CN108503690B - Repair peptide for promoting tissue repair and regeneration after trauma and application thereof - Google Patents

Abstract

The invention discloses a repair peptide for promoting tissue repair and regeneration after trauma and application thereof, belonging to the field of biological medicine. The repair peptide is a linear peptide or cyclic peptide containing 4 continuous GPANVET core sequences and more than 4-15 amino acid lengths. The repair peptide provided by the invention has good effects of promoting skin wound repair and nerve injury repair, has small molecular weight, is greatly reduced in degradation degree compared with recombinant proteins which can be used for skin wound repair and nerve injury repair, such as growth factors like bFGF, NGF and BDNF, and the like, and is lower in required production cost and use cost, thereby being a product for promoting skin wound repair and nerve injury repair with a prospect. Moreover, the experimental results disclosed by the invention prove that the repair peptide can continuously act on the treatment part, so that the repair peptide has the technical advance.

Description

Repair peptide for promoting tissue repair and regeneration after trauma and application thereof

Technical Field

The invention belongs to the field of biological medicine, and particularly relates to a repair peptide for promoting tissue repair and regeneration after trauma and application thereof, which can promote tissue repair and cell proliferation at trauma parts, particularly application in tissue regeneration and repair in the aspects of skin injury and nerve injury.

Background

Many factors can cause damage to cells and tissues. After injury, the surviving healthy cells at and near the tissue site continue to divide and proliferate to replace dead cells and repair the damaged tissue, and this physiological function of the organism is called regeneration and repair.

Normally, the repair and regeneration abilities of different organs of the human body are different. Epidermal cells (e.g., mucous membrane-coated epithelia of respiratory tract, digestive tract, and genitourinary tract), lymphocytes, hematopoietic cells, and the like are highly regenerative cells, and these cells undergo aging and neogenesis at all times, and have a strong regenerative repair ability to cope with damage, and thus are highly regenerative cells. And other cells such as vascular endothelial cells, periosteal cells, cells of various glandular organs such as liver, pancreas, endocrine glands, sweat glands, sebaceous glands, renal tubular epithelial cells and the like belong to secondary cells with strong regenerative power, and can also show certain regenerative capacity when being damaged. The method is specifically reflected in that when the normal cells of local tissues are destroyed, the residual similar cells divide and supplement; if the local cells are completely destroyed and the cells are completely necrotic, the site cannot be repaired. In addition, cells with weak or no regeneration ability, such as central nerve cells and ganglion cells, are weak in regeneration and extremely difficult to recover the original functions after being damaged; the regeneration capability of the myocardial cells is extremely weak, and the damaged myocardial cells are replaced by fibrous connective tissues, so that the original structure and function are difficult to recover.

There are many factors that affect tissue regeneration. In addition to the regenerative capacity of tissues and cells themselves, there are several factors: (1) degree and extent of tissue damage: the larger the size, the longer the repair and regeneration time, because after extensive cell death, it is difficult to replace a considerable number of the same cells, healthy cells are required to produce new cells, and the time required for the new cells to regenerate into new cells is long. (2) Age factors: the tissue regeneration capacity of children and teenagers is strong, and the wound is healed quickly; the old people have weak tissue regeneration capability and slow healing. (3) The nutritional status: adequate protein, vitamin C, E and mineral calcium and zinc supplies promote repair of injuries and, in turn, delay repair of various injuries. (4) Drug effects: some drugs such as adrenocortical hormone and pituitary corticotropin can inhibit inflammation, but are not beneficial to the body to eliminate wound infection, and can also inhibit granulation tissue growth and collagen synthesis, and accelerate collagen decomposition; also, cytotoxic drugs in anticancer drugs can delay healing. (5) Blood supply: after tissue injury, local capillaries are also damaged, or angiosclerosis and the like are caused by other factors, which can cause insufficient blood supply, cause tissue malnutrition and prevent healing. (6) Innervation: tissue that is denervated can lose its ability to regenerate.

From the above, after the tissue of the body is lost, in addition to objective factors such as age and the like which cannot be intervened by the outside, the repair of the tissue can be accelerated by increasing the nutrition supply of the damaged part, intervening by medicines to promote the proliferation of the survival cells, promoting the neogenesis of the capillary vessels and the regeneration of nerve cells, and the like.

