A functional polypeptide for promoting repair of clitoral sensitive nerves by endometrial stem cells and application thereof

Abstract

The application discloses a functional polypeptide for promoting endometrial stem cells to repair clitoral sensitive nerves and application, and belongs to the technical field of stem cell repair. The functional polypeptide is used for modifying endometrial stem cells, Schwann cells and local slow-release gel delivery to realize clitoral sensitive nerve repair. The functional polypeptide can activate Laminin expression and control ECM degradation balance, so as to reduce collagen deposition in scar tissue. The functional polypeptide can also effectively inhibit HOXA1 expression, enhance axon extension capacity and strengthen axon microtubule protein polymerization capacity at a nerve injury site, and significantly improves nerve repair efficiency and functional recovery quality.

Description

Technical Field

[0001] This invention belongs to the field of stem cell repair technology, specifically relating to a functional polypeptide that promotes the repair of clitoral sensitive nerves by endometrial stem cells and its application. Background Technology

[0002] The clitoris is the most sensitive sexual organ in the female external genitalia. Its nerve supply is primarily provided by the dorsal clitoral nerve, a branch of the pudendal nerve, which is mainly responsible for transmitting tactile, pain, and sexual pleasure signals to the clitoral mucosa and body. The functional state of the clitoral sensory nerves directly affects a woman's sexual perception, arousal, and orgasm ability, and is a key structure for maintaining normal sexual psychology and reproductive health.

[0003] However, due to their superficial anatomical location (under the clitoral prepuce and submucosa) and delicate course, clitoral sensitive nerves are easily damaged by various factors. Common clitoral sensitive nerve injuries include mechanical injuries during childbirth, accidental injury during gynecological surgery, chronic infectious inflammation, and trauma. Currently, repair methods for clitoral sensitive nerve injuries include surgical repair, drug treatment, and physical and rehabilitation therapy. However, although these methods can promote nerve repair to some extent, their effect on repairing already severed or severely damaged nerve fibers is weak. Furthermore, trauma itself can also cause some damage to the clitoral tissue, which may lead to new complications such as infection and tissue necrosis.

[0004] Stem cell therapy, as an emerging treatment approach, has shown great potential in the field of nerve repair. Stem cells possess self-renewal and multi-lineage differentiation characteristics, capable of differentiating into various cell types, including nerve cells. However, despite the potential of stem cell therapy in nerve repair, some challenges exist in the repair of clitoral sensitive nerves. Commonly used stem cell sources, such as bone marrow mesenchymal stem cells (BMSCs) and adipose-derived stem cells (ADSCs), have relatively complex acquisition processes and may involve donor site damage. In contrast, endometrial stem cells are adult stem cells with multi-lineage differentiation potential. They can differentiate into various cell types, including nerve cells, endothelial cells, and smooth muscle cells. Furthermore, endometrial stem cells have a strong self-renewal capacity, allowing for long-term in vitro culture and expansion, providing a sufficient cell source for applications. Moreover, the acquisition of endometrial stem cells is relatively simple, as they can be obtained through routine gynecological procedures such as curettage. This method of obtaining the clitoral nerve causes less damage to the donor and allows for faster regeneration of the endometrial tissue. However, in current procedures for repairing clitoral nerves, the nerves are located under the clitoral prepuce and submucosa, where the local tissue is loose and richly blood-supplied. Damage to the nerve fibers easily leads to secondary inflammation, oxidative stress, and scarring. These microenvironmental factors inhibit the survival and differentiation of endometrial stem cells into nerve cells. To address these issues, we propose a functional polypeptide that promotes the repair of clitoral nerves by endometrial stem cells and its application. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing a functional polypeptide that promotes the repair of clitoral sensitive nerves by endometrial stem cells and its application.

