CN110790822B

CN110790822B - Polypeptide derivative capable of simulating biological activity of platelet-derived factor, nanofiber and application of polypeptide derivative and nanofiber

Info

Publication number: CN110790822B
Application number: CN201911146406.XA
Authority: CN
Inventors: 杨志谋; 王玲; 商宇娜; 王忠彦; 高洁
Original assignee: Nankai University
Current assignee: Jiangxi Jifa Medical Instrument Co ltd
Priority date: 2019-11-21
Filing date: 2019-11-21
Publication date: 2021-04-06
Anticipated expiration: 2039-11-21
Also published as: CN110790822A; WO2021098399A1

Abstract

The invention provides a polypeptide derivative capable of simulating the biological activity of platelet-derived factor, a nanofiber and application thereof, wherein the polypeptide derivative can simulate the biological activity of PDGF protein, namely promoting the proliferation and migration of fibroblast, and has the advantages of simple production method, small molecular weight, high yield, low cost and definite chemical structure of the product. The polypeptide derivative can be self-assembled in an aqueous solution to form nano-fibers, and can effectively repair skin damage caused by radiation. The sequence of the polypeptide derivative capable of simulating the biological activity of the platelet-derived factor is X-Phe-Phe-Gly-Val-Arg-Lys-Lys-Pro, wherein the terminal group X is an aromatic group-containing terminal group.

Description

Polypeptide derivative capable of simulating biological activity of platelet-derived factor, nanofiber and application of polypeptide derivative and nanofiber

Technical Field

The invention relates to a polypeptide derivative capable of simulating the bioactivity of platelet-derived factor, a nanofiber and application thereof.

Background

The skin is the largest organ of the human body and is composed mainly of the epidermis, dermis and subcutaneous tissue. Generally, a damaged epidermis layer is easily repaired without scarring, but is difficult to heal once the dermis layer is damaged. In the radiotherapy of cancer, ionizing radiation can damage normal tissues of a human body while killing cancer cells, and about 95 percent of patients have side effects of skin damage and the like caused by ionizing radiation. There are two main methods for treating ionizing radiation damage: cytokine therapy and stem cell therapy. Cytokine therapy is the primary strategy capable of preventing or reducing acute radiation syndrome, and hematopoietic growth factors are particularly effective in stimulating hematopoietic recovery. However, since radiation damage leads to massive apoptosis of stem and progenitor cells, cytokine therapy is limited to only a moderate radiation dose range (3-7 Gy of systemic radiation). Stem cell therapy is a promising method for laboratory development, can be used to treat severe radiation injury, and generally employs Mesenchymal Stem Cells (MSCs) extracted from various human tissues such as bone marrow, adipose tissue, umbilical cord, etc., to treat radiation injury through substitution and paracrine effects. However, both of the two treatment modes have the defects of poor tissue specificity, unclear action mechanism, unstable curative effect and the like, and the clinical application of the two treatment modes is greatly limited. There is therefore a great need to develop a simple and effective drug for repairing skin damage caused by radiation.

Platelet derived factor (PDGF) is an important mitogenic factor and has the ability to stimulate the proliferation of specific cell populations such as fibroblasts, vascular smooth muscle cells, mouse myoblasts, mesenchymal stem cells, etc. Are involved in regulating numerous cellular processes including cell migration, differentiation and apoptosis. Plays an important role in the formation and development of gastrulation, early hematopoiesis and angiogenesis. When a tissue is damaged in the human body, platelets release several growth factors, the most prominent of which is platelet derived factor (PDGF), which stimulates the growth of adjacent connective tissue cells. These connective tissue cells are the pioneering team for reconstructing damaged tissue, healing wounds. Therefore, PDGF is a good medicine for patients with severe burns, wound healing, skin cancer and the like.

