Functional polypeptide for promoting repair of endometrial stem cells to clitoris sensitive nerves and application

Abstract

The invention discloses a functional polypeptide for promoting repair of endometrial stem cells to clitoris sensitive nerves and application, and belongs to the technical field of stem cell repair, the functional polypeptide is used for modifying the endometrial stem cells and Schwann cells through the functional polypeptide, and the clitoris sensitive nerves are repaired in a local slow-release gel delivery mode. The functional polypeptide can activate Laminin expression and regulate and control ECM degradation balance, so that collagen deposition in scar tissues is reduced; in addition, the functional polypeptide can effectively inhibit HOXA1 expression, enhance the axon extension capacity and strengthen the axon microtubulin polymerization capacity of the nerve injury part, and the nerve repair efficiency and the function recovery quality are remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of stem cell repair, and specifically relates to a functional polypeptide that promotes endometrial stem cells to repair clitoral sensitive nerves and its application. Background Art

[0002] The clitoris is the most sensitive of the female external genitalia. Its innervation is primarily through the dorsal clitoral nerve, a branch of the pudendal nerve. This nerve is primarily responsible for transmitting signals of touch, pain, and sexual pleasure to the clitoral mucosa and body. The function of the clitoral sensitive nerve directly influences a woman's sexual perception, arousal, and orgasm, and is a key structure for maintaining normal sexual psychology and reproductive health.

[0003] However, the clitoral sensitive nerve is easily damaged by various factors due to its superficial anatomical position (located under the clitoral hood and mucosa) and slender course. Common clitoral sensitive nerve injuries include mechanical injury during childbirth, accidental injury during gynecological surgery, chronic infectious inflammation, and trauma. Currently, the repair methods for clitoral sensitive nerve damage include surgical repair, drug therapy, physical and rehabilitation therapy. Although the above methods can promote nerve repair to a certain extent, they have a weak effect on repairing broken or severely damaged nerve fibers. In addition, the trauma itself will cause certain damage to the clitoral tissue, which may cause new complications such as infection and tissue necrosis.

[0004] Stem cell therapy, as an emerging treatment approach, has shown tremendous potential in the field of neural repair. Stem cells possess the properties of self-renewal and multipotency, capable of differentiating into a variety of cell types, including neurons. While stem cell therapy holds promise for neural repair, its application to clitoral sensitive nerve repair presents challenges. Commonly used stem cell sources, such as bone marrow mesenchymal stem cells (BMSCs) and adipose-derived stem cells (ADSCs), are complex to obtain and may pose a risk of donor site damage. Endometrial stem cells, on the other hand, are a type of adult stem cell with multipotential differentiation potential. They can differentiate into a variety of cell types, including neurons, endothelial cells, and smooth muscle cells. Endometrial stem cells also possess robust self-renewal capacity and can be cultured and expanded long-term in vitro, providing a sufficient cell source for these applications. Furthermore, endometrial stem cells are relatively easy to obtain through routine gynecological procedures, such as curettage. This method of harvesting is less damaging to the donor, and the endometrial tissue has a strong regenerative capacity, allowing for rapid recovery. However, current approaches to repair the clitoral sensitive nerve present challenges. Because the clitoral sensitive nerve is located beneath the clitoral hood and mucosa, the local tissue is loose and richly supplied with blood. However, nerve fiber damage can lead to secondary inflammation, oxidative stress, and scarring. These microenvironmental factors can inhibit the survival and differentiation of endometrial stem cells into neural cells. To address these issues, we have developed a functional peptide that promotes the repair of the clitoral sensitive nerve by endometrial stem cells and its application. Summary of the Invention

[0005] The purpose of the present invention is to address the deficiencies of the existing technology and provide a functional polypeptide and its application for promoting the repair of clitoral sensitive nerves by endometrial stem cells.

[0006] One aspect of the present invention provides endometrial stem cells for clitoral sensitive nerve repair. Currently, endometrial stem cells have multipotential differentiation potential, capable of differentiating into neuron-like cells and glial cells. These cells can directly replace damaged nerve cells in clitoral sensitive nerve repair, promoting nerve fiber regeneration and functional recovery. Endometrial stem cells can also differentiate into Schwann cells. Schwann cells play a key role in the myelination process of the peripheral nervous system, wrapping around nerve fibers to form myelin, thereby accelerating the conduction of nerve signals. In clitoral sensitive nerve repair, the generation of Schwann cells facilitates nerve fiber myelination, improving nerve conduction efficiency and promoting nerve function recovery. Laminin (LN) is a core functional component of the basement membrane (BM). As a key component of the extracellular matrix (ECM), laminin plays an irreplaceable role in tissue development, injury repair, and nerve regeneration by mediating cell adhesion, migration, differentiation, and signal transduction. High laminin expression has multiple biological implications for clitoral sensitive nerve repair. It significantly accelerates nerve repair by maintaining basement membrane structure, promoting neuronal cell adhesion and axonal growth, and synergizing with other ECM components and regulating signaling pathways. Functional peptides, endometrial stem cells, and Schwann cells can collectively improve the local microenvironment, providing more favorable conditions for neuronal growth and repair. This synergistic effect can reduce inflammation, alleviate oxidative stress, and promote angiogenesis, thereby providing comprehensive support for nerve repair. Inhibiting the expression of the homeobox gene HOXA1 (Homeobox A1) plays a key regulatory role in clitoral sensitive nerve repair. HOXA1, a core transcription factor in early embryonic development, significantly delays nerve regeneration in adult neural tissue by inhibiting Schwann cell function, interfering with neuroinflammatory regulation, and hindering myelination. HOXA1 inhibits Schwann cell function by arresting the cell cycle and suppressing the expression of myelin-related genes. The present invention uses functional polypeptides to cooperate with endometrial stem cells and Schwann cells to express laminin while inhibiting HOXA1 expression, thereby achieving effective repair of clitoral sensitive nerves.