[0006] In one aspect, this invention provides endometrial stem cells for the repair of clitoral sensitive nerves. Currently, endometrial stem cells possess multi-directional 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. Furthermore, endometrial stem cells can differentiate into Schwann cells. Schwann cells play a crucial role in myelination of the peripheral nervous system, encapsulating nerve fibers to form myelin sheaths, thereby accelerating nerve signal transmission. In clitoral sensitive nerve repair, the generation of Schwann cells contributes to nerve fiber myelination, improves nerve conduction efficiency, and promotes the recovery of nerve function. 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 and damage repair (including nerve regeneration) by mediating cell adhesion, migration, differentiation, and signal transduction. In the repair of clitoral sensitive nerves, high expression of laminin has multiple biological significances. It can significantly accelerate the repair process of nerve injury by maintaining basement membrane structure, promoting nerve cell adhesion and axonal growth, synergizing with other ECM components, and regulating signaling pathways. Functional peptides, endometrial stem cells, and Schwann cells can jointly improve the local microenvironment, providing more favorable conditions for nerve cell growth and repair. This synergistic effect can reduce inflammation, alleviate oxidative stress, and promote angiogenesis, thus providing comprehensive support for nerve repair. Inhibition of the expression of the homeobox gene HOXA1 (Homeobox A1) plays a key regulatory role in the repair of clitoral sensitive nerves. As a core transcription factor in early embryonic development, abnormal expression of HOXA1 in adult neural tissue can significantly delay nerve regeneration by inhibiting Schwann cell function, interfering with neuroinflammatory regulation, and hindering myelination. HOXA1 inhibits Schwann cell function by arresting the cell cycle and inhibiting the expression of myelin-related genes. In this invention, functional peptides are used in synergy 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. The functional polypeptide includes an NGF mimic peptide and an antioxidant peptide. The amino acid sequence of the NGF mimic peptide is shown in SEQ ID NO: 1, and the amino acid sequence of the antioxidant peptide is shown in SEQ ID NO: 2. The NGF mimic peptide and the antioxidant peptide are obtained through library construction and screening.

[0008] The functional peptides provided by the present invention can also be modified in a manner conventional in the art, and the modification can maintain the corresponding activity of the functional peptides or give them better anti-digestion properties.

[0009] To achieve this goal, we can generate diverse libraries with desired properties by altering one or more specific amino acids at certain positions within a functional peptide. During library construction, each individual position within the loop sequence is individually screened and optimized. If necessary, positions where alteration would clearly reduce the activity of the functional peptide will be excluded from modification to ensure the core function of the peptide remains unaffected.

[0010] In practice, those skilled in the art can optimize peptides using a "conservative" amino acid substitution strategy. This conservative substitution refers to replacing a single amino acid in a peptide sequence with another amino acid of similar chemical structure. Because the amino acids before and after the substitution are quite similar in properties, this change usually has little, or even almost no, impact on the overall function, activity, and other biological characteristics of the peptide. This strategy is widely recognized and applied in related research fields. Technicians can select appropriate types and combinations of conservative amino acid substitutions based on specific needs, thereby optimizing and improving the peptide without significantly altering its key properties.

[0011] In this invention, the method for preparing the functional polypeptide includes:

[0012] A diverse peptide library was designed and synthesized based on the target functions, including NGF mimicry and antioxidant functions. The library construction techniques for the diverse peptide library were synthetic peptide library or phage display peptide library.

[0013] Affinity screening and antioxidant function screening were performed on diverse peptide libraries, with the screening strategy consisting of primary screening, secondary screening, and final screening.

[0014] The amino acid sequences of the NGF mimic peptide and antioxidant peptide after final screening were obtained, and amino acids were coupled one by one based on the designed sequence numbers. The peptide chain was then constructed stepwise using the Fmoc protection strategy. After each coupling step, the Fmoc protecting groups were removed using reagents such as piperidine to obtain the NGF mimic peptide and antioxidant peptide.

[0015] The prepared NGF mimic peptide and antioxidant peptide were mixed in a ratio of 1:2, and the mixed functional peptide solution was sterilized to ensure sterility, thus obtaining the functional peptide.

[0016] Furthermore, the present invention also provides a pharmaceutical composition for the repair of clitoral sensitive nerves, the pharmaceutical composition comprising endometrial stem cells, Schwann cells and functional peptides, the functional peptides including NGF mimic peptides and antioxidant peptides, the NGF mimic peptide sequence being as shown in SEQ ID NO: 1, and the antioxidant peptide sequence being as shown in SEQ ID NO: 2.