The following three major application areas from current tissue repair: the wound repair of skin, nerve and bone tissue is described in more detail:

first, wound repair of skin tissue

The skin is the largest area of the human body, is the most directly contacted tissue with the outside, and is the organ which is most easily damaged by the external stimulation. The healing of normal wound is important for maintaining the body homeostasis, and abnormal healing such as non-healing or late healing caused by large-area burns and scalds, healing of chronic wounds caused by other diseases, abnormal healing such as the formation of pathological scars and the like not only affect the beauty, but also cause structural and functional disorders, often cause heavy psychological stress on patients and seriously affect the physical and mental health of the patients. Therefore, how to rapidly achieve the best healing effect in the treatment of skin wounds is a hot point of research.

The healing process of the skin is a complex and orderly tissue repair and regeneration process, which is a process completed by the combined action of a plurality of cells and cytokines, and can be roughly divided into an inflammatory reaction phase, a proliferation phase and a remodeling phase, wherein the three phases are intersected with each other without strict boundaries, and a large number of cells and cytokines are involved in the healing process, the cells involved in the healing process comprise keratinocytes, Vascular endothelial cells, fibroblasts, neutrophils, monocytes/macrophages, mast cells and the like, the cytokines involved in the healing process comprise Fibroblast Growth Factors (FGF), Vascular endothelial Growth factors (Vascular endothelial Growth factors, VEGF), platelet-derived factors (planar induced Growth factors, PDGF), Tumor necrosis Factor- α (Vascular endothelial Factor- α - α), Transforming Growth factors (transformingfactor, TGF-like), Insulin-like Growth factors (Insulin-like Growth factors, IGF) and various factors involved in healing processes of the skin, the healing process of the skin, the skin diseases caused by the abnormal blood ionizing, the abnormal Growth factors, the abnormal healing process of the blood coagulation, the skin diseases and the like, and the skin diseases caused by the abnormal radiation.

At present, clinically, different medicines and treatments are mainly performed on acute skin soft tissue wounds and chronic skin soft tissue wounds for skin wound repair. Based on the advantages of the traditional Chinese medicine in China, a plurality of simple prescriptions such as rehabilitation new liquid emerge in the field of wound repair, and a compound prescription is prepared by matching four or five traditional Chinese medicines with less prescriptions and twenty thirty traditional Chinese medicines with more prescriptions, such as 'moist burn ointment', 'Jingwanhong' and the like for treating burns and scalds. After modern medicine development, particularly after the development of biological medicines with intervention of genetic engineering technology, the production of recombinant Growth factors for promoting cell Growth is also the vitality for the development of wound repair medicines, for example, a biological technology medicine which is clinically applied to wounds, burns and scalds and the like, namely, external recombinant basic fibroblast Growth Factor (bFGF), which is the second new gene engineering class I medicine in China (note: basic fibroblast Growth Factor is also called fibroblast Growth Factor-2, FGF 2).

In the aspect of polypeptide drug development of skin repair, the inventor's Kunming animal research institute in the department of sciences in 2014 reports that a potent skin repair peptide Tylotoxin derived from lizards contains 12 amino acid residues, shows strong wound healing promotion activity, can promote proliferation and migration of keratinocyte epithelium and fibroblast, and can also promote generation of TGF-b and IL-6. Also, Chinese patent inventions disclose that polypeptides capable of promoting skin repair, which are 16 peptides, 13 peptides, 24 peptides and 11 peptides, are extracted from Rana huananensis, newt scrofula and Rana grahami, respectively.

Second, wound repair of nerve injury

The nervous system is a functional regulatory system that plays a leading role in the human body. The functions of various organs and systems of the human body and various physiological processes are mutually related, mutually influenced and closely matched under the direct or indirect regulation and control of a nervous system, so that normal life activities are realized and maintained. Meanwhile, the nervous system of the human body can sense the change of the external environment, receive the change information of the internal and external environments, and continuously and rapidly and perfectly adjust various functions in the human body, so that the human body adapts to the change of the internal and external environments of the human body.