The PDGF family is composed of five members of PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC and PDGF-DD formed by the dimerization of four different monomeric polypeptide chains of PDGF-A, PDGF-B, PDGF-C and PDGF-D. Among them, PDGF-AA and PDGF-BB have a wide variety of biological activities such as promotion of cell proliferation, migration, resistance to apoptosis, wound repair and the like. PDGF-BB is the most characterized member of the PDGF family, and has received extensive attention from researchers in recent years, and the crystal structure of X-ray diffraction shows that each polypeptide chain is composed of two antiparallel beta sheets, and the left and right ends are connected by 3 LOOP rings, wherein the LOOP1 and LOOP3 rings are important for the binding of the PDGF family to a receptor. The research of He et al in 2010 finds that the LOOP of LOOP1 on the free PDGF-B chain is originally disordered, and an ordered large LOOP is further formed after PDGF-BB contacts with a receptor, and then the PDGF receptor is firmly fixed together with the LOOP of LOOP3 like a clamp.

Inspired by the above findings, researchers have attempted to mimic the biological activity of the PDGF-BB polypeptide chain by truncating a portion of the amino acid sequence associated with receptor binding. For example, Heldint et al segmented and synthesized 97 th to 180 th amino acid sequences on PDGF-B chain in 1992 to obtain 18 independent polypeptide chains, and then used radiolabel to determine the binding capacity of the polypeptide chains with PDGF receptor, and finally screened two sequences, namely 116 th 121 amino acid sequence ANFLVW and 157 th 163 amino acid sequence EIVRKKP, which are directly related to the receptor binding. They then simply ligated the two fragments together to synthesize ANFLVWEIVRKKP directly, the 13 peptide. Unfortunately, it was finally found that this is a PDGF receptor antagonist which does not cause receptor dimerization and autophosphorylation and thus has no biological activity. Up to 2007 Zamora et al synthesized PDGF agonists for the first time by selecting amino acid sequence VRKIEIVRKK at 153 th and 162 th on PDGF-B chain and a region RKRKLERIAR capable of binding heparin (heparin is capable of assisting growth factor binding to receptor), and further mimicking PDGF by adding cysteine C before the sequence and forming disulfide bonds using sulfhydryl groups on its side chain to promote polypeptide chain cyclization. The design is really skillful, but the final molecular weight of the sequence reaches 4497.63Da due to the longer sequence, and the preparation process is more complicated and the yield is greatly reduced because two cysteines are required to be connected into a ring. Although the finally synthesized polypeptide has stronger acting force with a PDGF receptor, the cell proliferation experiment of the finally synthesized polypeptide reflects that the finally synthesized polypeptide can reach the level equivalent to the biological activity of the PDGF protein under the concentration of 1 mu g/ml. In view of the above, it is not ideal to actually obtain a research concept that can mimic the biological activity of PDGF by simply connecting PDGF to a receptor-binding related sequence and/or mimicking a cyclic structure that plays an important role in PDGF-receptor binding.

The PDGF proteins that have been commercialized to date are mainly recombinant human platelet-derived factor (rhPDGF) extracted from Escherichia coli or yeast. However, it has a number of problems: the production cost is high, the production flow is difficult to be strictly controlled, the quality among batches is difficult to be ensured, and the industrialization is difficult; easy inactivation, harsh preservation conditions and need of cold chain transportation; because the molecular weight is too large and is difficult to be absorbed through the skin, the medicine can be only administered by intramuscular injection and intravenous injection, and meanwhile, the medicine is easy to degrade after being directly injected into the body, the tissue retention rate is low, the half life period is short, and the like, and the further practical application of the medicine is greatly limited by the problems.

Disclosure of Invention

In view of the above, the invention provides a polypeptide derivative, a nanofiber and applications thereof, wherein the polypeptide derivative can simulate the biological activity of a platelet derived factor, i.e., promote proliferation and migration of fibroblasts, and has the advantages of simple production method, small molecular weight, high yield, low cost and definite chemical structure of a product. The polypeptide derivative can be self-assembled in an aqueous solution to form nano-fibers, and can effectively repair skin damage caused by radiation.