[0017] In this invention, the pharmaceutical composition further includes: a neural differentiation inducing factor, a homing modification molecule, and a HOXA1 regulatory agent, wherein the neural differentiation inducing factor is NGF or BDNF, and the dosage form of the pharmaceutical composition is a suspension, a lyophilized powder, or a hydrogel.

[0018] Furthermore, the present invention also provides the use of functional peptides in the preparation of pharmaceutical compositions for the repair of clitoral sensitive nerves, wherein the functional peptides include NGF mimic peptides and antioxidant peptides.

[0019] Furthermore, the present invention also provides the use of endometrial stem cells, Schwann cells and functional peptides in the preparation of a pharmaceutical composition for the repair of clitoral sensitive nerves.

[0020] Furthermore, the present invention also provides the use of endometrial stem cells and functional peptides in the preparation of a pharmaceutical composition for the repair of clitoral sensitive nerves.

[0021] Furthermore, the present invention also provides the use of Schwann cells and functional peptides in the preparation of a pharmaceutical composition for the repair of clitoral sensitive nerves.

[0022] Furthermore, the present invention provides a pharmaceutical composition that further contains a pharmaceutically acceptable carrier.

[0023] Furthermore, the present invention also provides a method for repairing clitoral sensitive nerves using endometrial stem cells, the method comprising:

[0024] Endometrial tissue samples were obtained, washed, and then mixed with 0.3% trypsin. After centrifugation, the supernatant was discarded to obtain digested endometrial tissue samples.

[0025] Endometrial stem cells were isolated and identified from endometrial tissue samples, and the endometrial stem cells were pretreated.

[0026] After separation, endometrial stem cells were centrifuged, the supernatant was discarded, and the stem cells were washed with PBS. The endometrial stem cells were then induced to differentiate into Schwann cells. The surface of the endometrial stem cells was modified to enhance their homing ability to the site of clitoral nerve injury. The inducing factors for Schwann cell differentiation included nerve growth factor, sound hedgehog factor, or small nucleic acid factor. The induction temperature was 35-40℃, with 7.5% carbon dioxide, and the induction time was 7-15 days.

[0027] Endometrial stem cells and Schwann cells were modified using functional peptides, and the pre-prepared functional peptides were introduced into endometrial stem cells and Schwann cells for stable expression using electroporation.

[0028] Then the quantities are 2×10 5 Endometrial stem cells with introduced functional peptides, Schwann cells, and 25 mg sodium alginate were dissolved in 120 mg physiological saline. Then, 20 mg calcium chloride aqueous solution was added to the physiological saline and mixed evenly to obtain a combined gel.

[0029] The prepared combined gel was injected into the submucosal layer of the clitoris using either local injection or scaffold loading.

[0030] Furthermore, the pharmaceutical composition provided by this invention also contains a pharmaceutically acceptable carrier.

[0031] In this invention, useful solid carriers include finely fragmented solids such as talc, clay, microcrystalline cellulose, silica, alumina, etc. Useful liquid carriers include water, hydroxyalkyl or glycol derivatives, or water-alcohol / diol blends, wherein the functional peptides of this invention can be dissolved or dispersed at an effective level, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and other antimicrobial agents can be added to optimize properties for a given application. The resulting liquid composition can be applied via absorbent pads, for impregnated bandages and other dressings, or sprayed onto the infected area using a pump or aerosol sprayer.

[0032] Furthermore, the present invention provides pharmaceutical compositions in the form of tablets, pills, granules, capsules, etc.

[0033] The tablets, pills, pellets, and capsules may also contain binders, lubricants, disintegrants, solubilizers, diluents, stabilizers, suspending agents, colorants, flavoring agents, etc., for injectable formulations; preservatives, solvents, stabilizers, etc., for topical formulations; and bases, diluents, lubricants, preservatives, etc., for topical formulations. In addition to the substances of the types mentioned above, it may also contain liquid carriers, such as vegetable oils or polyethylene glycol. Various other substances may be present as coatings or to further modify the physical form of the solid unit dosage form. For example, tablets, pills, or capsules may be coated with gums, waxes, shellac, or sugars. 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. Furthermore, the functional polypeptides of the present invention can be incorporated into sustained-release formulations and devices.