The nervous system is composed of a central part and its peripheral parts. The central part comprises brain and spinal cord, which are respectively located in cranial cavity and vertebral canal, and the cranial cavity and the vertebral canal are closely connected in structure and function to form a central nervous system. The peripheral portion includes 12 pairs of cranial nerves and 31 pairs of spinal nerves, which make up the peripheral nervous system. Peripheral nerves are distributed throughout the body, linking the brain and spinal cord with other organs throughout the body, so that the central nervous system can sense the changes of internal and external environments (transmitting sensory information through afferent nerves), and can regulate various functions in the body (transmitting regulating instructions through efferent nerves), thereby ensuring the integrity and unity of the human body and the adaptation to the environment.

The central nervous system composed of the human brain and spinal cord lacks the ability to self-regenerate and repair, and there is no effective treatment means at present due to cell death, tissue destruction and permanent loss of nerve function caused by the injury.

Peripheral nerve lesions are divided into open lesions and non-open lesions. The former is generally associated with open lesions of the soft tissue, causing partial or total truncation of the nerve; the latter is associated with blunt, non-open injury to soft tissues, causing contusion, compression or stretch of nerve trunks, small bleedings and edema, myelin edema and degeneration within the nerve. The result of peripheral nerve injury is clinically manifested mainly as nerve paralysis, sensory disturbance, dyskinesia, muscular atrophy, and the like in the region innervated by the nerve.

The general drugs for nerve injury in clinic are: a nerve growth factor enhancer: 1. prionin potassium, a cognitive enhancer, is used in the treatment of mild and moderate Alzheimer's Disease (AD), enhances neuronal cell function by increasing the level of neurotrophic growth factors in damaged or degenerated neurons, stimulates axonal growth, improves memory, and is the first drug to enter phase iii clinical trials for enhancing nerve regeneration. 2. Acetyl L-carnitine: is a cholinergic agonist, can actively pass through a blood brain barrier, can protect central and peripheral nerve synapses in a neurodegenerative and aging model, improve the level of nerve growth factors and improve cognitive defects of old rats, and is currently in phase III clinical trials in the United states. Second, the neurotrophic medicines are provided; 1. gangliosides (GM1) promote nerve remodeling including nerve cell survival, axon elongation and synaptic growth, and play important roles in cell differentiation, development, nerve tissue repair, neuronal plasticity, etc. 2. The brain protein hydrolysate is a new medicine for improving brain metabolism, is easy to penetrate through blood brain barrier to enter brain nerve cells, influences protein synthesis and respiratory chain, enhances the oxidation resistance of the brain cells, protects the nervous system from being damaged by toxic substances, delays death of the brain cells and promotes survival of the brain cells. 3. The cytidine disodium triphosphate injection is a nucleotide medicine, can promote protein synthesis, regulate and promote synthesis and construction of cell membranous structures of nerve cells, glial cells and vascular walls, and can resist nerve cell damage caused by excitatory amino acids and free radicals, so that the cytidine disodium triphosphate injection has the functions of supporting survival, enhancing activity, delaying death, improving cell damage resistance and repair capacity, promoting nerve axon regrowth, improving the nerve function of blood vessels and resisting angiosclerosis. 4. Oxiracetam is a novel central nervous system drug capable of promoting learning and memory ability, can selectively act on cerebral cortex and hippocampus, and can activate, protect or promote the functional recovery of nerve cells.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention mainly aims to provide a small molecule polypeptide for promoting the tissue repair and regeneration after trauma, which is named as repair peptide. The invention provides a small molecular polypeptide consisting of 4-15 amino acid residues, wherein the polypeptide is a plurality of linear peptides and cyclic peptides derived based on a sequence GPANVET, and the linear peptides and the cyclic peptides are temporarily named as repair peptides, and the polypeptide sequence is obtained by taking the outer membrane segment of a fibroblast growth factor receptor 2(FGFR2) as a target spot through a phage peptide library (7 peptide library) and carrying out multi-round screening and identification. No document reports that the sequence polypeptide can be used for repairing skin wounds and nerve injuries.

Another object of the present invention is to provide the use of the above-mentioned repair peptide in the repair and regeneration of skin wounds and nerve injuries.