Specifically, the method comprises the following technical scheme:

according to a first aspect of the present invention, there is provided a polypeptide derivative having a sequence of X-Phe-Phe-Gly-Val-Arg-Lys-Lys-Pro (hereinafter referred to as X-FFGVHRKKP) wherein the terminal group X is an aromatic group-containing terminal group, which can mimic the biological activity of platelet-derived factor. According to the common general knowledge in the art, the amino acid configuration is not limited, but is considered to be the L configuration.

Preferably, the terminal group X is Nap, Fbp, Car or Npx. Nap, Fbp, Car, Npx are common end groups known in the art. More preferably, the terminal group X is Nap.

Specifically, when the terminal group X is Nap, the structural formula of the short peptide is as follows:

the structural formula of the short peptide when X is Fbp is as follows:

when X is Car, the structural formula of the short peptide is as follows:

the structural formula of the short peptide when X is Npx is as follows:

the polypeptide derivative is synthesized by adopting a known FMOC-short peptide solid phase synthesis method. Specifically, the preparation method of the polypeptide derivative comprises the following steps (taking the terminal group X as Nap as an example):

(1) the C-terminus of the Fmoc-amino acid is bound to the resin;

(2) removing and washing the Fmoc protecting group;

(3) coupling the C end of the next Fmoc-amino acid with the N end of the amino acid or polypeptide on the resin, and washing;

(4) repeating the steps (2) to (3) until the last amino acid coupling is finished, removing the Fmoc protecting group, and washing;

(5) coupling and washing 2-naphthylacetic acid and the N end of the polypeptide on the resin;

(6) the polypeptide derivative is cut off from the resin to obtain a crude product;

(7) the crude product was purified using high performance liquid chromatography.

According to a second aspect of the present invention, there is provided nanofibers of said polypeptide derivative formed by heating and cooling an aqueous mixture of said polypeptide derivative. When the concentration of the aqueous mixture of the polypeptide derivative reaches millimolar scale, a supramolecular hydrogel can be formed. As is common knowledge in the art, a supramolecular hydrogel is a gel formed by non-covalent interactions between small molecule compounds with molecular weights less than 2000, which aggregate, self-assemble to give a network structure and encapsulate water molecules.

Further, the specific method for forming the nano-fiber by heating and cooling the water mixture of the polypeptide derivative comprises the following steps: adding the polypeptide derivative into a PBS (phosphate buffer solution) with the pH value of 5.0-9.0, adjusting the pH value of the solution to 6.0-7.0 by using a sodium carbonate solution or a hydrochloric acid solution, heating to boiling to completely dissolve the compound, and cooling to room temperature to obtain a mixture of the nanofiber containing the polypeptide derivative with the concentration of 5 nM-1 MuM.

According to a third aspect of the present invention, there is provided a use of the nanofiber for the preparation of a medicament for repairing skin damage.

The inventor finds that the polypeptide derivative can be combined with a PDGF protein receptor, stimulate a downstream signal path and promote cell proliferation and migration. The cell may be mouse fibroblast (NIH 3T3), Vascular Smooth Muscle Cell (VSMC), mouse myoblast (C2C12), Mesenchymal Stem Cell (MSC).

The drug may be treated by intramuscular injection as is commonly used in the art. Meanwhile, since the polypeptide derivative of the present invention has a small molecular weight, it can be treated by transdermal absorption according to common knowledge.

The technical scheme provided by the embodiment of the invention has the beneficial effects that at least:

1. the preparation process is simple, the chemical structure of the product is definite, the used raw materials are all amino acids necessary for human bodies every day, the polypeptide derivative can be prepared by a solid-phase synthesis method, the molecular weight is small, the production process is simple, the yield is high, the cost is low, and the biocompatibility is good;

2. the polypeptide derivative can be combined with a PDGF protein receptor, excite a downstream signal channel and promote the proliferation and migration of mouse fibroblasts;

3. the polypeptide derivative can effectively repair skin injury caused by radiation, and experimental results show that the treatment effect is unexpectedly superior to PDGF protein. The inventor analyzes that the microstructure of the polypeptide derivative nanofiber possibly contributes to the result, the nanofiber microstructure can be slowly released in vivo, the degradation of protease in vivo is effectively resisted, the in vivo half-life period of the nanofiber is remarkably prolonged, and the tissue retention rate is improved.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.