[0034] This invention may also include water-soluble additives for purposes such as optimizing release rate or stabilizing the drug. The water-soluble additives used herein in the in vivo environment are solid at room temperature, and 1g of them is soluble in less than 100mL of water, preferably less than 5mL. There are no limitations on the water-soluble additives used herein, as long as they are medically / pharmaceutically acceptable, and they 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; and glucose, mannitol, and lactose are preferred. Salts used herein include, for example, sodium chloride, potassium chloride, and calcium chloride; and sodium chloride is preferred. Amino acids used herein may be leucine, isoleucine, valine, proline, phenylalanine, methionine, tryptophan, serine, glutamine, threonine, cysteine, asparagine, tyrosine, aspartic acid, glutamic acid, lysine, arginine, and histidine; and lysine and arginine are preferred. The 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 lithochate; and conjugate 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.

[0035] Those skilled in the art can determine the appropriate dosage and range of the functional peptide 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 skilled in the art and can be resolved simply through routine experiments. In some embodiments, a maximum dose is used, i.e., the highest safe dose based on reasonable medical judgment.

[0036] Compared with the prior art, the embodiments of this application have the following main advantages:

[0037] The present invention provides a functional polypeptide and its application for promoting the repair of clitoral sensitive nerves by endometrial stem cells. This is achieved through the modification of endometrial stem cells and Schwann cells with the functional polypeptide and delivery via local sustained-release gel. The functional polypeptide can activate laminin expression and regulate ECM degradation balance, thereby reducing collagen deposition in scar tissue. Furthermore, the functional polypeptide can promote the secretion of matrix metalloproteinases by endometrial stem cells and Schwann cells to further degrade scar fibers. Simultaneously, the functional polypeptide can effectively inhibit HOXA1 expression, enhancing axonal elongation capacity and strengthening the axonal microtubule polymerization capacity at the nerve injury site. This systematically solves the problems of inflammation, oxidative stress, and scar inhibition in the microenvironment after clitoral nerve fiber injury, significantly improving nerve repair efficiency and functional recovery quality. Attached Figure Description

[0038] Figure 1 The results of testing the relative expression levels of Laminin and HOXA1 in endometrial stem cells by different types of peptides are shown.

[0039] Figure 2 The results of testing the relative expression levels of Laminin and HOXA1 in endometrial stem cells at different concentrations of functional peptides are shown.

[0040] Figure 3 The results of testing the relative expression levels of Laminin and HOXA1 in Schwann cells at different concentrations of functional peptides are shown.

[0041] Figure 4 The results of behavioral reflex rate tests of rats in Tests 1-6, the positive control group, and the model group in this embodiment of the invention are shown.

[0042] Figure 5 The results of the maximum scar thickness tests for tests 1-6, the positive control group, and the model group in embodiments of the present invention are shown.

[0043] Figure 6 The results of the Laminin positive area test in Tests 1-6, the positive control group, and the model group in the embodiments of the present invention are shown.

[0044] Figure 7 The results of axonal density tests for tests 1-6, the positive control group, and the model group in embodiments of the present invention are shown. Detailed Implementation

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; the terminology used herein in the specification of the application is for the purpose of describing particular embodiments only and is not intended to limit the application; the terms “comprising” and “having” and any variations thereof in the specification, claims and foregoing description of the drawings are intended to cover non-exclusive inclusion.

[0046] Example 1

[0047] Preparation of functional peptides:

[0048] A diverse peptide library was designed and synthesized based on target functions, including NGF mimicry and antioxidant functions. The library construction techniques for the diverse peptide library were synthetic peptide library or phage display peptide library. The design of the antioxidant peptide was based on the characteristics of known antioxidant peptides (glutathione GSH, superoxide dismutase mimicry peptide), and the design of the "NNK" degenerate codon was carried out. A phage display peptide library was constructed to cover a wide range of amino acid combinations to screen for highly active sequences. The diverse peptide library of NGF mimicry peptide was synthesized in solid phase using Wang resin. When constructing the phage display peptide library of antioxidant peptide, M13 phage was selected as the vector and a phage library was designed and constructed.