[0007] Furthermore, the present invention provides a functional polypeptide that promotes the repair of clitoral sensitive nerves by endometrial stem cells, wherein the functional polypeptide includes an NGF mimetic peptide and an antioxidant peptide, wherein the amino acid sequence of the NGF mimetic peptide is shown in SEQ ID NO: 1, and the amino acid sequence of the antioxidant peptide is shown in SEQ ID NO: 2, and the NGF mimetic peptide and the antioxidant peptide are obtained by library construction and screening.

[0008] The functional polypeptide provided by the application can also be modified in a conventional manner in the art, which can maintain the corresponding activity of the functional polypeptide or have better anti-digestion properties.

[0009] To achieve this goal, we can generate a diversity library with desired properties by changing the amino acids at one or more specific positions in the functional polypeptide. In the process of constructing the library, each independent position in the loop sequence is screened and optimized one by one. If necessary, if the change of some positions obviously leads to the decrease of the activity of the functional polypeptide, these positions will be excluded from the change range to ensure that the core function of the polypeptide is not affected.

[0010] In actual operation, the skilled person in the art can optimize the polypeptide by using the "conservative" amino acid substitution strategy. Such conservative substitution refers to replacing an amino acid in a polypeptide sequence with another amino acid with similar chemical structure. Since the amino acids before and after the replacement are relatively close in nature, such change usually has little or even no effect on the overall function, activity and other biological properties of the polypeptide. This strategy has been widely recognized and applied in the relevant research field. The skilled person can select the appropriate type and combination of conservative amino acid substitution according to the specific needs, so as to optimize and improve the polypeptide without significantly changing its key properties.

[0011] In the application, the preparation method of the functional polypeptide comprises: Design and synthesize a diversity peptide library based on the target function, wherein the target function includes NGF mimetic function and antioxidant function, and the library construction technology of the diversity peptide library is a synthetic peptide library or a phage display peptide library; Affinity screening and antioxidant function screening are performed on the diversity peptide library, wherein the screening strategy is preliminary screening, rescreening and final screening; Obtain the amino acid sequence numbers of the NGF mimetic peptide and the antioxidant peptide after the final screening, respectively, and couple the amino acids one by one based on the designed sequence numbers. And use Fmoc protection strategy to gradually construct the peptide chain, after each coupling step, use piperidine and other reagents to remove the Fmoc protecting group, to prepare the NGF mimetic peptide and the antioxidant peptide; Mix the prepared NGF mimetic peptide and antioxidant peptide, and the ratio of the NGF mimetic peptide and the antioxidant peptide is 1:2, and sterilize the mixed functional polypeptide solution to ensure sterility, to prepare the functional polypeptide.

[0012] Furthermore, the present invention also provides a pharmaceutical composition for repairing clitoral sensitive nerves, which contains endometrial stem cells, Schwann cells and functional polypeptides. The functional polypeptides include NGF mimetic peptides and antioxidant peptides. The sequence of the NGF mimetic peptide is shown in SEQ ID NO: 1, and the sequence of the antioxidant peptide is shown in SEQ ID NO: 2.

[0013] In the present invention, the pharmaceutical composition further comprises: a neural differentiation inducing factor, a homing modifying molecule, and a HOXA1 regulating agent, wherein the neural differentiation inducing factor is NGF or BDNF, and the pharmaceutical composition is in the form of a suspension, a lyophilized powder, or a hydrogel.

[0014] Furthermore, the present invention also provides the use of a functional polypeptide in preparing a pharmaceutical composition for repairing clitoral sensitive nerves, wherein the functional polypeptide includes an NGF mimetic peptide and an antioxidant peptide.

[0015] Furthermore, the present invention also provides the use of endometrial stem cells, Schwann cells and functional polypeptides in preparing a pharmaceutical composition for repairing clitoral sensitive nerves.

[0016] Furthermore, the present invention also provides the use of endometrial stem cells and functional polypeptides in preparing a pharmaceutical composition for repairing clitoral sensitive nerves.

[0017] Furthermore, the present invention also provides the use of Schwann cells and functional polypeptides in preparing a pharmaceutical composition for repairing clitoral sensitive nerves.

[0018] Furthermore, the pharmaceutical composition provided by the present invention further comprises a pharmaceutically acceptable carrier.