FIG. 1 is a high resolution mass spectrum of Nap-FFGVRKP.

FIG. 2 is a graph showing the determination of binding constants of Nap-FFGVRKP and PDGF receptor protein.

FIG. 3 is a graph showing the effect of Nap-FFGVRKP in promoting the proliferation of mouse fibroblasts (NIH 3T 3).

FIG. 4 is a graph showing the effect of Nap-FFGVRKP in promoting the migration of mouse fibroblasts (NIH 3T 3).

FIG. 5 is a graph showing the effect of Nap-FFGVRKP in repairing radiation skin damage in mice.

Detailed Description

In order to make the technical solutions and advantages of the present invention clearer, the following will describe embodiments of the present invention in further detail with reference to the accompanying drawings.

According to a first aspect of the invention, the invention provides a polypeptide derivative which can simulate the biological activity of platelet derived factor, the sequence of the polypeptide derivative is X-FFGVRKP, wherein, the terminal group X is an aromatic group-containing terminal group.

Specifically, when X is Nap, the structural formula of the short peptide is as follows:

the structural formula of the short peptide when X is Fbp is as follows:

when X is Car, the structural formula of the short peptide is as follows:

the structural formula of the short peptide when X is Npx is as follows:

(1) the C-terminus of the Fmoc-amino acid is bound to the resin;

(2) removing and washing the Fmoc protecting group;

The sources of the formulations referred to in the following examples are as follows:

the 2-Cl-Trt resin is purchased from Tianjin Nankai and science and technology Limited and has the activity of 1.2 mmol/mL;

n, N-diisopropylethylamine (DIEPA below) was purchased from Adamas, Inc. (Adamas) with a purity of 99%;

benzotriazole-N, N, N ', N' -tetramethyluronium hexafluorophosphate (hereinafter HBTU) was purchased from Sigma Aldrich (Sigma-Aldrich) with a purity of 98%;

trifluoroacetic acid (hereinafter indicated as TFA), purchased from Sigma Aldrich (Sigma-Aldrich), 99% pure;

triisopropylsilane (hereinafter referred to as TIS), purchased from Sigma Aldrich (Sigma-Aldrich) with a purity of 99%;

all amino acids were purchased from gill biochemical (shanghai) ltd with a purity of 98%;

EdU staining kit, 1% crystal violet staining solution, hematoxylin-eosin staining solution, and mahalanobis staining solution were purchased from Sigma Aldrich (Sigma-Aldrich);

DMEM high-sugar medium and fetal bovine serum were purchased from Tianjin Zhi Biotech limited;

mouse fibroblast cells NIH3T3, purchased from santa haiensis;

6-8 week old female Balb/c mice, all purchased from Beijing Wittiulihua laboratory animal technology, Inc.;

PDGF protein, purchased from Acro biosystems, 98% pure.

Preparation example 1:

synthesis and preparation of polypeptide derivative Nap-FFGVRKP and its nano fiber

(1) Solid phase synthesis of Nap-FFGVRKP

The method comprises the following specific steps:

1) weighing 0.5mmol of 2-Cl-Trt resin in a solid phase synthesizer, adding 10mL of anhydrous dichloromethane (hereinafter represented by DCM), placing on a shaking table, shaking for 5min, and fully swelling the 2-Cl-Trt resin;

2) removing DCM from the solid phase synthesizer containing 2-Cl-Trt resin by washing the ear with an ear bulb;

3) dissolving 0.75mmol of Fmoc-Pro in 10mL of anhydrous DCM, adding 0.75mmol of DIPEA, then transferring to the solid phase synthesizer, supplementing 0.75mmol of DIPEA, and reacting for 1h at room temperature;

