Hydrogel containing 7-amino acid phosphorylated peptide and application thereof in neural restoration

Abstract

The invention discloses hydrogel containing 7-amino acid phosphorylated peptide and application of the hydrogel in neural restoration. The hydrogel comprises acellular matrix hydrogel, 7-amino acid phosphorylated peptide, 7-amino acid phosphorylated peptide, 7-amino acid phosphorylated peptide and 7-amino acid phosphorylated peptide, and a 7-amino acid phosphorylated peptide supported in the acellular matrix hydrogel. The 2, 7-amino acid phosphorylated peptide can play a multi-dimensional regulation role in nerve injury microenvironment remodeling, realizes dynamic targeted regulation of peripheral nerve injury microenvironment, and promotes rapid regeneration and recovery of injured peripheral nerves.

Description

Technical Field

[0001] This application belongs to the field of tissue engineering and regenerative medicine technology, and in particular relates to a hydrogel containing a 7-amino acid phosphorylated peptide and its use in nerve repair. Background Technology

[0002] Peripheral nerve injury (PNI) refers to the damage to the structure or function of peripheral nerves caused by trauma (such as cutting or compression), disease (such as diabetes or autoimmune diseases), or iatrogenic factors (such as surgical injury), which in turn leads to sensory, motor, and autonomic nerve dysfunction.

[0003] Traditional single-function treatment strategies often struggle to adapt quickly to the unbalanced injury microenvironment and fail to achieve dynamic targeted regulation of the peripheral nerve injury microenvironment, resulting in slow nerve regeneration and limited functional recovery. Summary of the Invention

[0004] This application provides a hydrogel containing a 7-amino acid phosphorylated peptide and its use in nerve repair, which can achieve the regeneration and recovery of damaged peripheral nerves.

[0005] In a first aspect, this application provides a hydrogel for repairing nerve defects, the hydrogel comprising: a decellularized matrix hydrogel; and a 7-amino acid phosphorylated peptide loaded in the decellularized matrix hydrogel.

[0006] In any embodiment of this application, the concentration of the 7-amino acid phosphorylated peptide in the hydrogel used for repairing nerve defects is 30 µg / ml to 70 µg / ml, based on the total mass of the hydrogel used for repairing nerve defects.

[0007] In any embodiment of this application, the 7-amino acid phosphorylated peptide repairs neural defects through at least one of the following mechanisms: (1) Promotes macrophage polarization towards the anti-inflammatory M2 phenotype; (2) Promote the reconstruction of the vascular matrix network, wherein promoting the reconstruction of the vascular matrix network includes at least one of enhancing the proliferative activity of vascular endothelial cells and promoting the migration of vascular endothelial cells; (3) Enhance Schwann cell function, wherein enhancing Schwann cell function includes improving at least one of Schwann cell proliferation capacity, migration capacity and secretion function.

[0008] In any embodiment of this application, the 7-amino acid phosphorylated peptide is loaded in a decellularized matrix hydrogel by at least one of covalent bonding, non-covalent bonding, and physical embedding.

[0009] In any embodiment of this application, the decellularized matrix hydrogel includes a decellularized matrix, and the concentration of the decellularized matrix in the decellularized matrix hydrogel is 0.3% (w / v) to 0.9% (w / v).

[0010] Secondly, this application provides the use of a 7-amino acid phosphorylated peptide in the preparation of products for repairing nerve defects.

[0011] In any embodiment of this application, the product includes at least one of the following products: (1) hydrogel; (2) three-dimensional scaffold; (3) kit.

[0012] In any embodiment of this application, the 7-amino acid phosphorylated peptide repairs neural defects through at least one of the following mechanisms: (1) promoting macrophage polarization to the anti-inflammatory M2 phenotype; (2) promoting vascular matrix network reconstruction, wherein promoting vascular matrix network reconstruction includes at least one of enhancing the proliferative activity of vascular endothelial cells and promoting the migration of vascular endothelial cells; (3) enhancing Schwann cell function, wherein enhancing Schwann cell function includes at least one of improving the proliferative capacity, migration capacity and secretory function of Schwann cells.

[0013] In any embodiment of this application, the nerve defect includes at least one of spinal nerve injury, cranial nerve injury, and autonomic nerve injury.

[0014] In any embodiment of this application, the therapeutically effective amount of the 7-amino acid phosphorylated peptide is 0.002 mg / kg body weight to 0.008 mg / kg body weight.

[0015] The hydrogel containing 7-amino acid phosphorylated peptides in this application and its use in nerve repair: 7-amino acid phosphorylated peptides can play a multi-dimensional regulatory role in the remodeling of the microenvironment of nerve injury. By promoting the proliferation of Schwann cells, promoting the expression of neurotrophic factors in Schwann cells, promoting the polarization of macrophages to the repair phenotype, and promoting angiogenesis, it can achieve dynamic targeted regulation of the microenvironment of peripheral nerve injury, and promote the rapid regeneration and recovery of damaged peripheral nerves. Attached Figure Description

[0016] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments of this application will be briefly introduced below. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 The results are the CCK-8 assay results of different intervention groups and treatment concentrations on the proliferation of SCs after 3 days in Example 1 of this application.

[0018] Figure 2 This is a schematic diagram illustrating the effects of different intervention groups on the vertical migration of SCs in Example 1 of this application (where "*" indicates a significant difference compared to group 7A or group 7S, ***). p < 0.001).

[0019] Figure 3 In Example 1 of this application, after 24 h of treatment in different intervention groups, the expression levels of neurotrophic factor-related genes at the mRNA level in each group were detected by RT-qPCR (where A shows the mRNA expression level of NGF gene in SCs; B shows the mRNA expression level of BDNF gene in SCs; C shows the mRNA expression level of VEGF gene in SCs; in the figures, "*" indicates a significant difference compared to the Control group, ****) p < 0.0001).

[0020] Figure 4 This is a bubble chart showing the GO enrichment analysis of short peptide 7AP and blank control group in Example 1 of this application.

[0021] Figure 5 This is the expression of M1-related genes in macrophages in Example 2 of this application (where Figure A shows the expression of the CD86 gene; Figure B shows the expression of the TNF-α gene; Figure C shows the expression of the IL-1β gene; in the figures, "*" indicates a significant difference compared to the Control group, *... p < 0.05,** p < 0.01).