[0019] Furthermore, the present invention also provides a method for repairing clitoral sensitive nerves using endometrial stem cells, the method comprising: obtaining an endometrial tissue sample, washing the endometrial tissue sample, then mixing the endometrial tissue sample with 0.3% trypsin, centrifuging, and discarding the supernatant to obtain a digested endometrial tissue sample; Isolating and identifying endometrial stem cells from endometrial tissue samples, and pre-treating endometrial stem cells; The separated endometrial stem cells are centrifuged, the supernatant is discarded, and the endometrial stem cells are washed with PBS. The endometrial stem cells are induced to differentiate into Schwann cells, and the surface of the endometrial stem cells is modified to enhance the homing ability of the endometrial stem cells to the site of clitoral sensitive nerve damage. The induction factors for inducing the endometrial stem cells to differentiate into Schwann cells include nerve growth factor, sonic hedgehog factor, or small nucleic acid factor. The differentiation induction temperature is 35-40°C, 7.5% carbon dioxide, and the differentiation induction time is 7-15 days. Endometrial stem cells and Schwann cells are transformed with functional peptides, and pre-prepared functional peptides are introduced into endometrial stem cells and Schwann cells by electroporation for stable expression; Then the quantity is 2×10 5 Endometrial stem cells and Schwann cells introduced with functional peptides were dissolved in 120 mg of normal saline with 25 mg of sodium alginate, and 20 mg of calcium chloride aqueous solution was added to the normal saline and mixed evenly to prepare a combined gel; The prepared combined gel is taken and injected into the submucosal layer of the clitoris by local injection or stent loading.

[0020] Furthermore, the pharmaceutical composition provided by the present invention also contains a pharmaceutically acceptable carrier.

[0021] Among the present invention, useful solid carrier comprises the solid of fine fragmentation, such as talc, clay, microcrystalline cellulose, silica, alumina etc.Useful liquid carrier comprises water, hydroxyalkane or glycols or water-alcohol / glycol admixture, wherein functional polypeptide of the present invention can dissolve or disperse with effective level, optionally with the help of nontoxic surfactant.Can add adjuvant such as flavoring and other antimicrobial agent, so that the characteristic of given use is optimized.The liquid composition obtained can be used for impregnated bandage and other dressing by absorbent pad application, perhaps uses pump type or aerosol sprayer to be sprayed into the infected area.

[0022] Furthermore, the present invention provides pharmaceutical compositions in the form of tablets, troches, pills, capsules, and the like.

[0023] The tablets, troches, pills, and capsules may also contain the following: binders, lubricants, disintegrants, solubilizers, diluents, stabilizers, suspending agents, pigments, flavorings, etc.; preservatives, solubilizers, stabilizers, etc. for injectable formulations; and bases, diluents, lubricants, preservatives, etc. for topical formulations. In addition to the above-mentioned types of substances, they may also contain liquid carriers, such as vegetable oils or polyethylene glycols. Various other substances may be present as coatings or otherwise modify the physical form of the solid unit dosage form. For example, tablets, pills, or capsules may be coated with glue, wax, shellac, or sugar. Syrups or elixirs may contain the functional polypeptides of the present invention, sucrose or fructose as sweeteners, methylparaben and propylparaben as preservatives, dyes, and flavorings such as cherry or orange flavorings. Of course, any substance used in the preparation of any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amount used. In addition, the functional polypeptides of the present invention may be incorporated into sustained-release formulations and devices.

[0024] The present invention may also include water-soluble additives, for example, to optimize release rate or stabilize the drug. In vivo, the water-soluble additives used herein are solid at room temperature, and 1 gram of the additives can be dissolved in less than 100 mL of water, preferably less than 5 mL. Water-soluble additives used herein are not limited, as long as they are medically / pharmaceutically acceptable, and include, for example, sugars, salts, amino acids, and bile salts. Specifically, sugars used herein include, for example, glucose, mannitol, lactose, trehalose, sucrose, erythritol, sorbitol, and xylitol; preferably, glucose, mannitol, and lactose. Salts used herein include, for example, sodium chloride, potassium chloride, and calcium chloride; preferably, sodium chloride. Amino acids used herein may include leucine, isoleucine, valine, proline, phenylalanine, methionine, tryptophan, serine, glutamine, threonine, cysteine, asparagine, tyrosine, aspartic acid, glutamic acid, lysine, arginine, and histidine; preferably, lysine and arginine. Bile salts used herein include, for example, primary bile salts such as sodium cholate and sodium chenodeoxycholate; secondary bile salts such as sodium deoxycholate and sodium lithocholate; and conjugated bile salts such as sodium glycocholate and sodium taurocholate; and preferably sodium cholate, sodium deoxycholate and sodium glycocholate. More preferably, the water-soluble additive is sodium chloride and / or sodium deoxycholate.

[0025] Those skilled in the art can determine the appropriate dosage and range of the functional polypeptide used, for example, based on in vitro and / or in vivo testing and / or other knowledge of compound dosage. These factors are well known to those of ordinary skill in the art and can be solved only by routine experimentation. In some embodiments, a maximum dose is used, i.e., the highest safe dose based on reasonable medical judgment.