The purpose of the invention is realized by the following technical scheme:

the invention aims to provide a repair peptide for promoting tissue repair and regeneration after trauma, which contains 4 continuous linear peptides or cyclic peptides with amino acid length of 4-15 or more of GPANVET core sequence.

Specifically, the R1 peptide sequence of the repair peptide is GPANVET,

the sequence of the R2 peptide is KVKVGPANVKVKV,

the sequence of the R3 peptide is GPANVEKEKEK,

the peptide sequence of R4 is CGPANVETC.

Wherein the R1 peptide contains only the core sequence gpanet; the R2 peptide contains 5 consecutive amino acids of the core sequence GPANV; the R3 peptide contains the 6 consecutive amino acids gpgave of the core sequence; the R4 peptide contains all 7 amino acids of the core sequence and forms a cyclic peptide through the disulfide bond of two cysteines at both ends.

The repair peptides can be obtained by adopting a chemical synthesis method.

The invention aims to provide application of the repair peptide in preparing products for tissue regeneration and repair in the aspects of skin injury and nerve injury, in particular to application in preparing products for nerve repair and regeneration of central nerve injury caused by skin trauma, burn and scald, chronic wound healing, skin cell repair and regeneration, cerebral trauma, cerebral apoplexy, cerebral edema, cerebral anoxia and the like, sensory disorder, dyskinesia and dystrophy caused by peripheral nerve injury.

The product is preferably a medical product, a skin care product or a cosmetic product.

The formulation type of the product is preferably, but not limited to, a solution, a lyophilizate, an emulsion, a cream, a gel, a mask or a dressing, and the like.

The invention relates to an experiment on animal models of mouse fibroblast balb/c 3T3, human vascular endothelial cell HUVEC, chick embryo allantoic membrane, mouse and rat.

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

compared with the prior similar product of externally used recombinant bFGF (comprising 155 amino acids), the repair peptide provided by the invention can be combined with a membrane receptor FGFR2 to play a role, has equivalent effects of promoting skin wound repair and nerve injury repair and regeneration, but has smaller molecular weight, and adopts a chemical synthesis method rather than a genetic engineering method, so that the required production cost and use cost are lower under the use condition of the same molar concentration, and the repair peptide is a good product for replacing the wound repair and cell regeneration and proliferation of the recombinant bFGF. The recombinant bFGF has harsh in-vitro storage and transportation conditions and is easy to degrade, so that the accumulation of a treatment part is low, and the sustained action is difficult, so that the use and the development of related dosage forms are limited and influenced to some extent.

Drawings

FIG. 1 is a graph showing the results of ITC method for detecting the affinity of repair peptides R1 and bFGF for FGFR 2.

FIG. 2 is a diagram showing the results of CCK-8 method for detecting the cell proliferation promoting effect of repair peptides R1, R2, R3, R4 and bFGF on mouse fibroblast Balb/c 3T 3.

FIG. 3 is a diagram showing the results of CCK-8 assay for the cell proliferation-promoting effect of repair peptides R1, R2, R3, R4 and bFGF on Human Umbilical Vein Endothelial Cells (HUVEC).

FIG. 4 is a graph showing the results of repair peptide R1 promoting angiogenesis in chick embryo allantoic membrane.

FIG. 5 is a graph showing the results that repair peptides R1, R2, R3 and R4 promote angiogenesis in chick chorioallantoic membrane at the same concentration.

FIG. 6 is a graph showing the results of experiments on the repair peptides R1, R2, R3 and R4 at the same concentration for promoting the healing of the skin wound of rats

FIG. 7 is a schematic diagram of the establishment of a rat dorsal root nerve injury model and drug therapy.

Fig. 8 is a graph of the withdrawal thresholds of rats to mechanical stimulation measured after repair peptides R1, R2, R3, R4 repaired dorsal root ganglion injury: the ordinate represents the threshold for the number of times the right hand lifts the paw compared to the left hand. A larger value indicates a greater number of paw lifts, a greater sensitivity to mechanical pain stimuli and a better recovery of sensory function. The label sham is a sham operation group, PBS is a group treated by PBS solution after trauma, and R1, R2, R3 and R4 are groups to be detected and treated by different repair peptides.