[0022] Figure 6 This is the expression of M2-related genes in macrophages in Example 2 of this application (where Figure A shows the expression of the CD163 gene; Figure B shows the expression of the CD206 gene; Figure C shows the expression of the IL-10 gene; in the figures, "*" indicates a significant difference compared to the Control group, ** p < 0.01, *** p < 0.001).

[0023] Figure 7 The results of CCK-8 assay on HUVEC proliferation after treatment with 7AP at a concentration of 100 ng / mL for 1, 3, and 5 days in Example 3 of this application are as follows (where "*" indicates a significant difference compared to group 7A or group 7S). p < 0.05,** p < 0.01, *** p < 0.001).

[0024] Figure 8This is a schematic diagram illustrating the effects of different intervention groups on HUVEC migration in Example 3 of this application (where "*" indicates a significant difference compared to group 7A or group 7S, ***). p < 0.001).

[0025] Figure 9 These are the statistical results of the ischial function index of rats in each group at 4, 8, and 12 weeks post-surgery in Example 4 of this application (where "*" indicates a significant difference compared to the 7AP group, **). p < 0.01).

[0026] Figure 10 In Example 4 of this application, the schematic diagram shows the axonal regeneration length results of each intervention group of rats 2 weeks after surgery (where "*" indicates a significant difference compared to the Control group). p < 0.05; "#" indicates a significant difference compared to the 7AP group. # p < 0.05).

[0027] Figure 11 Statistical results of sciatic function index of rats in each group at 4, 8 and 12 weeks after surgery in Example 5 of this application (where "*" indicates a significant difference compared with the Control group, **) p < 0.01).

[0028] Figure 12 This is a schematic diagram showing the regenerated myelin density results of each intervention group in Example 5 of this application at 12 weeks post-surgery (where "*" indicates a significant difference compared to the 7AP / Matrigel group, **). p < 0.01; ns indicates no significant difference compared to the 7AP / Matrigel group). Detailed Implementation

[0029] To make the purpose, technical solution, and beneficial technical effects of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the implementation details described in this specification are merely for illustrative purposes and are not intended to limit the scope of this application.

[0030] For simplicity, this application only explicitly discloses some numerical ranges. However, any lower limit can be combined with any upper limit to form a range not explicitly stated; and any lower limit can be combined with other lower limits to form a range not explicitly stated, just as any upper limit can be combined with any other upper limit to form a range not explicitly stated. Furthermore, although not explicitly stated, every point or individual value between the endpoints of the range is included within that range. Therefore, each point or individual value can be used as its own lower or upper limit and combined with any other point or individual value or with other lower or upper limits to form a range not explicitly stated.

[0031] It should be noted that the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0032] Unless otherwise stated, the values ​​of the parameters mentioned in this application can be measured using various measurement methods commonly used in the art (e.g., they can be tested according to the methods given in the embodiments of this application). Unless otherwise stated, the test temperature for all parameters mentioned in this application is 25°C and the test pressure is standard atmospheric pressure.

[0033] The foregoing description of this application is not intended to describe every disclosed implementation or method. Instead, the following description provides more specific examples of exemplary embodiments. Throughout the application, guidance is provided through a series of embodiments, which can be used in various combinations. The examples listed are representative only and should not be construed as exhaustive.

[0034] In the treatment of peripheral nerve injury, the single mechanism of action of short peptides in existing technologies cannot meet the differentiated regulatory needs of different stages (inflammatory phase, proliferative phase, and remodeling phase) in the process of nerve regeneration. In particular, they lack the ability to synergistically regulate the timing of macrophage phenotypic transformation (pro-inflammatory to anti-inflammatory within 72 hours), angiogenesis (critical period of 7 days after injury), and Schwann cell activation (lasting 2-4 weeks). Therefore, it is difficult to accurately regulate the microenvironment of nerve injury, resulting in slow nerve regeneration rate and poor functional recovery.

[0035] To address the problems of the prior art, this application provides a hydrogel containing a 7-amino acid phosphorylated peptide and its use in nerve repair, which can achieve regeneration and repair of damaged peripheral nerves.

[0036] A first aspect of this application provides a hydrogel for repairing nerve defects, the hydrogel comprising: a decellularized matrix hydrogel; and a 7-amino acid phosphorylated peptide loaded in the decellularized matrix hydrogel.

[0037] 7-amino acid phosphorylated peptides can precisely regulate the microenvironment of nerve injury and promote the regeneration and recovery of damaged peripheral nerves through a triple synergistic mechanism of temporal regulation of macrophage polarization toward an anti-inflammatory phenotype (M2 type), promotion of vascular matrix network reconstruction, and enhancement of Schwann cell function.

[0038] As an example, decellularized matrix hydrogels can be fat-derived decellularized matrix hydrogels.

[0039] Acellular matrix hydrogels derived from fat are ideal materials for nerve regeneration because they are readily available for tissue acquisition, have strong pro-angiogenic ability, high soft tissue compatibility, and are rich in bioactive substances, providing a suitable microenvironment for nerve regeneration. Glycosamines such as hyaluronic acid retained in acellular matrix hydrogels can stably load peptides through charge interactions. The protein components in the acellular matrix are rich in reactive functional groups such as amino and carboxyl groups, which can provide a molecular basis for the directional coupling of 7-amino acid phosphorylated peptides.

[0040] In some embodiments, the concentration of the 7-amino acid phosphorylated peptide in the hydrogel used for repairing nerve defects is 30 µg / ml to 70 µg / ml, based on the total mass of the hydrogel used for repairing nerve defects.

[0041] As an example, based on the total mass of the hydrogel used to repair nerve defects, the concentration of 7-amino acid phosphorylated peptide in the hydrogel can be 30 µg / ml, 35 µg / ml, 40 µg / ml, 45 µg / ml, 50 µg / ml, 55 µg / ml, 60 µg / ml, 65 µg / ml, or 70 µg / ml.