[0026] Compared with the prior art, the embodiments of the present application have the following beneficial effects: The functional polypeptides and applications provided by the present invention that promote the repair of clitoral sensitive nerves by endometrial stem cells achieve clitoral sensitive nerve repair by modifying endometrial stem cells and Schwann cells with functional polypeptides and delivering them through local sustained-release gel. The functional polypeptides can activate Laminin expression and regulate the balance of ECM degradation, thereby reducing collagen deposition in scar tissue. The functional polypeptides can also promote the secretion of matrix metalloproteinases by endometrial stem cells and Schwann cells to further degrade scar fibers. At the same time, the functional polypeptides can effectively inhibit HOXA1 expression to enhance axon extension ability, while strengthening the axon microtubule polymerization ability at the nerve injury site, systematically solving the problems of inflammation, oxidative stress and scar inhibition in the microenvironment after clitoral nerve fiber injury, and significantly improving the efficiency of nerve repair and the quality of functional recovery. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The results show the effects of different peptides on the relative expression of Laminin and HOXA1 in endometrial stem cells.

[0028] Figure 2 The results show the effects of different concentrations of functional peptides on the relative expression of Laminin and HOXA1 in endometrial stem cells.

[0029] Figure 3 The results show the effects of different concentrations of functional peptides on the relative expression of Laminin and HOXA1 in Schwann cells.

[0030] Figure 4 Shown are the behavioral reflectivity test results of rats in Tests 1 to 6, the positive control group, and the model group in the embodiments of the present invention.

[0031] Figure 5 The maximum scar thickness test results of Tests 1 to 6, the positive control group, and the model group in the embodiments of the present invention are shown.

[0032] Figure 6 The results of the Laminin positive area tests in Tests 1 to 6, the positive control group, and the model group in the embodiments of the present invention are shown.

[0033] Figure 7 The results of axon density tests in tests 1 to 6, the positive control group, and the model group in the embodiments of the present invention are shown. DETAILED DESCRIPTION

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this application belongs; the terms used in the specification of the application herein are only for the purpose of describing specific embodiments and are not intended to limit this application; the terms "including" and "having" and any variations thereof in the specification and claims of this application and the above-mentioned figure descriptions are intended to cover non-exclusive inclusions.

[0035] Example 1 Preparation of functional peptides: A diverse peptide library is designed and synthesized based on target functions, including NGF mimetic function and antioxidant function. The diverse peptide library is constructed using synthetic peptide library or phage display peptide library. The design of antioxidant peptides is based on the characteristics of known antioxidant peptides (glutathione GSH, superoxide dismutase mimetic peptides), the "NNK" degenerate codon is designed, and a phage display peptide library is constructed to cover a wide range of amino acid combinations to screen for highly active sequences. The diverse peptide library of NGF mimetic peptides is synthesized using Wang resin for solid phase synthesis, while the M13 phage is used as the carrier for the construction of the antioxidant peptide phage display peptide library, and a phage library is designed and constructed. The diverse peptide library was screened for affinity and antioxidant function, with the screening strategy consisting of primary screening, secondary screening, and final screening; In an embodiment of the present invention, the purpose of affinity screening of a diverse peptide library is to screen NGF mimetic peptides, and the screening method for NGF mimetic peptides is as follows: screen candidate NGF mimetic peptide sequences with high affinity to a receptor (which may be Laminin); in the initial screening, the receptor fusion protein is coated in a 96-well plate and coated overnight at 4°C, and then the candidate NGF mimetic peptide is added and incubated at 37°C for 1-1.5 hours, and the unbound candidate NGF mimetic peptide is washed away with 0.2% PBS solution; in the rescreening, the concentration of the receptor fusion protein is reduced to gradually screen candidate NGF mimetic peptides with high affinity; in the final screening, 48 groups of candidate NGF mimetic peptides are selected and their binding activity with the receptor fusion protein is detected, thereby obtaining the final selected NGF mimetic peptide sequence with the highest binding activity.

[0036] The amino acid sequence numbers of the final screened NGF mimetic peptide and antioxidant peptide were obtained, and amino acids were coupled one by one based on the designed sequence numbers. The peptide chain was gradually constructed using the Fmoc protection strategy. After each coupling step, the Fmoc protecting group was removed using reagents such as piperidine to obtain the NGF mimetic peptide and antioxidant peptide; In the embodiment of the present invention, when amino acids are coupled one by one based on the designed sequence number, the coupling conditions are activation with 0.5M HBTU, 0.2M HOBT, and 0.3M DIPEA, a temperature of 35-37° C., and a time of 25-30 min.