[0049] Affinity screening and antioxidant function screening were performed on diverse peptide libraries, with the screening strategy consisting of primary screening, secondary screening, and final screening.

[0050] In this embodiment of the invention, the purpose of affinity screening of the diverse peptide library is to screen for NGF mimic peptides. The screening method for NGF mimic peptides is as follows: Candidate NGF mimic peptide sequences with high affinity for the receptor (which may be Laminin) are screened. In the initial screening, the receptor fusion protein is coated onto a 96-well plate and incubated overnight at 4°C. Then, candidate NGF mimic peptides are added and incubated at 37°C for 1-1.5 hours. Unbound candidate NGF mimic peptides are washed away with 0.2% PBS solution. In the secondary screening, the concentration of the receptor fusion protein is reduced to gradually screen for candidate NGF mimic peptides with high affinity. In the final screening, 48 groups of candidate NGF mimic peptides are selected, and their binding activity with the receptor fusion protein is detected, thereby obtaining the final NGF mimic peptide sequence with the highest binding activity.

[0051] The amino acid sequences of the NGF mimic peptide and antioxidant peptide after final screening were obtained, and amino acids were coupled one by one based on the designed sequence numbers. The peptide chain was then constructed stepwise using the Fmoc protection strategy. After each coupling step, the Fmoc protecting groups were removed using reagents such as piperidine to obtain the NGF mimic peptide and antioxidant peptide.

[0052] In this embodiment of the invention, when amino acids are coupled one by one based on the designed sequence number, the coupling conditions are as follows: activation with 0.5 M HBBTU, 0.2 M HOBT, and 0.3 M DIPEA, at a temperature of 35-37 °C, for a time of 25-30 min.

[0053] The prepared NGF mimic peptide and antioxidant peptide were mixed in a 1:2 ratio, and the resulting functional peptide solution was sterilized to ensure sterility, thus obtaining the functional peptide. The sterilization process involved filtration through a 0.1 μm sterile membrane and sterilization with 20 kGy gamma rays. The solvent for the final functional peptide solution was physiological saline at pH 7.3. The functional peptide includes an NGF mimic peptide and an antioxidant peptide. The NGF mimic peptide sequence is shown in SEQ ID NO: 1, and the antioxidant peptide sequence is shown in SEQ ID NO: 2.

[0054] Example 2

[0055] Endometrial stem cell culture and identification: 120 female adult rats were selected and divided into 6 groups: experimental group 1, experimental group 2, experimental group 3, associated group 1, associated group 2, and blank group. The rats were fed for 14 days to acclimatize to their environment and fasted for 10 hours before surgery. A longitudinal incision was made in the midline of the rat's abdomen under sterile conditions using a sterilized scalpel to expose the uterus. Endometrial tissue samples were obtained from the cervix. The endometrial tissue samples were washed 3-5 times with PBS solution, and the endometrial adipose tissue was removed. The endometrial tissue samples were then minced using sterile ophthalmic scissors. Tissue samples were mixed with 0.3% trypsin, centrifuged, and the supernatant was discarded. When endometrial tissue samples were mixed with 0.3% trypsin, the mixing conditions were 37°C and 5% CO2 incubator for 10 min, after which the trypsin was discarded. Tissue fragments were then digested with preheated 0.2% collagenase and 0.02% EDTA, and the digestion was promoted by stirring with a magnetic stirrer. The digestion was terminated by using an equal volume of DMEM complete medium containing 20% ​​FBS. The digested mixture was collected and centrifuged at 5°C for 1500 rpm for 10 min. The supernatant was discarded to obtain the digested endometrial tissue samples.