[0042] In some implementations, the 7-amino acid phosphorylated peptide repairs neural deficits through at least one of the following mechanisms: (1) Promote macrophage polarization to the anti-inflammatory M2 phenotype, including downregulating the expression of M1 macrophage-related inflammatory factors (such as the mRNA of CD86 gene, TNF-α gene and IL-1β gene) and upregulating the expression of M2 macrophage-related markers and anti-inflammatory factors (such as the mRNA of CD163 gene, CD206 gene and IL-10 gene); (2) Promote the reconstruction of the vascular matrix network, wherein promoting the reconstruction of the vascular matrix network includes at least one of enhancing the proliferative activity of vascular endothelial cells, promoting the migration of vascular endothelial cells and improving the angiogenesis efficiency, wherein the vascular endothelial cells can be human umbilical vein endothelial cells, wherein promoting the migration of vascular endothelial cells includes increasing the migration area of ​​vascular endothelial cells. (3) Enhance Schwann cell function, wherein enhancing Schwann cell function includes improving at least one of Schwann cell proliferation capacity, migration capacity and secretion function, wherein improving Schwann cell secretion function includes promoting the expression of neurotrophic factors in Schwann cells, such as upregulating the mRNA expression levels of NGF gene, BDNF gene and VEGF gene in Schwann cells; improving Schwann cell migration capacity includes increasing the number of Schwann cells that migrate.

[0043] In some embodiments, the 7-amino acid phosphorylated peptide is loaded in a decellularized matrix hydrogel via at least one of covalent interaction, non-covalent interaction, and physical embedding.

[0044] Preferably, the 7-amino acid phosphorylated peptide is loaded in the decellularized matrix hydrogel via covalent bonding.

[0045] Optionally, the 7-amino acid phosphorylated peptide is loaded in the decellularized matrix hydrogel via an amide bond. Further, the carboxyl group of the decellularized matrix hydrogel is activated using N-hydroxysuccinimide-maleimide, and then reacted with the primary amino group (-N) of the 7-amino acid phosphorylated peptide. The reaction forms an amide bond.

[0046] Optionally, the 7-amino acid phosphorylated peptide is loaded in a decellularized matrix hydrogel via a one-step chemical bond coupling method.

[0047] By using tissue decellularized matrix hydrogels and chemical bond modification, a gel scaffold sustained-release system capable of in-situ loading of 7-amino acid phosphorylated peptides was constructed. This gel scaffold sustained-release system can achieve uniform distribution of 7-amino acid phosphorylated peptides within the gel scaffold sustained-release system while maintaining the activity of 7-amino acid phosphorylated peptides, significantly enhance the stability of 7-amino acid phosphorylated peptides, and maintain an effective concentration in the peripheral nerve injury area, thereby promoting the rapid regeneration and recovery of injured peripheral nerves.

[0048] In some embodiments, the decellularized matrix hydrogel includes a decellularized matrix, the concentration of which is 0.3% (w / v) to 0.9% (w / v), preferably 0.6% (w / v).

[0049] As an example, the concentration of decellularized matrix in decellularized matrix hydrogel can be 0.3% (w / v), 0.35% (w / v), 0.4% (w / v), 0.45% (w / v), 0.5% (w / v), 0.55% (w / v), 0.6% (w / v), 0.65% (w / v), 0.7% (w / v), 0.75% (w / v), 0.8% (w / v), 0.85% (w / v), or 0.9% (w / v).

[0050] Excessive concentration of decellularized matrix in decellularized matrix hydrogels can lead to increased viscosity, reduced porosity of the hydrogel's three-dimensional network, and compromised handling convenience. Conversely, excessively low concentrations can result in difficulty maintaining adequate mechanical strength and excessive looseness, leading to rapid loss of the encapsulated 7AP. Therefore, selecting a decellularized matrix concentration of 0.3% (w / v) to 0.9% (w / v), especially 0.6% (w / v), is a key parameter for balancing handling performance and functional activity.

[0051] A second aspect of this application provides the use of a 7-amino acid phosphorylated peptide in the preparation of a product for repairing nerve defects.

[0052] In some embodiments, the product includes at least one of the following: (1) a hydrogel; (2) a three-dimensional scaffold; and (3) a kit.

[0053] In some implementations, the three-dimensional scaffold includes: a catheter; and a hydrogel filled in the catheter.

[0054] In some embodiments, the inner diameter of the catheter is 0.1 mm to 4 mm; and / or, the amount of hydrogel filling the catheter is 15 μL to 25 μL.

[0055] As an example, the inner diameter of the catheter can be 0.1 mm, 0.2 mm, 0.4 mm, 0.6 mm, 0.8 mm, 1.0 mm, 1.5 mm, 2.0 mm, 2.5 mm, 3.0 mm, 3.5 mm, or 4 mm.

[0056] As an example, the amount of hydrogel filling the catheter can be 15 μL, 16 μL, 17 μL, 18 μL, 19 μL, 20 μL, 21 μL, 22 μL, 23 μL, 24 μL, or 25 μL.

[0057] Furthermore, the catheter can be made of at least one of polycaprolactone (PCL), silicone, polylactic acid-glycolic acid copolymer, polylactic acid, and chitin.

[0058] In some implementations, the 7-amino acid phosphorylated peptide repairs neural deficits through at least one of the following mechanisms: (1) Promote macrophage polarization to the anti-inflammatory M2 phenotype, including downregulating the expression of M1 macrophage-related inflammatory factors (such as the mRNA of CD86 gene, TNF-α gene and IL-1β gene) and upregulating the expression of M2 macrophage-related markers and anti-inflammatory factors (such as the mRNA of CD163 gene, CD206 gene and IL-10 gene); (2) Promote the reconstruction of the vascular matrix network, wherein promoting the reconstruction of the vascular matrix network includes at least one of enhancing the proliferative activity of vascular endothelial cells, promoting the migration of vascular endothelial cells and improving the angiogenesis efficiency, wherein the vascular endothelial cells can be human umbilical vein endothelial cells, wherein promoting the migration of vascular endothelial cells includes increasing the migration area of ​​vascular endothelial cells. (3) Enhance Schwann cell function, wherein enhancing Schwann cell function includes improving at least one of Schwann cell proliferation capacity, migration capacity and secretion function, wherein improving Schwann cell secretion function includes promoting the expression of neurotrophic factors in Schwann cells, such as upregulating the mRNA expression levels of NGF gene, BDNF gene and VEGF gene in Schwann cells; improving Schwann cell migration capacity includes increasing the number of Schwann cells that migrate.

[0059] In some embodiments, the nerve deficit includes at least one of spinal nerve injury, cranial nerve injury, and autonomic nerve injury. Preferably, the spinal nerve injury is a sciatic nerve injury.

[0060] In some embodiments, the therapeutically effective amount of the 7-amino acid phosphorylated peptide is 0.002 mg / kg body weight to 0.008 mg / kg body weight.