[0037] The prepared NGF mimetic peptide and antioxidant peptide are mixed, and the ratio of the NGF mimetic peptide and the antioxidant peptide can be 1:2, and the mixed functional polypeptide solution is sterilized to ensure sterility to obtain a functional polypeptide. Wherein, when the mixed functional polypeptide solution is sterilized, it is filtered with a 0.1μm sterile filter membrane and sterilized with 20kGy gamma rays. The solvent of the functional polypeptide solution finally obtained is physiological saline with a pH of 7.3. The functional polypeptide includes an NGF mimetic peptide and an antioxidant peptide, the NGF mimetic peptide sequence is shown in SEQ ID NO: 1, and the antioxidant peptide sequence is shown in SEQ ID NO: 2.

[0038] Example 2 Endometrial stem cell culture and identification: 120 female adult rats were selected and divided into 6 groups, namely experimental group 1, experimental group 2, experimental group 3, association group 1, association group 2, and blank group. The rats were raised for 14 days to adapt to the environment and fasted for 10 hours before surgery. A sterilized skin preparation knife was used to make a longitudinal incision in the middle of the rat abdomen under sterile conditions to expose the position of the rat uterus. Endometrial tissue samples were obtained from the rat cervix and cleaned. During the cleaning process, PBS solution was used to wash 3-5 times, and the endometrial fat tissue was removed. The endometrial tissue samples were cut into pieces with sterile ophthalmic scissors, and then the endometrium was The tissue sample was mixed with 0.3% trypsin, centrifuged, and the supernatant was discarded. When the endometrial tissue sample was mixed with 0.3% trypsin, the mixing conditions were 37°C, 5% carbon dioxide incubator for digestion for 10 minutes, after which the trypsin was discarded and the tissue fragments were digested with preheated 0.2% collagenase and 0.02% EDTA. A magnetic stirrer was used to promote digestion, and an equal volume of DMEM complete medium containing 20% ​​FBS was used to terminate the digestion. The digested mixture was collected and placed in a 5°C centrifuge, centrifuged at 1500 rpm for 10 minutes, and the supernatant was discarded to obtain the digested endometrial tissue sample. Endometrial stem cells were isolated and identified from endometrial tissue samples. The endometrial stem cells were pretreated. When isolating endometrial stem cells, the digested endometrial tissue samples were filtered through 100 μm, 80 μm, and 50 μm filter membranes to collect the endometrial stem cells. The cells were then resuspended in 2 mL of DMEM complete medium, which consisted of DMEM / F12, 20% FBS, 150 U / mL penicillin, 200 μg / mL streptomycin, and a functional peptide. The resuspended cells were plated in a 20 cm culture dish and cultured in a 37°C, 5% carbon dioxide incubator. After 14 days, adherent cells showing clonal growth were collected and transferred to 48-well plates for culture. When the adherent cells reached nearly 85% confluence, they were passaged. The passaged cells were labeled P1 and the cells passaged again were labeled P2. Morphological observation and cell surface antigen determination were performed after culturing to the fourth passage. The endometrial stem cell suspension was then counted at 2×10 3 The endometrial stem cell suspension was dispensed into a 96-well plate at a density of 150 μL per well, and the culture plate was sterilized and placed in a carbon dioxide incubator for culture at 37°C and 5% carbon dioxide concentration. The NGF mimetic peptide, antioxidant peptide, functional peptide, associated peptide 1, associated peptide 2, and ineffective peptide prepared in Example 1 were introduced into the endometrial stem cell suspension of experimental group 1, experimental group 2, experimental group 3, associated group 1, associated group 2, and blank group at an addition amount of 5 μg / mL per well. The cells were cultured for 4 days, and the cells in each well were collected into a 4 mL EEP tube. The relative expression levels of Laminin and HOXA1 (based on the blank group) were detected by Western blot. Table 1 and Figure 1 The results show the effects of different peptides on the relative expression of Laminin and HOXA1 in endometrial stem cells.

[0039] Table 1 As can be seen from Table 1, the functional polypeptides provided by the present invention can synergistically cooperate with endometrial stem cells to significantly increase the expression of Laminin and effectively inhibit the expression of HOXA1. Among them, the antioxidant peptide can reduce oxidative stress, thereby downregulating the expression of HOXA1, and the NGF mimetic peptide can activate the PI3K / Akt pathway through the TrkA receptor, thereby inhibiting the transcription of HOXA1 mediated by caspase-3. At the same time, the combination of NGF mimetic peptide and antioxidant peptide in the functional polypeptide realizes the synergistic effect of NGF simulation + antioxidant. The combination of NGF mimetic peptide and antioxidant peptide is better than the use of NGF mimetic peptide and antioxidant peptide alone in increasing the expression of Laminin and inhibiting the expression of HOXA1. The functional polypeptide effectively promotes basement membrane repair by combining NGF mimetic peptide and antioxidant peptide, and provides a stable physical scaffold for nerve regeneration.