Fig. 9 shows the recovery experiment of thermal pain stimulation in rats after repairing dorsal root ganglion injury by repair peptides R1, R2, R3 and R4: the ordinate represents the threshold for the right hand to paw lifting time compared to the left hand. Smaller values indicate a shorter time to lift the paw, more sensitive to thermal irritation and better recovery of sensory function. The label sham is a sham operation group, PBS is a group treated by PBS solution after trauma, and R1, R2, R3 and R4 are groups to be detected and treated by different repair peptides.

FIG. 10 shows the detection of neurite outgrowth 2 weeks after the dorsal root ganglion was injured by the treatment with the repair peptide by indirect immunofluorescence, using an antibody against the marker protein NF200 and a fluorescent secondary antibody. NF200 is a marker protein for nerve fibers.

FIG. 11 shows the detection of neurite outgrowth 2 weeks after the dorsal root ganglion was injured by the treatment with the repair peptide by indirect immunofluorescence, using an antibody against the marker protein Lamin and a fluorescent secondary antibody. Laminin is a marker protein of peripheral nerve fibers.

FIG. 12 shows the detection of neurite outgrowth 2 weeks after the dorsal root ganglion was injured by the treatment with the repair peptide by indirect immunofluorescence, using an antibody against the marker protein CGRP and a fluorescent secondary antibody. The CGRP proteins are involved in pain receptors that transmit pain.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

The experimental methods used in the following examples are all conventional methods unless otherwise specified, and the experimental materials, reagents and the like used in the examples are all commercially available unless otherwise specified. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1

1. ITC method for detecting affinity of repair peptide R1 and FGFR2

The results of affinity detection by isothermal microcalorimetry (ITC) are shown in fig. 1, and the affinity constant K of repair peptide R1 and FGFR2 is 7.35E8 ± 2.10E8M^-1Dissociation constant Kd 1.37nM, affinity constant of bFGF to FGFR2 is 1.62E5 + -2.50E 4M^-1The dissociation constant Kd ≈ 6.17. mu.M. The results show that the affinity of the repair peptide R1 to FGFR2 is much higher than that of bFGF to FGFR2, and the former is about 4500 times that of the latter. (K represents affinity constant, Kd represents dissociation constant, and the larger the K, the smaller the Kd, the stronger the affinity).

2. CCK-8 method for detecting proliferation promoting effect of repair peptides R1, R2, R3, R4 and bFGF on fibroblast Balb/c 3T3

According to the result, the repair peptides R1, R2, R3 and R4 have the proliferation promoting activity equivalent to that of bFGF in a lower concentration group, while the bFGF group has the cell proliferation inhibiting phenomenon in a high concentration group, but the repair peptides R1, R2, R3 and R4 still have the proliferation promoting activity, and the repair peptides and the bFGF can both promote the proliferation of fibroblasts, and the high-concentration proliferation inhibiting phenomenon does not occur, so that the action persistence is good.

3. CCK-8 method for detecting proliferation promoting effect of repair peptides R1, R2, R3, R4 and bFGF on Human Umbilical Vein Endothelial Cells (HUVEC)

HUVEC cells are taken and laid on a 96-plate according to 4000 cells per well, after starvation is carried out for 24 hours after wall attachment, repair peptides R1, R2, R3, R4 and bFGF are taken and treated according to the multiplication concentration of 5 × for 24 hours, then CCK-8 reagent is adopted for treatment, and an enzyme-labeling instrument is adopted to detect the OD value of each well, as shown in figure 3, in a lower concentration group (less than or equal to 0.16 mu M), the proliferation promoting activity of the repair peptides R1, R2, R3 and R4 is equivalent to that of the bFGF, while in a high concentration group, the bFGF has the phenomenon of cell proliferation inhibition, but the proliferation promoting activity of the repair peptides R1, R2, R3 and R4 still has the proliferation promoting activity.

4. The repair peptide R1 can promote the growth of blood vessels in chick embryo allantoic membrane

Taking a fertilized egg cultured for 5 days, removing an eggshell with a big head facing upwards, exposing an allantoic membrane of a chicken embryo, placing a silica gel ring in the middle, dripping 10 mu L of each R1 with the multiplication concentration of 25 × into the silica gel ring, sealing an opening by clean filter paper, setting a PBS solution treatment group as a blank control, culturing for 3 days, then stripping the allantoic membrane of the chicken embryo, observing under a stereoscope, taking a picture, and analyzing and processing the image by software ImageJ plus, as shown in figure 4, after the R1 is added, the capillary vessels in the allantoic membrane of the chicken embryo are obviously increased and are increased along with the increase of the dosage, which indicates that the R1 obviously promotes the generation of the blood vessels.