[0061] As an example, the therapeutically effective dose of 7-amino acid phosphorylated peptide can be 0.002 mg / kg body weight, 0.003 mg / kg body weight, 0.004 mg / kg body weight, 0.005 mg / kg body weight, 0.006 mg / kg body weight, 0.007 mg / kg body weight, or 0.008 mg / kg body weight.

[0062] The term "HDAC7" refers to histone deacetylase 7.

[0063] The term "7A" is a cleavage isomer of HDAC7, generated by specific cleavage of HDAC7; the amino acid sequence of 7A is MHSPGAD, as shown in SEQ ID NO:1.

[0064] The term "7AP" refers to a 7-amino acid phosphorylated peptide, which can be formed by phosphorylation of serine residues 7A mediated by MEKK1 kinase; the amino acid sequence of 7AP is MH[pS]PGAD, and its amino acid sequence is shown in SEQ ID NO:2.

[0065] The term "7S" is another cleavage isomer of HDAC7, generated by HDAC7 specific cleavage; the amino acid sequence of 7S is MPHASGD, and its amino acid sequence is shown in SEQ ID NO:3.

[0066] Example The following embodiments describe the disclosure of this application in more detail. These embodiments are for illustrative purposes only, as various modifications and variations will be apparent to those skilled in the art within the scope of the disclosure of this application. Unless otherwise stated, all parts, percentages, and ratios reported in the following embodiments are based on mass, and all reagents used in the embodiments are commercially available or synthesized by conventional methods and can be used directly without further processing, and the instruments used in the embodiments are commercially available.

[0067] The sources of some of the reagents and instruments used in Examples 1 to 5 are as follows: 1. [Key reagents and instruments used in Example 1]: Type I collagenase was purchased from Sigma-Aldrich (catalog number: C9891). The pancreatic enzyme was purchased from Sigma-Aldrich (catalog number: T4049). CCK-8 was purchased from Tongren Chemical Research Institute (item number: CK04); Crystal violet was purchased from Sigma-Aldrich (product number: 32675); Primers were purchased from Qingke Biotechnology Co., Ltd. SD rats were purchased from Beijing Vital River Co., Ltd.

[0068] The centrifuge is manufactured by Eppendorf (model: 5810R). The manufacturer of the clean bench is Harbin Donglian Company (model: DL-CJ-1N). The cell incubator was manufactured by Thermofisher (model: 3111). The microscope is manufactured by Nikon Corporation (model: ECLIPSE-TS2). The Q-PCR instrument is manufactured by Bio-Rad (model: CFX Duet).

[0069] 2. [Key reagents and instruments used in Example 2] F4 / 80-PE-Cy7 flow cytometry antibody was purchased from Invitrogen (catalog number: 25-4801-82). M-CSF was purchased from Peprotech (part number: 315-02). Primers were purchased from Qingke Biotechnology Co., Ltd.

[0070] The microscope is manufactured by Nikon Corporation (model: ECLIPSE-TS2). The Q-PCR instrument is manufactured by Bio-Rad (model: CFX Duet).

[0071] 3. [Key reagents and instruments used in Example 3]: CCK-8 was purchased from Tongren Chemical Research Institute (item number: CK04).

[0072] The microscope is manufactured by Nikon Corporation (model: ECLIPSE-TS2).

[0073] 4. [Key reagents and instruments used in Example 4]: Isopropanol was purchased from Sinopharm Group (product number: 40064360). Pepsin was purchased from Sigma-Aldrich (catalog number: P7000). The nuclease was purchased from Xinhai Gene Company (Catalog No.: C2001). Concentrated hydrochloric acid was purchased from Sinopharm Group (item number: 10011008). The 10×MEM was purchased from Gibco (part number: 11430030). Sodium hydroxide was purchased from Sinopharm Group (item number: 53100160). DAPI was purchased from Solarbio (item number: C0065); Polycaprolactone (PCL) was purchased from Sigma-Aldrich (catalog number: 440744); Sodium pentobarbital was purchased from Sigma-Aldrich (product number: P3761). S100 antibody was purchased from Abcam (catalog number: ab52642). The NF200 antibody was purchased from Sigma-Aldrich (catalog number: N0142).

[0074] The cryogenic grinder is manufactured by SPEX Sample Prep (model: 6875D). The laser confocal microscope is manufactured by Nikon Corporation (model: A1). The Catwalk gait analyzer is manufactured by Noldus (model: Version 10.6). 5. [Key reagents and instruments used in Example 5]: Toluidine blue staining reagent was purchased from Solarbio (product number: G1032). Type I bovine collagen was purchased from Sigma-Aldrich (product number: C8919). Matrigel was purchased from Corning (part number: 354248).

[0075] 6. The extraction method for Schwann cells (SCs) is as follows: (1) Take 3-day-old SD suckling mice. Sacrifice them by cervical dislocation, then immerse them in 75% alcohol for 5 minutes for disinfection, and transfer them to a clean bench.

[0076] (2) Fix the SD suckling mice on a sterilized wax table, cut and expose the bilateral sciatic nerves, and aseptically harvest approximately 1 cm of the bilateral sciatic nerves using sterile ophthalmic instruments. Remove the epineurium under a microscope and wash three times in PBS with 5% penicillin-dextrose antibody. Transfer to a sterile glass bottle and cut the adipose tissue into uniformly sized fragments.

[0077] (3) Add DMEM / F 12 medium containing 0.2% NB4 type collagenase, put in magnetic beads, and use a magnetic stirrer to digest the adipose tissue in a CO2 incubator for about 10 minutes.

[0078] (4) After the nerve tissue has been completely digested, add an equal volume of serum-containing culture medium to stop the digestion and filter it using a cell sieve.

[0079] (5) Collect the suspension and transfer it to a centrifuge. Digest at 1500 r / min for 5 min, discard the supernatant and resuspend the precipitate.

[0080] Inoculate into T25 culture flasks and incubate in a cell culture incubator.

[0081] (6) Observe the cell morphology and growth under a microscope. When the cell fusion reaches 80%, digest with NB4 type collagenase and passage at 1:2 for later use.