[0040] Example 3 Effect of functional polypeptide concentration on the expression of Laminin and HOXA1 in endometrial stem cells: The endometrial stem cells prepared in Example 2 were taken and 2×10 3 The endometrial stem cell suspension was dispensed into a 96-well plate at a density of 150 μL per well. The culture plate was sterilized and placed in a carbon dioxide incubator for culture at 37°C and 5% carbon dioxide. The functional polypeptide prepared in Example 1 was introduced into the endometrial stem cell suspension at an addition rate of 0 μg / mL (blank group), 1 μg / mL, 3 μg / mL, 6 μg / mL, 8 μg / mL, and 12 μg / mL per well, respectively. The cells were cultured for 4 days, and the cells in each well were collected into a 4 mL LEP tube. The relative expression levels of Laminin and HOXA1 (based on the blank group) were detected by Western blot. Table 2 and Figure 2 The results show the effects of different concentrations of functional peptides on the relative expression of Laminin and HOXA1 in endometrial stem cells.

[0041] Table 2 As can be seen from Table 2, with respect to Laminin expression, relative to the blank group, as the concentration of the functional polypeptide continued to increase, the expression level of Laminin showed a trend of dependent increase, and even in the case of extremely high concentrations of the functional polypeptide, no obvious toxicity caused by the functional polypeptide was shown. Therefore, it can be seen that the addition of the functional polypeptide in this example has a promoting effect on Laminin expression in endometrial stem cells, thereby providing a microenvironment for the differentiation of endometrial stem cells into neurons and Schwann cells, and also facilitating the repair of basement membrane during clitoral sensitive nerve repair. With respect to HOXA1 expression, relative to the blank group, as the concentration of the functional polypeptide continued to increase, the expression level of HOXA1 showed a trend of decreasing inhibition, and even in the case of extremely high concentrations of the functional polypeptide, no obvious toxicity caused by the functional polypeptide was shown.

[0042] Example 4 Effect of functional polypeptide concentration on the expression of Laminin and HOXA1 in Schwann cells: The endometrial stem cells separated in Example 2 were taken, and after centrifugation, the supernatant was discarded, and the endometrial stem cells were washed with PBS to induce the endometrial stem cells to differentiate into Schwann cells, modify the surface of the endometrial stem cells, and enhance the homing ability of the endometrial stem cells to the site of clitoral sensitive nerve damage; wherein, when inducing the differentiation of endometrial stem cells into Schwann cells, the induction factors include nerve growth factor, sonic hedgehog factor or small nucleic acid factor, the induction differentiation temperature is 35-40 ° C, 7.5% carbon dioxide, and the induction differentiation time is 7-15 days. Then, 2×10 3 The endometrial stem cell suspension was dispensed into a 96-well plate at a density of 150 μL per well. The culture plate was sterilized and placed in a carbon dioxide incubator for culture at 37°C and 5% carbon dioxide. The functional polypeptide prepared in Example 1 was introduced into the endometrial stem cell suspension at an addition rate of 0 μg / mL (blank group), 1 μg / mL, 3 μg / mL, 6 μg / mL, 8 μg / mL, and 12 μg / mL per well, respectively. The cells were cultured for 4 days, and the cells in each well were collected into a 4 mL LEP tube. The relative expression levels of Laminin and HOXA1 (based on the blank group) were detected by Western blot. Table 3 and Figure 3 The results show the effects of different concentrations of functional peptides on the relative expression of Laminin and HOXA1 in Schwann cells.

[0043] Table 3 As can be seen from Table 3, similar to Example 3, in this example, the functional polypeptide cooperates with Schwann cells to achieve the effects of activating Laminin transcription and inhibiting HOXA1 expression. Schwann cells are the core cells of the basement membrane and can secrete Laminin to participate in the construction of the basement membrane around the nerve. In this example, with the increase of the concentration of the functional polypeptide, the expression level of Laminin gradually increases. When the concentration of the functional polypeptide increases to a certain concentration, the expression level of Laminin shows a gentle and stable trend. Compared with the mode of cooperation between endometrial stem cells and functional polypeptides in Example 3, the synergistic cooperation between the functional polypeptide and Schwann cells has a more efficient responsiveness and expression level. As for the HOXA1 test, with the increase of the concentration of the functional polypeptide, the expression level of HOXA1 gradually decreases. In this example, the functional polypeptide can block the positive regulation of inflammatory factors by inhibiting HOXA1 expression, and the oxidative activity characteristics of the functional polypeptide can reduce the damage of oxidative stress to Schwann cells, thereby ensuring the anti-inflammatory function of Schwann cells. The functional polypeptide acts on Schwann cells to promote the secretion of matrix metalloproteinases by Schwann cells, thereby degrading collagen fibers in scar tissue and promoting ECM remodeling; and inhibiting excessive proliferation of fibroblasts.

[0044] Example 5 Functional peptide safety test: In this example, 120 SD female rats were randomly divided into 6 groups, 20 in each group, and the six groups of SD rats were divided into high-, medium- and low-dose groups of functional peptides in turn, and 3 groups of PBS solvent control groups were used to observe the abnormal toxic reactions (body weight, general condition) of rats in each group. 24 hours after administration, skin pathology, blood biochemical indicators, and toxic effects on major organs such as the liver, kidney and spleen were examined. The results showed that as the treatment time increased, the high-dose peptide group showed better therapeutic effects, which were significantly better than the therapeutic effects of other groups. The above results show that the functional peptide has a strong characteristic of promoting the expression of Laminin and inhibiting the expression of HOXA1 in endometrial stem cells. At the same time: Skin pathological examination of rats was normal: no pathological conditions such as edema and erythema were found on the skin. Skin pathological examination showed that the squamous endothelial cell layer was intact, the cell layers were clear and arranged neatly, the hair follicle structure and glandular morphology were clear, and no abnormal reactions such as basal cell changes, hyperplasia and hemorrhage were found, indicating that the functional polypeptide had no obvious irritation and toxicity to skin tissue.