4) and (3) sealing: removing reaction liquid in the solid phase synthesizer by using an aurilave, washing with 10mL of anhydrous DCM for 1min each time for 5 times, adding 20mL of prepared solution with the volume ratio of anhydrous DCM to DIPEA to methanol being 17: 1: 2, and reacting at room temperature for 10 min;

5) removing reaction liquid in the solid phase synthesizer by using an aurilave, washing by using anhydrous DCM for 5 times, washing by using N, N-dimethylformamide (hereinafter referred to as DMF) for 10mL each time for 1min, washing for 5 times, adding 10mL of DMF containing 20% by volume of piperidine for reaction for 25min, reacting by using 10mL of DMF containing 20% by volume of piperidine for 5min, washing by using DMF for 1min, washing for 5 times, and carrying out next reaction by using 10mL of DMF for 10mL each time for 1 min;

6) adding Fmoc-Lys 1mmol, HBTU 1.5mmol, DIPEA 2mmol and 10mLDMF, adding the prepared solution into the solid phase synthesizer, and reacting for 2 h;

7) repeating the steps 5) and 6), sequentially adding Fmoc-Lys, Fmoc-Arg, Fmoc-Val, Fmoc-Gly, Fmoc-Phe and 2-naphthylacetic acid, washing with DMF for 5 times, washing with DCM for 5 times, and carrying out the next reaction;

8) as 95% TFA, 2.5% TIS, 2.5% H₂Adding 10mL of solution consisting of O in volume percent into the solid phase synthesizer, reacting for half an hour (or preparing a TFA solution with the volume percent concentration of 1% by volume by using the TFA and DCM in a volume ratio of 1: 99), adding 3mL of the TFA solution into the solid phase synthesizer every time, adding ten times for 1min each time, cutting the product from the 2-Cl-Trt resin, concentrating in vacuum, removing the solvent to obtain a crude product, and then separating and purifying by using HPLC (high performance liquid chromatography) to obtain the polypeptide derivative Nap-FFGVRKP with the yield of 80%. FIG. 1 is a high resolution mass spectrum of Nap-FFGVRKP. The molecular weight of Nap-FFGVRKP obtained by calculating a molecular structure drawing software Chemdraw is 1146.3821, the molecular weight of a main peak shown in a figure is 1146.6449, the difference with the calculation result is less than 1, the results show that the molecular weight of the compound synthesized by using the method is consistent with the calculated molecular weight, and the successful synthesis of Nap-FFGVRKP is proved.

(2) Preparation of nanofibers of polypeptide derivatives

Weighing 1.0mg of purified polypeptide derivative Nap-FFGVRKP, placing in a 2mL glass bottle, adding 500 μ L of PBS solution (pH 7.4), adjusting pH to 7.4 with sodium carbonate solution, heating to boil to completely dissolve the compound, and cooling to room temperature to obtain colorless transparent invertible hydrogel. Whether or not the gel is formed is determined by methods known in the art, such as by inverting the bottle, leaving a hydrogel at the bottom of the bottle and a liquid if flowable.

Weighing 1.1mg of purified polypeptide derivative Nap-FFGVRKP, placing in a 2mL glass bottle, adding 1mL of PBS solution (pH 7.4), adjusting the pH value to 7.4 with sodium carbonate solution, heating to boiling to completely dissolve the compound, sucking 1 μ L of the compound with a microsampler, adding into 999 μ L of the PBS solution, heating to boiling to completely dissolve the compound to obtain 1 μ M Nap-FFGVRKP solution; mu.L of Nap-FFGVRKP solution with a concentration of 1. mu.M was pipetted into 995. mu.L of PBS and heated to boiling to completely dissolve the compound, thus obtaining 5nM Nap-FFGVRKP solution. The electron micrograph shows that the internal structures of the Nap-FFGVRKP solutions with the concentrations of 1 mu M and 5nM are all nanofibers.