[0082] 7. The extraction method for Bone Marrow-Derived Macrophages (BMDMs) is as follows: Primary macrophage extraction method: Female 6-8 week old C57BL / 6 mice were euthanized by cervical dislocation. Using aseptic techniques, the skin from the proximal hind limb to the foot was removed, the tibia was dissected, and the ankle and knee joints were severed. The femur was dissected in the same manner. Excess tissue was removed with forceps and ophthalmic scissors. The femur and tibia were kept on ice and washed twice with sterile PBS. Then, under aseptic conditions, both ends of the bone were cut open, and the bone marrow cavity was flushed with complete macrophage culture medium (DMEM / F12, 10% fetal bovine serum, 50 ng / ml mouse M-CSF, 100 U / ml penicillin, and 100 U / ml streptomycin) using a 1 ml syringe. The collected cells were filtered through a 70 μm diameter filter, centrifuged at 400 g for 5 min, resuspended in erythrocyte lysis buffer, placed on ice for 15 min, centrifuged again, washed with PBS, and counted. After centrifugation again, the cells were resuspended in complete macrophage culture medium to a concentration of 7.5-10 × 10⁻⁶ cells / mL. 5 Cells were seeded at a concentration of / ml into suitable cell culture containers and induced to differentiate into primitive macrophages (n) at 37℃ and 5% CO2 for 6-7 days. Macrophages (MCS) were cultured for 7 days. After characterization confirmed successful induction of macrophages, subsequent experiments were conducted (Reference: Tian Guangzhao. Exploration of the immune regulation mechanism of extracellular matrix of chondrocytes and in vivo study on the promotion of osteochondral repair by combination with interleukin-4 [D]. Nankai University, 2021.).

[0083] 8. The extraction method for human umbilical vein endothelial cells (HUVECs) is as follows: Using a sterile umbilical cord from a healthy newborn, repeatedly clean the cord with sterile, pre-cooled PBS solution to remove blood stains. Separate both ends of the umbilical vein and rinse the blood clot within the vessel with PBS solution. Then, slowly inject 20 ml of 0.1% type I collagenase solution into the vein lumen, and place the tube in a cell culture incubator for digestion for 15 minutes. Aspirate the digestion fluid from the umbilical vein and place it in a 50 ml centrifuge tube. Centrifuge at 1200 rpm for 5 minutes at 4°C. Seed the centrifuged pellet into T25 vials coated with 0.1% gelatin containing EGM-2 complete medium. Incubate the cells in an adherent manner, observing a cobblestone-like arrangement.

[0084] Example 1 Investigating the effects of 7A, 7AP, and 7S short peptides on Schwann cell function 1. Experimental Materials 1.1 Short peptide compounds Synthesis of 7A, 7AP, and 7S Short peptide compounds were obtained using the FMOC solid-phase chemical synthesis method.

[0085] The targeted synthesis of short peptide compounds was achieved through solid-phase synthesis, stepwise condensation of amino acids from the C-terminus to the N-terminus, combined with FMOC protection / deprotection cycling and efficient cleavage and purification steps. The specific synthetic steps are shown below: (1) First amino acid coupling and blocking with resin Coupling reaction: Starting with 0.45 mmol (1.3 eq) FMOC-AA1-COOH as the starting amino acid, CTC-resin was coupled with 0.15 mmol DIEA (N,N-diisopropylethylamine) and 3 mL DCM (dichloromethane) as solvent at room temperature for 1.5 h to obtain FMOC-AA1-resin.

[0086] Blocking unreacted sites: Add 2 mmol methanol, 10 mL DCM solution and DIEA, and react for 20 min to block unreacted amino sites on the resin.

[0087] (2) De-FMOC protection Deprotection reagent: DMF (N,N-dimethylformamide) solution containing 20% ​​piperidine was used to remove FMOC groups by reacting at room temperature for 20 min.

[0088] Washing: After removal, the resin was washed 4 times with DMF (1 min / time) to obtain NH2-AA1-resin (free amino resin peptide).

[0089] (3) Subsequent amino acid condensation (cyclic operation) Condensation reagents and conditions: Using 0.45 mmol FMOC-AA2-OH (3 eq) as the next amino acid, the resin peptide was dehydrated and condensed with the condensation reagent 0.45 mmol DIC (N,N'-diisopropylcarbodiimide) + 0.45 mmol HOBT (1-hydroxybenzotriazole) and 3 mL DMF as solvents to obtain FMOC-AA2-AA1-resin by condensation at room temperature for 1 h.

[0090] Cyclic process: Repeat the “FMOC removal (2) → amino acid condensation (3)” operation, condensing the remaining amino acids sequentially from the C-terminus to the N-terminus until the target peptide chain sequence is complete. See Table 1 for specific parameters.

[0091] Table 1 (4) Peptide cleavage The lysis buffer composition is: 95% TFA (trifluoroacetic acid) + 1% H2O + 2% EDT (1,2-ethylenedithiol) + 2% TIS (triisopropylsilane).

[0092] Lysis and sedimentation: The peptide was lysed at room temperature by adding 10 ml of lysis buffer to 1 g of peptide. After lysing at room temperature for 2 h, the lysis buffer was filtered into ice-cold ether. The peptide was then precipitated by adding 10 mL of ether to 1 mL of lysis buffer and centrifuged to obtain crude peptide.

[0093] (5) Polypeptide purification and preparation Purification method: The crude polypeptide was lyophilized and then separated by HPLC (high performance liquid chromatography).

[0094] HPLC conditions: acetonitrile gradient 15%-40%, elution time 40 min.

[0095] Purity requirement: Collect components with a purity >95%, freeze-dry them to obtain pure peptides.

[0096] 2. Experimental Methods 2.1 Schwann cells (SCs) were isolated from the sciatic nerve of SD rats (8 weeks old, 200-220 g, female) using mechanical separation combined with type I collagenase / trypsin digestion. After purification by differential adhesion, they were used for subsequent experiments.

[0097] 2.2 In vitro construction of co-culture systems of 7A, 7AP, and 7S short peptides (0, 50 ng / mL, and 100 ng / mL) with SCs: The effects of 7A, 7AP, and 7S short peptides on the proliferation ability of SCs were compared by CCK-8 assay; the effects of 7A, 7AP, and 7S short peptides on the migration ability of SCs were evaluated by Transwell assay; the effects of 7A, 7AP, and 7S short peptides on the secretory function of SCs were analyzed by real-time quantitative PCR (RT-qPCR); and the molecular mechanism of action of 7AP short peptide on SCs was analyzed by transcriptome sequencing (RNA-seq).