[0045] Normal blood biochemical indicators: liver and kidney function (ALT, AST, BUN, CRE), electrolytes (Na + , K + ), blood sugar (GLU) and blood lipids are normal, indicating that the use of functional peptides will not have adverse effects on the body's metabolism and physiological functions, and will not cause systemic toxic reactions.

[0046] Normal major organs: Major organs such as the liver, kidneys, and spleen were all normal. Hematoxylin and eosin staining of liver tissue revealed intact hepatic lobules in all groups, with regular hepatocyte arrangement and no obvious ballooning, necrosis, or inflammatory cell infiltration. Hematoxylin and eosin staining of kidney tissue revealed clear glomerular and tubular structures, with no tubular necrosis, proteinaceous casts, or inflammatory cell infiltration. Hematoxylin and eosin staining of spleen tissue revealed intact splenic corpuscles, evenly distributed lymphocytes, and no necrosis or fibrosis.

[0047] Example 6 The repair effect of functional peptides was verified in a rat dorsal clitoral nerve transection model: 6-week-old SD female rats were housed for 2 weeks to acclimate to the environment and fasted 10 hours before treatment. Rats were anesthetized by injection of 0.8 mL / 100 g of chloral hydrate. The abdomen and perineum of the rats were shaved and disinfected three times with povidone-iodine (1:20 dilution) and 75% ethanol alternately. Sterile drapes were laid to expose the surgical area. A longitudinal incision (2-3 cm in length) was made along the midline of the rat's lower abdomen. The skin, subcutaneous fat, and anterior rectus abdominis sheath were incised layer by layer to expose the extraperitoneal fat. The rats were then separated along the side walls of the vagina to expose the suspensory ligaments of the clitoris. The clitoral body was pulled cranial to expose the neurovascular bundle on the dorsal surface of the clitoris. The dorsal clitoral nerve was then severed with microscissors. The clitoral body was rinsed with normal saline. After hemostasis, the anterior rectus abdominis sheath, subcutaneous tissue, and skin were sutured layer by layer. Immediately after surgery, 7 mg / kg of tramadol was injected intraperitoneally to relieve pain. The model was successfully established after behavioral testing and histological examination. The rats were divided into the following groups for testing: Test 1: Starting on the seventh day after surgery, 0.4 mL of endometrial stem cells were injected into the clitoral area of ​​the rats, with injections every three days for six weeks. Test 2: Endometrial stem cells were modified with functional peptides and introduced into the endometrial stem cells via electroporation for stable expression. The endometrial stem cells, which had been injected with the functional peptides, were then dissolved with 25 mg of sodium alginate in 120 mg of normal saline. 20 mg of calcium chloride aqueous solution was then added to the saline and mixed thoroughly to produce a combined gel. Starting on the seventh day after surgery, 0.4 mL of the combined gel was injected into the clitoral region of the rats, every three days for six weeks. Test 3: Starting on the seventh day after surgery, 0.4 mL of prepared Schwann cells were injected into the clitoral area of ​​the rats, with injections every three days for a total of six weeks; Test 4: Schwann cells were modified with functional peptides and introduced into the Schwann cells via electroporation for stable expression. The Schwann cells containing the functional peptides were then dissolved with 25 mg of sodium alginate in 120 mg of normal saline. 20 mg of calcium chloride aqueous solution was added to the saline and mixed thoroughly to produce a combined gel. Starting on the seventh day after surgery, 0.4 mL of the combined gel was injected into the clitoral region of the rats, with injections occurring every three days for a total of six weeks. Test 5: Starting on the seventh day after surgery, 0.4 mL of endometrial stem cells and Schwann cells were injected into the clitoral area of ​​the rats, with injections every three days for a total of six weeks. Test 6: Endometrial stem cells and Schwann cells were modified with functional peptides, and the functional peptides were introduced into the endometrial stem cells and Schwann cells by electroporation for stable expression. The endometrial stem cells and Schwann cells introduced with the functional peptides were then dissolved with 25 mg of sodium alginate in 120 mg of normal saline, and 20 mg of calcium chloride aqueous solution was added to the normal saline and mixed evenly to prepare a combined gel. Starting on the seventh day after surgery, 0.4 mL of the combined gel was injected into the clitoral area of ​​the rats, with injections every three days for a total of six weeks.

[0048] Positive control group: Starting from the seventh day after surgery, 0.4 mL of nerve growth factor (NGF) was injected into the injured clitoral area of ​​rats, and the injection was given every three days for a total of six weeks.