The binding constant of the polypeptide derivative Nap-FFGVRKP and PDGF receptor protein is measured by adopting a micro-scale thermophoresis method, and the PDGF receptor protein is marked by using a single-molecule NT protein marking kit and a fluorescent dye NT-647. PBS buffer containing 0.05% Tween-20 (pH 7.4) was used as the detection buffer. The concentration of the PDGF receptor protein with the fluorescent label was kept constant at 10. mu.M, while the concentration of the hydrogel formed by Nap-FFGVRKP was diluted from 2. mu.M to 0.054nM to give a series of concentration-gradient solutions. The fluorescent protein solution was then mixed with solutions of different concentrations at a 1:1 volume ratio. After 1 minute of incubation, the samples were loaded into nt.115 standard glass capillaries and analyzed using nt.115 monomer system. KD values were calculated using the nanotemperer software package. FIG. 2 is a graph showing the determination of binding constants of Nap-FFGVRKP and PDGF receptor protein. The binding constant shown in the figure is 117.96nM, indicating specific binding of Nap-FFGVRKP to the PDGF receptor protein.

Comparative preparation example 1

Sterile 1 XPBS

Weighing 8g NaCl, 0.2g KCl and 1.44g Na₂HPO₄And 0.24g KH₂PO₄Dissolving in 800ml of distilled water, adjusting the pH of the solution to 7.4 with HCl, and addingAnd (5) adding distilled water to a constant volume of 1L. Sterilizing with autoclave, and storing in refrigerator at room temperature or 4 deg.C.

Comparative preparation example 2

(1) Referring to preparation example 1, a control polypeptide derivative Ac-Val-Arg-Lys-Lys-Pro (hereinafter abbreviated as Ac-VRKKP) was synthesized by FMOC-short peptide solid phase synthesis: except that Fmoc-Lys, Fmoc-Arg, Fmoc-Val, Fmoc-Gly, Fmoc-Phe and 2-naphthylacetic acid are added in sequence in the step 7) of solid phase synthesis in the step (1), and Fmoc-Lys, Fmoc-Arg, Fmoc-Val and acetic anhydride are added in sequence instead.

(2) 0.66mg of purified control polypeptide derivative Ac-VRKKP was weighed into a 2mL glass vial, 1mL of PBS solution (pH 7.4) was added, the pH was adjusted to 7.4 with sodium carbonate solution, the solution was boiled to completely dissolve the compound, and after cooling down the vial was inverted to find a solution that did not form a hydrogel but flowed, to obtain a 1mM Ac-VRKKP solution. Sucking 1 mu L of Ac-VRKKP solution with the concentration of 1mM by using a micro-sampler, adding the Ac-VRKKP solution into 999 mu L of PBS solution, and heating to boil to completely dissolve the compound to obtain 1 mu M Ac-VRKKP solution; and (3) sucking 5 mu L of Ac-VRKKP solution with the concentration of 1 mu M by using a microsampler, adding the solution into 995 mu L of PBS solution, and heating to boil to completely dissolve the compound to obtain 5nM Ac-VRKKP solution.

Comparative preparation example 3

1.23mg of PDGF protein powder was weighed into a 2mL glass bottle, and 1mL of PBS solution (pH 7.4) was added thereto, and the pH was adjusted to 7.4 with sodium carbonate solution to obtain a PDGF protein solution with a concentration of 50 μ M. Sucking 20 μ L PDGF protein solution with concentration of 50 μ M with a microsampler, adding into 980 μ L PBS solution, heating to boiling to completely dissolve the compound to obtain 1 μ M PDGF protein solution; mu.L of a PDGF protein solution at a concentration of 1. mu.M was pipetted into 995. mu.L of PBS using a microsampler and heated to boiling to completely dissolve the compound, thereby obtaining a PDGF protein solution of 5 nM.