[0098] 3. Experimental Results 3.1 Proliferative capacity of SCs The results are as follows Figure 1 As shown, different intervention groups all exhibited a promoting effect on SC proliferation at each treatment concentration; among them, the 100 ng / mL 7AP treatment group had a more significant promoting effect on SC proliferation than other treatment groups, that is, the 100 ng / mL 7AP group was significantly different from the 100 ng / mL Control group, the 100 ng / mL 7A group and the 100 ng / mL 7S group.

[0099] 3.2 SCs migration function Transwell assays were used to analyze the effects of 7A, 7AP, and 7S peptides on the vertical migration of stem cells (SCs). Crystal violet staining results showed that after 24 hours of intervention with 7A, 7AP, and 7S peptides, more migrating cells were observed on the lower surface of the upper chamber cells. Statistical analysis of cell numbers is shown below. Figure 2 Over time, the number of migrating cells increased in groups 7A, 7AP, and 7S, with the 7AP group showing the highest number of migrating cells. The 7AP group was significantly different from the Control group, 7A group, and 7S group.

[0100] 3.3 Secretory function of SCs Statistical results of real-time PCR detection are as follows: Figure 3 As shown, after treatment at different concentrations in different intervention groups, the mRNA expression levels of NGF, BDNF, and VEGF genes in SCs were significantly upregulated. Among the intervention groups, the effect of 7AP was more significant, that is, the 7AP group was significantly different from the Control group, 7A group, and 7S group, suggesting that it can effectively promote the expression of neurotrophic factors in SCs.

[0101] 3.4 Molecular mechanism of action of 7AP short peptide on SCs Transcriptome sequencing (RNA-seq) was performed on SCs treated with 100 ng / mL or an equal volume of PBS for 3 days. Figure 4 As shown, GO enrichment analysis suggests that 7AP may be involved in the positive regulation of neural development and neural regeneration.

[0102] Example 2 Investigating whether 7AP can reprogram macrophages to polarize towards a repair phenotype 1. Experimental Materials 1.1 Short peptide compounds The synthesis of 7AP is the same as in Example 1.

[0103] 2. Experimental Methods 2.1 Bone marrow-derived macrophages (BMDMs) were extracted from SD rats (8 weeks old, 200-220g, female) and their phenotypic characteristics were identified by flow cytometry. Cells with a purity of ≥90% were used for subsequent experiments.

[0104] 2.2 Evaluation of whether the short peptide 7AP can affect macrophage phenotypic polarization by in vitro co-culture system: The experiment was divided into 4 groups, namely, adding 7A, 7AP, 7S short peptide (100 ng / mL) or an equal volume of PBS. After co-culturing for 24 h, the expression of M1 macrophages (CD86, TNF-α, IL-1β) and M2 macrophages (CD163, CD206, IL-10) was detected by RT-qPCR.

[0105] 3. Experimental Results 3.1 Expression of M1 macrophages (CD86, TNF-α, IL-1β) RT-qPCR test results are as follows Figure 5 As shown, compared with the untreated BMDMs, 7AP treatment significantly reduced the mRNA expression levels of CD86, TNF-α, and IL-1β genes. The 7AP group showed significant differences compared with the Control, 7A, and 7S groups. This indicates that 7AP can exert its effect by downregulating the expression of M1 macrophage-related inflammatory factors.

[0106] 3.2 Expression of M2 macrophages (CD163, CD206, IL-10) In addition, such as Figure 6 As shown, compared with the untreated BMDMs, 7AP treatment significantly upregulated the mRNA expression levels of CD163, CD206, and IL-10 genes. The 7AP group showed significant differences compared to the Control, 7A, and 7S groups. These results suggest that 7AP may exert its effect by upregulating the expression of M2 macrophage-related markers and anti-inflammatory factors.

[0107] Example 3 Investigating the pro-angiogenic effect of 7AP in an in vitro model 1. Experimental Materials 1.1 Short peptide compounds The synthesis of 7AP is the same as in Example 1.

[0108] 2. Experimental Methods 2.1 Human umbilical vein endothelial cells (HUVECs) were extracted, passaged, and used in subsequent experiments.

[0109] 2.2 Evaluation of the effect of short peptide 7AP on HUVEC proliferation in an in vitro co-culture system: The experiment was divided into four groups, with the addition of 7A, 7AP, 7S short peptide (100 ng / mL) or an equal volume of PBS. Cell proliferation activity was detected by CCK-8 assay. The scratch assay was used to evaluate the effect of 7A, 7AP, and 7S short peptide on the migration ability of HUVECs.

[0110] 3. Experimental Results 3.1 Proliferation capacity of HUVECs The results are as follows Figure 7 As shown, compared with other treatment groups (7A group, 7S group) and the control group, 100 ng / mL 7AP treatment can significantly promote the proliferation of HUVECs. The 7AP group is significantly different from the control group, 7A group and 7S group.

[0111] 3.2 Migration Function of HUVECs The effects of short peptides 7A, 7AP, and 7S on the migration of HUVECs were investigated using a scratch assay. Statistical analysis of the scratch spacing confirmed the experimental results as follows: Figure 8 As shown, the 7AP group had the best scratch healing effect, and the 7AP group was significantly different from the Control group, 7A group and 7S group.

[0112] Example 4 1. Preparation of decellularized fat matrix hydrogel (DAM-gel) Sterile granular adipose tissue was collected from healthy women (aged 18-35 years; BMI <30; hemoglobin >110 g / L) who underwent liposuction of the abdomen or chest. The collected adipose tissue was thoroughly rinsed with 0.9% sodium chloride solution, aliquoted into 50 mL centrifuge tubes, and immediately decellularized or frozen at -80°C. The adipose tissue obtained from liposuction was processed using a combination of enzymatic hydrolysis and organic solvent extraction to obtain flocculent white adipose tissue-derived decellularized matrix (DAM). The extracted DAM was frozen overnight at -80°C and then freeze-dried for at least 24 hours. The freeze-dried DAM was ground into powder using a high-speed cryogenic tissue homogenizer and purified through an 80-mesh sieve. 200 mg of DAM powder and 20 mg of 250 U / mg pepsin were added to 10 mL of 0.01 M HCl and shaken continuously at room temperature for 48-72 hours to prepare a DAM pregel. Taking the preparation of 6 mg / ml DAM-gel as an example, 6 ml of the DAM digest (DAM pregel) prepared by the above steps was taken, and 1 ml of 10×MEM and 2.94 ml of ddH2O were added at low temperature and mixed thoroughly. Then, 60 μl of 1M NaOH was added to obtain the DAM pregel solution. The DAM pregel solution was incubated at 37°C for 30 min to prepare 6 mg / ml DAM-gel. The DAM pregel solution or 0.6% w / v DAM-gel was used for in vitro and in vivo studies.

