having a concentration of 0.5% (v/v);
the dosage of the 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride is calculated according to the final concentration of 50 mmol/L;
the dosage of the N-hydroxysuccinimide is calculated according to the final concentration of 50 mmol/L;
the molar ratio of the chitosan to the cysteine is 1:1.
4. A use according to claim 3, wherein: the large-area skin defect is skin ulcer caused by trauma, burn, tumor excision or diabetes; the dosage of the histamine 1 polypeptide is 10-1000 mug/day/person, and 1 time per week.
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| WO2023005945A1 (en) * | 2021-07-27 | 2023-02-02 | 杭州彗搏科技有限公司 | Cyclic peptide, preparation method therefor and use thereof |
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