Cell example 1:

activity test of nanofiber of polypeptide derivative Nap-FFGVRKP at cell level

(1) Fibroblast proliferation assay

1) Heating the water bath to 37 deg.C in advance, preheating culture medium and serum in the water bath, and simultaneously turning on ultra-clean bench ultraviolet lamp to irradiate for half an hour;

2) the frozen mouse fibroblasts NIH3T3 were removed from the liquid nitrogen tank, immediately placed in a 37 ℃ water bath to thaw the cells, and then immediately transferred to a clean bench for the following operations: carefully transferring the solution containing the cells into a centrifuge tube containing a culture medium by using a pipette, centrifuging for 3min, removing a supernatant, resuspending the cell in a DMEM culture medium containing 10% fetal calf serum, transferring the cell into a culture dish, and then putting the cell into an incubator at 37 ℃ for culture;

3) observing the cell state on the next day, and carrying out the following experiment after the cell state is good and first generation;

4) adding 2mL pancreatin, gently shaking, uniformly covering, tapping the bottom of the culture dish, digesting at 37 ℃ for 3min, observing most cell suspension under a microscope, blowing and beating 5-10 times by using a 1mL gun, adding 2mL culture medium, uniformly blowing and beating, collecting cell culture solution, sucking into a centrifuge tube, centrifuging at the rotating speed of 1000rpm for 3min, then discarding supernatant solution, adding DMEM culture medium containing 10% fetal calf serum, uniformly blowing and beating by using a gun, counting by using a cell counting plate to obtain the cell suspension containing 5 multiplied by 10 per milliliter⁷And (4) cells.

5) The cells were resuspended in 96-well plates and 100. mu.L of DMEM medium containing 10% fetal bovine serum per well, and cultured overnight in a 37 ℃ incubator. The following day, the DMEM medium was aspirated and replaced with serum-free DMEM medium, starvation was performed for 4h, 100. mu.L of serum-free DMEM medium containing different concentrations of Nap-FFGVRKKKP was added to each well, and 1ml of serum-free DMEM medium without any polypeptide derivative was added to the Control group. After incubation in an incubator at 37 ℃ for 48h, 10. mu.L of CCK-8 and 90. mu.L of serum-free DMEM medium were added to each well, and after 4h, OD at 450nm was measured using a microplate reader.

FIG. 3 is a graph showing the effect of Nap-FFGVRKP in promoting the proliferation of mouse fibroblasts (NIH 3T 3). In the figure, the Control bar graph represents the cell number (set as 100%) of NIH3T3 after 48 hours of culture in the cell culture medium without Nap-FFGVRKP, and the other bar graphs represent the cell number (percentage of Control group) of NIH3T3 after 48 hours of culture in the medium with different concentrations of Nap-FFGVRKP, and the cell number of NIH3T3 in the medium with Nap-FFGVRKP is larger than that in the Control group in the concentration range of 1nM to 100nM, which indicates that Nap-FFGVRKP has the function of promoting the proliferation of NIH3T 3.

(2) Experiment for promoting mouse fibroblast migration by PDGF self-assembled polypeptide hydrogel

1) After repeating the above steps 1) -4) of the cell proliferation experiment, the cells were resuspended in six-well plates, 10 ten thousand cells per well and 1mL of DMEM medium containing 10% fetal bovine serum, and cultured overnight in an incubator at 37 ℃.

2) On the next day, scratches were manually made by marking the same width line on the bottom of the well plate straight with a pipette tip, the cells were starved for 12 hours with a serum-free medium, 1mL of each serum-free DMEM medium containing 5nM of Ac-VRKKP, Nap-FFGVRKKP, and PDGF protein was added, and 1mL of a serum-free DMEM medium containing no polypeptide derivative and no serum was added to the Control group. After further culturing for 16h, the cells were stained with 1% crystal violet, and the cells were observed for migration and growth to the scratched area.

3) And randomly selecting 3 visual fields for photographing in each group, counting the cells migrated into the scratch, and counting the difference among the groups.

FIG. 4 is a graph showing the effect of Nap-FFGVRKP in promoting the migration of mouse fibroblasts (NIH 3T 3). After further culturing in a serum-free medium for 16 hours after the scratches were artificially made, the cells were stained with 1% crystal violet, and the cells were observed for migration and growth into the scratched area. In the figure, a Control bar graph represents the cell migration number of NIH3T3 after being cultured for 16 hours in a serum-free cell culture medium, other bar graphs sequentially represent the cell migration number after being cultured for 16 hours in a serum-free culture medium containing Ac-VRKKP, Nap-FFGVRKKKP and PDGF protein of 5nM, the migration number of NIH3T3 in a culture medium containing Nap-FFGVRKKKP can be found to be the maximum, and the function of promoting the migration of NIH3T3 by the Nap-FFGVRKKKP is shown.