[0113] 2. Construction of conduits for nerve regeneration and repair 2.1 Synthesis of Catheters The catheter was prepared using electrospinning technology. The synthesis method is as follows: 8% (w / v) polycaprolactone (PCL) was electrospinned into a tubular material with an inner diameter of 1.5 mm. The tubing was then cut into 12 mm lengths as needed, sterilized by cobalt irradiation, and frozen at -80°C until use.

[0114] 2.2 Synthesis of conduits for nerve regeneration and repair The previously prepared 0.6% w / v DAM-gel was coupled with N-hydroxysuccinimide-maleimide in a solution system at a mass ratio of 1:10. The reaction temperature was 4°C. Unreacted reagents were removed by dialysis and the mixture was purified. 7AP was then thoroughly mixed with the above system in a specific ratio to ensure a final concentration of 50 μg / ml in the gel product, thus preparing a chemically cross-linked 7AP-loaded gel-release scaffold material. Three-dimensional tomography was performed using laser confocal microscopy to observe the loading of DAM-gel and 7AP (Cy5-labeled). The mixture of 7AP and DAM-gel was injected at low temperature into 12 mm long commercial catheters to construct nerve-guided conduits (NGCs). In the catheters, the final concentration of DAM-gel was 0.6% w / v, and the final concentration of 7AP was 50 μg / ml (based on the total mass of DAM-gel loaded with 7AP). The catheter has an inner diameter of approximately 1.2 mm, and each catheter contains 20 μl of an injection volume (a mixture of 7AP and DAM-gel). After gelation at 37°C, it is used for animal experiments.

[0115] 3. Establishment of animal models 3.1 Animal type: SD rats, 8 weeks old, 200-220 g, female.

[0116] 3.2 Animal grouping: A total of 60 animals were randomly divided into 4 groups of 15 animals each, and sciatic nerve defect surgery was performed.

[0117] 3.3 Animal model group settings: Control group: Normal saline was injected into the catheter.

[0118] Experimental group 1 (7AP): 7AP dissolved in physiological saline was injected into the catheter, and the final concentration of 7AP in the catheter was 50 ug / ml.

[0119] Experimental group 2 (7AP / DAM-gel): DAM-gel loaded with 7AP was injected into the catheter. The final concentration of DAM-gel in the catheter was 0.6% w / v, and the final concentration of 7AP in the catheter was 50 ug / ml (based on the total mass of DAM-gel loaded with 7AP).

[0120] Positive control group (Autograft): Autologous nerve reverse grafting 4. Experimental Methods: After weighing, the SD rats in each group were anesthetized by intraperitoneal injection of 1% (40 mg / kg) sodium pentobarbital. After complete anesthesia, the surgical area of ​​the right hind limb of the SD rats was prepared and disinfected with iodine. The rats were placed in the left lateral decubitus position, and the skin was incised 0.5 cm below the right femur. The tissue was bluntly dissected layer by layer to fully expose the sciatic nerve. The right sciatic nerve was removed to create a 10 mm defect, while the left side served as a self-control and was not treated. According to the above grouping, the nerve ends were bridged. In the blank control group, a simple PCL catheter was used, and in the experimental group, the nerve ends were bridged using the prepared scaffold. The nerve ends on both sides were fixed with two sutures each using 11-0 microsutures at a depth of about 1 mm inside the catheter. In the Autograft group, the cut nerve was rotated 180° and four sutures were used to close each end of the nerve with 11-0 microsutures. After complete hemostasis, the surgical incision was closed layer by layer. Tests were conducted at 4, 8 and 12 weeks postoperatively to calculate the sciatic nerve index (SFI); tissue samples were taken 2 weeks postoperatively, and the regeneration rate of nerves was evaluated by immunofluorescence staining.

[0121] The sciatic nerve index (SFI) test method was as follows: Fifteen rats in each group were used to analyze and assess the recovery of motor function using a Cat Walk XT 10.6 small animal gait analyzer, and the sciatic nerve function index (SFI) was calculated. The rats were placed at the beginning of the measurement platform and allowed to move to the end. The walking process was recorded by the software system using fluorescence capture, leaving 5-6 footprints on each hind limb. The software then analyzed the obtained data, and the SFI was measured and calculated using the following formula: formula: Note: E represents the experimental side, N represents the control side, TS represents the toe width, PL represents the footprint length, and IT represents the middle toe width.

[0122] 5. Experimental Results 5.1 Functional Science Footprints of rats in each group were collected at 4, 8 and 12 weeks after surgery using CatWalk, and the SFI was calculated by analysis. Figure 9 The results showed that the SFI (Self-Range Function Index) in all groups initially decreased and then increased. Starting at 8 weeks post-surgery, the SFI value in the 7AP / DAM-gel group was significantly higher than that in the other groups, gradually approaching that of the Autograft group. Furthermore, the SFI in the 7AP / DAM-gel group was significantly higher than that in the 7AP-only group and the Control group. These results indicate that the motor function of rats in the 7AP / DAM-gel group was significantly restored.

[0123] 5.2 Morphology Longitudinal frozen sections and immunofluorescence staining were performed on the regenerated nerve tissue in each group 2 weeks after surgery. Figure 10 The results showed that the Autograft group had the longest axon regeneration distance, at 6438.53 ± 422.6 μm. The 7AP / DAM-gel group had the second fastest axon regeneration rate, and compared with the 7AP and Control groups, it still had a significant advantage in axon growth and Schwann cell migration.

[0124] Example 5 1. Construction of conduits for nerve regeneration and repair 7AP was mixed with type I bovine collagen (Sigma Aldrich), Matrigel (Corning), and the decellularized adipose matrix hydrogel prepared in Example 4, respectively. The mixture was then injected at low temperature into 12 mm long catheters (the synthesis method of the catheters is described in 2.1 of Example 4) to construct neural guiding conduits (NGCs). The final concentrations of type I bovine collagen, Matrigel, DAM-gel, and 7AP in the catheters were 6 mg / ml, 6 mg / ml, 0.6% w / v, and 50 μg / ml, respectively. The inner diameter of the catheter was approximately 1.2 mm, and the injection volume per catheter was 20 μl. After gelation at 37°C, the mixture was used for animal experiments.