[0049] Model group: Starting from the seventh day after surgery, 0.4 mL of PBS solution was injected into the injured clitoral area of ​​rats, and the injection was performed every three days for a total of six weeks.

[0050] Functional and histological tests were performed on Tests 1 to 6, the positive control group, and the model group in this embodiment. The functional test included stimulating the rat clitoral tissue at 8 and 10 weeks after surgery and recording the rat hip lifting and urination reflex frequencies. Each rat was tested 3 times, and the rat behavioral reflex rate was calculated. The rat behavioral reflex rate test results are shown in Tables 4 and Figure 4 When the rats were subjected to histological examination, the maximum thickness of the scar at the site of clitoral sensitive nerve injury was recorded by HE staining. The test results are shown in Table 4 and Figure 5 At the same time, the Laminin-positive area ratio of the clitoral sensitive nerve injury site in rats at 8 weeks after surgery was tested to verify the integrity of the basement membrane after surgery. The test results are shown in Table 4 and Figure 6 As shown. Axon density was calculated for test 1 to test 6, positive control group and model group (NF-200 + Area ratio, %), the test results are shown in Table 4 and Figure 7 shown.

[0051] Table 4 As can be seen from Table 4, compared with the model group and the positive control group, Tests 1 to 6 in this embodiment showed significant advantages in promoting the recovery of behavioral reflexes after clitoral sensitive nerve injury in rats, reducing the maximum thickness of scars at the injury site, promoting the recovery of the ratio (integrity) of Laminin-positive area of ​​the basement membrane, and increasing axon density. It can be seen that the functional polypeptides, endometrial stem cells, Schwann cells and their combination help to reduce scar formation and promote tissue repair. The test results show that the functional polypeptides relieve the inhibition of axon growth by inhibiting HOXA1 expression; at the same time, the functional polypeptides can cooperate with neurotrophic factors to further enhance the axonal tubulin polymerization ability.

[0052] In summary, the present invention provides a functional polypeptide and application that promotes the repair of clitoral sensitive nerves by endometrial stem cells. The repair of clitoral sensitive nerves is achieved by modifying endometrial stem cells and Schwann cells with functional polypeptides and delivering them through local sustained-release gel. The functional polypeptide can activate Laminin expression and regulate the balance of ECM degradation, thereby reducing collagen deposition in scar tissue; the functional polypeptide can also promote the secretion of matrix metalloproteinases by endometrial stem cells and Schwann cells to further degrade scar fibers. At the same time, the functional polypeptide can also effectively inhibit HOXA1 expression to enhance axon extension ability, while strengthening the axon microtubule polymerization ability of nerve injury sites, systematically solving the problems of inflammation, oxidative stress and scar inhibition in the microenvironment after clitoral nerve fiber injury, and significantly improving the efficiency of nerve repair and the quality of functional recovery.

[0053] It should be noted that for the aforementioned embodiments, for simplicity of description, they are all expressed as a series of action combinations. However, those skilled in the art should be aware that the present invention is not limited by the order of the actions described, because according to the present invention, certain steps may be performed in other orders or simultaneously. Secondly, those skilled in the art should also be aware that the embodiments described in this specification are all preferred embodiments, and the actions and modules involved are not necessarily required by the present invention.

[0054] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the scope of protection of the invention. Obviously, the embodiments described are only some embodiments of the present invention, rather than all embodiments. Based on these embodiments, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in this field can still combine, add, delete or make other adjustments to the features in the various embodiments of the present invention according to the circumstances without conflict, without making creative work, so as to obtain different other technical solutions that do not deviate from the concept of the present invention in essence, and these technical solutions also fall within the scope of protection of the present invention.

Claims

1. A functional polypeptide that promotes the repair of clitoral sensitive nerves by endometrial stem cells, characterized in that: The functional polypeptide includes an NGF mimetic peptide and an antioxidant peptide. The sequence of the NGF mimetic peptide is shown in SEQ ID NO: 1, and the sequence of the antioxidant peptide is shown in SEQ ID NO:

2.

2. A pharmaceutical composition for repairing clitoral sensitive nerves, comprising the functional polypeptide according to claim 1, characterized in that: The pharmaceutical composition contains endometrial stem cells, Schwann cells and functional polypeptides. The functional polypeptides include NGF mimetic peptides and antioxidant peptides. The sequence of the NGF mimetic peptide is shown in SEQ ID NO: 1, and the sequence of the antioxidant peptide is shown in SEQ ID NO:

2.

3. Use of the functional polypeptide according to claim 1 in preparing a pharmaceutical composition for repairing clitoral sensitive nerves, wherein: The functional polypeptides include NGF mimetic peptides and antioxidant peptides.

4. Use of the functional polypeptide, endometrial stem cells and Schwann cells according to claim 1 in the preparation of a pharmaceutical composition for repairing clitoral sensitive nerves.

5. Use of the functional polypeptide and endometrial stem cells according to claim 1 in the preparation of a pharmaceutical composition for repairing clitoral sensitive nerves.

6. Use of the functional polypeptide and Schwann cells according to claim 1 in preparing a pharmaceutical composition for repairing clitoral sensitive nerves.

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