Damage repair example 1:

PDGF self-assembled polypeptide hydrogel repair experiment on radiation skin injury

1) Establishing a model: a female BALB/c mouse of 6-8 weeks old is selected, 150 mu L of 4% chloral hydrate is narcotized, and the left hind limb skin is exposed to 40Gy X-ray irradiation at the dose rate of 2Gy/min for 20min (biological X-ray irradiator, Rad source, RS 2000).

2) Administration treatment and statistics: after successful modeling, the mice are randomly divided into four groups, 10 mice in each group, 100 mu L of solution containing 1 mu M of Ac-VRKKP, Nap-FFGVRKKKP and PDGF protein is respectively injected into the injured skin of the mice in different groups in situ, and 100 mu L of PBS is added into the Control group. And then observing the state of the damaged part every week, taking a picture, regularly monitoring the inflammation change condition of the surface by using a laser Doppler ultrasonic instrument, and counting the skin repair condition of the mouse. The mice in the non-irradiated group were not irradiated with X-rays, nor were they injected intramuscularly with any solution such as polypeptide or PBS.

3) Tissue section staining: after the experiment on the 60 th day, all mice are killed after neck removal, the skin at the damaged part is selected to prepare paraffin sections, the repairing condition of the damaged epidermis layer and the dermis layer of the skin is observed by hematoxylin-eosin staining, the synthesis and deposition condition of collagen fibers is observed by mahalanobis staining, and the inflammation condition is observed by CD68 labeled macrophage immunofluorescence staining.

FIG. 5 is a graph showing the effect of Nap-FFGVRKP in repairing radiation skin damage in mice. On day 0 of radiation injury, each group of mice was treated by in situ injection of 1 μ M of Ac-VRKKP, Nap-FFGVRKP, PDGF protein, followed by regular monitoring of surface blood perfusion using a laser Doppler ultrasound. Inflammation is a defense reaction of living tissues with vascular systems to injury factors, and vascular reaction is a central link of an inflammation process, so the degree of blood perfusion on the surface of skin can reflect the strength of inflammation. It can be seen from the figure that certain inflammation conditions are appeared at day 18 except for the non-irradiated group, the difference between the groups is small, the inflammation of the Control group is further increased at day 30, but the inflammation of the Nap-FFGKRKP group is greatly relieved, the difference between the non-irradiated group and the Nap-FFGKRKP group is small, the effect is even unexpectedly better than that of the PDGF group, and the Nap-FFGRKKP can effectively repair the radiation skin injury of the mice.

The above description is only for facilitating the understanding of the technical solutions of the present invention by those skilled in the art, and is not intended to limit the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A polypeptide derivative capable of simulating the biological activity of platelet-derived factor, wherein the sequence of the polypeptide derivative is X-Phe-Phe-Gly-Val-Arg-Lys-Lys-Pro, and the terminal group X is Nap.

2. A nanofiber comprising the polypeptide derivative of claim 1, wherein said nanofiber is formed by heating and cooling an aqueous mixture of said polypeptide derivative.

3. The nanofiber according to claim 2, wherein the nanofiber is formed by heating and cooling the aqueous mixture of the polypeptide derivative by the following specific method: adding the polypeptide derivative into a PBS (phosphate buffer solution) with the pH = 5.0-9.0, adjusting the pH value of the PBS to 6.0-7.0 by using a sodium carbonate solution or a hydrochloric acid solution, heating to boiling to completely dissolve the compound, and cooling to room temperature to obtain a mixture of the nanofiber containing the polypeptide derivative with the concentration of 5 nM-1 mu M.

4. Use of the nanofibers according to claim 2 or 3 in the preparation of a medicament for repairing skin damage.