[0125] 3. Establishment of animal models 3.1 Animal type: SD rats, 8 weeks old, 200-220g, female.

[0126] 3.2 Animal grouping: A total of 60 animals were randomly divided into 4 groups of 15 animals each, and sciatic nerve defect surgery was performed.

[0127] 3.3 Animal model group settings: Control group: Normal saline was injected into the catheter. Experimental group 1 (7AP / Type I bovine collagen): 7AP dissolved in type I bovine collagen was injected into the catheter. The final concentration of type I bovine collagen was 6 mg / ml, and the final concentration of 7AP was 50 ug / ml.

[0128] Experimental group 2 (7AP / Matrigel): Matrigel loaded with 7AP was injected into the catheter. The final concentration of Matrigel was 6 mg / ml and the final concentration of 7AP was 50 ug / ml.

[0129] Experimental group 3 (7AP / DAM-gel): DAM-gel loaded with 7AP was injected into the catheter at a concentration of 0.6% w / v and a final concentration of 7AP of 50 ug / ml.

[0130] 4. Experimental Methods: After weighing, SD rats in each group were anesthetized via intraperitoneal injection with 1% (40 mg / kg) sodium pentobarbital. After complete anesthesia, the surgical area of ​​the right hind limb was prepared and disinfected with povidone-iodine. The rats were placed in a left lateral decubitus position, and the skin was incised 0.5 cm below the right femur. The tissue was bluntly dissected layer by layer to fully expose the sciatic nerve. A 10 mm defect was created by removing the right sciatic nerve, while the left side served as a self-control and was left untreated. Based on the grouping, the nerve ends were bridged. In the blank control group, a simple PCL catheter was used, while in the experimental group, the prepared scaffold was used to bridge the nerve ends. Two sutures were used to fix each nerve end approximately 1 mm inside the catheter using 11-0 microsutures. After complete hemostasis, the surgical incision was closed layer by layer. Tests were performed at 4, 8, and 12 weeks post-surgery to calculate the sciatic nerve index (SFI). Tissue samples were taken at 12 weeks post-surgery, and the density of regenerated axons was evaluated using toluidine blue staining.

[0131] The sciatic nerve index (SFI) test method was as follows: Fifteen rats in each group were used to analyze and assess the recovery of motor function using a Cat Walk XT 10.6 small animal gait analyzer, and the sciatic nerve function index (SFI) was calculated. The rats were placed at the beginning of the measurement platform and allowed to move to the end. The walking process was recorded by the software system using fluorescence capture, leaving 5-6 footprints on each hind limb. The software then analyzed the obtained data, and the SFI was measured and calculated using the following formula: formula: Note: E represents the experimental side, N represents the control side, TS represents the toe width, PL represents the footprint length, and IT represents the middle toe width.

[0132] 5. Experimental Results 5.1 Functional Science Footprints of rats in each group were collected at 4, 8 and 12 weeks after surgery using CatWalk, and the SFI was calculated by analysis. Figure 11 The results showed that the SFI value of the 7AP / DAM-gel group was significantly higher than that of the other groups.

[0133] 5.2 Morphology Toluidine blue staining was used to assess the density of regenerated axons. At 12 weeks post-surgery, abundant well-formed myelin sheaths were only found in the 7AP / DAM-gel group, such as... Figure 12 As shown, the myelin density of the 7AP / DAM-gel group was significantly higher than that of the Control group, the 7AP / type I bovine collagen group, and the 7AP / Matrigel group.

Claims

1. A hydrogel for repairing nerve defects, characterized in that, The hydrogel comprises: Decellularized matrix hydrogels; and 7-amino acid phosphorylated peptide loaded in the decellularized matrix hydrogel.

2. The hydrogel according to claim 1, characterized in that, Based on the total mass of the hydrogel used for repairing nerve defects, the concentration of the 7-amino acid phosphorylated peptide in the hydrogel used for repairing nerve defects is 30 µg / ml to 70 µg / ml.

3. The hydrogel according to claim 1, characterized in that, The 7-amino acid phosphorylated peptide repairs nerve damage through at least one of the following mechanisms: (1) Promotes macrophage polarization towards the anti-inflammatory M2 phenotype; (2) Promoting the reconstruction of the vascular matrix network, wherein the promotion of the reconstruction of the vascular matrix network includes at least one of enhancing the proliferative activity of vascular endothelial cells and promoting the migration of vascular endothelial cells; (3) Enhance Schwann cell function, wherein the enhancement of Schwann cell function includes improving at least one of Schwann cell proliferation capacity, migration capacity and secretion function.

4. The hydrogel according to any one of claims 1 to 3, characterized in that, The 7-amino acid phosphorylated peptide is loaded in the decellularized matrix hydrogel through at least one of covalent bonding, non-covalent bonding, and physical embedding.

5. The hydrogel according to any one of claims 1 to 3, characterized in that, The decellularized matrix hydrogel includes a decellularized matrix, and the concentration of the decellularized matrix in the decellularized matrix hydrogel is 0.3% (w / v) to 0.9% (w / v).

6. Use of a 7-amino acid phosphorylated peptide in the preparation of products for repairing nerve defects.

7. The use according to claim 6, characterized in that, The product includes at least one of the following products: (1) Hydrogel; (2) Three-dimensional scaffold; (3) Kit.

8. The use according to any one of claims 6 to 7, characterized in that, The 7-amino acid phosphorylated peptide repairs nerve damage through at least one of the following mechanisms: (1) Promotes macrophage polarization towards the anti-inflammatory M2 phenotype; (2) Promoting the reconstruction of the vascular matrix network, wherein the promotion of the reconstruction of the vascular matrix network includes at least one of enhancing the proliferative activity of vascular endothelial cells and promoting the migration of vascular endothelial cells; (3) Enhance Schwann cell function, wherein the enhancement of Schwann cell function includes improving at least one of Schwann cell proliferation capacity, migration capacity and secretion function.

9. The use according to any one of claims 6 to 7, characterized in that, The neurological deficit includes at least one of spinal nerve injury, cranial nerve injury, and autonomic nerve injury.

10. The use according to any one of claims 6 to 7, characterized in that, The therapeutically effective dose of the 7-amino acid phosphorylated peptide is 0.002 mg / kg body weight to 0.008 mg / kg body weight.

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