5. Detection of repair peptides R1, R2, R3 and R4 for promoting angiogenesis in chick embryo allantoic membrane

Taking fertilized eggs cultured for 5 days, removing eggshells with the big heads facing upwards, exposing allantoic membranes of the chicken embryos, dripping 100 mu L of repair peptides R1, R2, R3 and R4 with the concentration of 100ng/mL on the fertilized eggs, sealing an opening by clean filter paper, and setting a PBS solution treatment group as a blank control. And culturing for 3 days, stripping the chick embryo allantoic membrane, observing under a stereoscope, photographing, and analyzing and processing the image by using a software ImageJ plus. As shown in FIG. 5, the addition of repair peptides R1, R2, R3, and R4, respectively, resulted in a significant increase in capillary blood vessels in the chick allantoic membrane, increasing with increasing dose. Thus, the repair peptide can promote angiogenesis.

6. Detection of repair peptides R1, R2, R3 and R4 for promoting healing of rat skin wound

Balb/c mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (100mg/kg), the backs were dehaired and sterilized, and 1% iodophor and 75% alcohol were sterilized conventionally. Cutting 1cm long incision on two symmetrical sides, sterilizing surgical operation, suturing two needles, dripping 10 μ L of R1, R2, R3 and R4 peptides of the repair peptide on the wound, leaving the wound open without covering cotton yarn, and setting PBS solution treatment group as control group. Then, the solution was added dropwise once a day while observing wound healing. As shown in FIG. 6, the healing rate was increased by adding R1, R2, R3 and R4 peptides as repair peptides. The repair peptide obviously promotes the healing of skin wound and promotes the generation of blood vessel at the healing part.

7. Establishment of rat dorsal root nerve injury model

The model is used for detecting the influence of the drug on the recovery of the peripheral nerve sensory function of the rat.

As shown in FIG. 7, the rat spinal cord segment C5 to T1 was exposed surgically. The right dorsal root was clipped with forceps under equal force to make the dorsal root injured but not broken. The rats were then injected with the test drug daily near the lesion and observed for 3 weeks for 21 days.

8. Measurement of rat response to mechanical stimulation after repair of dorsal root ganglion injury with repair peptides R1, R2, R3, R4

The mechanical pain stimulation test is a classical method widely used to examine sensory functions of neuropathic pain animals.

The specific method comprises the following steps: the rats stand on an elevated platform, the surface of which is a wide gauge wire mesh. Von-Frey fibers (which are very fine gauge wires) are inserted from below through the mesh and pierce the lower surface of the front paw. On the threshold, the animal quickly flung its paw away from the fiber. The mechanical withdrawal threshold is defined as the minimum gauge line stimulus that causes a withdrawal response. Each foot (left front and right front) was measured 5 times separately and the number of paw lifts recorded.

As shown in fig. 8, the ordinate represents the threshold for the number of times the right hand lifts the paw compared to the left hand. A larger value indicates a greater number of paw lifts, a greater sensitivity to mechanical pain stimuli and a better recovery of sensory function. The label sham is a sham operation group, PBS is a group treated by PBS solution after trauma, and R1, R2, R3 and R4 are groups to be detected and treated by different repair peptides.

9. Thermal pain stimulation recovery experiment of rats with repair peptides R1, R2, R3 and R4 after repairing dorsal root ganglion injury

Rats were placed in a clear plastic chamber (18 × 29 × 13cm, 2.2mm thick) and placed 20 minutes prior to testing to acclimate the animals, then a heat source was placed under the glass plate under the front right or left paw, the paw was withdrawn from the automatic activation timer with a 0.1 second delay, 3 measurements were taken at 15 minute intervals and the average paw withdrawal time was recorded.

As shown in fig. 9, the ordinate represents the threshold for the right hand to paw lifting time over the left hand. Smaller values indicate a shorter time to lift the paw, more sensitive to thermal irritation and better recovery of sensory function. The label sham is a sham operation group, PBS is a group treated by PBS solution after trauma, and R1, R2, R3 and R4 are groups to be detected and treated by different repair peptides.

10. Indirect immunofluorescence method for detecting growth condition of neuron axon

At the time point to be measured (2 weeks or 4 weeks), after the rat enters a deep anesthesia state, the thoracic cavity is opened, the heart is exposed, the right auricle is slightly cut, and a needle is inserted from the apical position to the left ventricle. And (3) opening a peristaltic pump, and perfusing with PBS and then 4% PFA stationary solution until the liver of the rat is peach yellow and the body is stiff.

The C5-T1 spinal cord tissue and dorsal roots were stripped with surgical instruments, post-fixed in 4% PFA for 24h, at 4 degrees in 15% sucrose overnight, and then at 4 degrees in 30% sucrose overnight. After embedding with OCT, cryosections were taken, 15 μm thick, and only sections of C7-segmented tissue were harvested for further experiments.

Taking the prepared tissue frozen section, slightly wiping off liquid outside the specimen by using filter paper, enclosing the tissue by using an immunohistochemical pen, and sealing the tissue by using 10% donkey serum in a wet box at room temperature for 1 hour. The blocking solution was then gently wiped off the specimen with filter paper, primary antibody (1: 100) diluted with 10% donkey serum was added dropwise, and incubated overnight at 4 ℃ in a wet box. The primary antibody was discarded and washed 3 times with PBS for 5min each. And wiping off liquid outside the specimen by using filter paper, dripping a fluorescence-labeled secondary antibody diluted by 10% donkey serum, and then placing in a wet box to incubate for 2 hours at room temperature in a dark place. The secondary antibody was aspirated and the PBS solution was gently removed 3 times for 5min each. After wiping off the liquid outside the specimen with filter paper, the anti-fluorescence quencher containing DAPI staining solution is dripped, and then the piece is sealed with a clean cover glass and stored in a place which is protected from light at 4 ℃.

The antibodies were: NF200 (available from abcam, cat # ab40796), Lamin (available from sigma, cat # L9393), CGRP (available from sigma, cat # C8198), counterstaining with a fluorescent secondary antibody (available from invitrogen), observation under a fluorescent microscope, and photographing.

Wherein: NF200 is a marker protein of nerve fibers, and the growth condition of neuron axons can be observed; laminin is a marker protein of peripheral nerve fibers, and the condition that the drug stimulates the growth of axons can be observed; CGRP is associated with pain receptors that transmit pain.

The label sham is a sham group, PBS is a group treated by PBS solution after trauma, and R1, R2, R3 and R4 are groups to be detected treated by different repair peptides. The results are shown in fig. 10-12, and experimental detection shows that the repair peptide treatment group can obviously promote the growth of the nerve synapse and promote the repair of the damaged dorsal root nerve.

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> river-south university

<120> repair peptide for promoting tissue repair and regeneration after trauma and application thereof

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<223> R3 peptide sequence of repair peptide

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Claims (6)

1. A repair peptide for promoting post-traumatic tissue repair and regeneration, comprising:

the sequence of the repair peptide is a linear peptide of GPANVET, KVKVGPANVKVKV, GPANVEKEKEK or CGPANVETC;

the cyclic peptide is formed by disulfide bonds of two cysteines at two ends.

2. Use of the peptide according to claim 1 for the preparation of a product for tissue regeneration and repair in the areas of skin injury, nerve injury.

3. Use according to claim 2, characterized in that:

the repair peptide for promoting tissue repair and regeneration after wound is applied to the preparation of products for skin wound, burn and scald, chronic wound healing, skin cell repair and regeneration, and nerve injury repair and regeneration.

4. Use according to claim 2 or 3, characterized in that:

the product is a medical product, a skin care product or a cosmetic.

5. Use according to claim 2 or 3, characterized in that:

the product is prepared from solution, lyophilized preparation, emulsion, cream, gel, facial mask or dressing.

6. Use according to claim 4, characterized in that:

the product is prepared from solution, lyophilized preparation, emulsion, cream, gel, facial mask or dressing.

Images