[0056] Endometrial stem cells were isolated and identified from endometrial tissue samples. The endometrial stem cells underwent pretreatment. During isolation, digested endometrial tissue samples were filtered through 100μm, 80μm, and 50μm filters to collect endometrial stem cells. The cells were then resuspended in 2mL of DMEM complete medium, which consisted of DMEM / F12, 20% FBS, 150U / mL penicillin, 200μg / mL streptomycin, and functional peptides. The resuspended cells were seeded in 20cm culture dishes and cultured at 37℃ in a 5% CO2 incubator. After 14 days, adherent cells exhibiting clonal growth were collected and transferred to 48-well plates. When the adherent cells reached approximately 85% confluence, they were passaged. Passaged cells were labeled P1, and subsequent passages were labeled P2. Morphological observation and cell surface antigen assays were performed after the fourth passage. Finally, the endometrial stem cell suspension was counted at a ratio of 2×10⁻⁶ cells / well. 3 Endometrial stem cell suspensions were aliquoted into 96-well plates at a density of 150 μL per well. The plates were aseptically treated and incubated in a CO2 incubator at 37°C and 5% CO2. NGF mimic peptides, antioxidant peptides, functional peptides, associated peptide 1, associated peptide 2, and ineffective peptides prepared in Example 1 were added to the endometrial stem cell suspensions of experimental groups 1, 2, 3, associated groups 1, associated groups 2, and the control group at a dosage of 5 μg / mL per well. After 4 days of incubation, cells from each well were collected into 4 mL LEP tubes. The relative expression levels of Laminin and HOXA1 (based on the control group) were detected by Western blot. (Table 1 and...) Figure 1 The results of testing the relative expression levels of Laminin and HOXA1 in endometrial stem cells by different types of peptides are shown.

[0057] Table 1

[0058]

[0059] As shown in Table 1, the functional peptides provided by this invention, in synergy with endometrial stem cells, can significantly increase the expression level of Laminin and effectively inhibit the expression level of HOXA1. Among them, the antioxidant peptides can reduce oxidative stress, thereby downregulating HOXA1 expression, while the NGF mimic peptides can activate the PI3K / Akt pathway through the TrkA receptor, thereby inhibiting caspase-3-mediated HOXA1 transcription. At the same time, the combination of NGF mimic peptides and antioxidant peptides in the functional peptides achieves the synergistic effect of NGF mimicry and antioxidation. The combination of NGF mimic peptides and antioxidant peptides is more effective than the use of NGF mimic peptides and antioxidant peptides alone in increasing the expression level of Laminin and inhibiting the expression level of HOXA1. The functional peptides using the combination of NGF mimic peptides and antioxidant peptides effectively promote basement membrane repair and provide a stable physical scaffold for nerve regeneration.

[0060] Example 3

[0061] Effects of functional peptide concentration on the expression levels of Laminin and HOXA1 in endometrial stem cells: Endometrial stem cells prepared in Example 2 were used at a concentration of 2 × 10⁻⁶. 3 Endometrial stem cell suspensions were aliquoted into 96-well plates at a density of 150 μL per well. The plates were aseptically treated and placed in a CO2 incubator at 37°C and 5% CO2. Functional peptides prepared in Example 1 were added to the endometrial stem cell suspensions at concentrations 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. After 4 days of culture, cells from each well were collected into 4 mL LEP tubes. 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 of testing the relative expression levels of Laminin and HOXA1 in endometrial stem cells at different concentrations of functional peptides are shown.

[0062] Table 2

[0063]

[0064] As shown in Table 2, regarding Laminin expression, compared to the control group, the expression level of Laminin showed a dependent increasing trend with the continuous increase of the concentration of functional peptides. Moreover, no significant toxicity was shown from the functional peptides even at extremely high concentrations. Therefore, it can be concluded that the addition of functional peptides in this embodiment has a promoting effect on the expression of Laminin in endometrial stem cells, thereby providing a microenvironment for the differentiation of endometrial stem cells into nerve cells and Schwann cells, and also facilitating the repair of the basement membrane during the repair of clitoral sensitive nerves. Regarding HOXA1 expression, compared to the control group, the expression level of HOXA1 showed a decreasing and inhibiting trend with the continuous increase of the concentration of functional peptides. Moreover, no significant toxicity was shown from the functional peptides even at extremely high concentrations.

[0065] Example 4

[0066] Effects of functional peptide concentrations on the expression levels of Laminin and HOXA1 in Schwann cells: Endometrial stem cells isolated in Example 2 were centrifuged, the supernatant was discarded, and the cells were washed with PBS to induce Schwann cell-directed differentiation. The surface of the endometrial stem cells was modified to enhance their homing ability to the clitoral nerve injury site. The inducing factors included nerve growth factor, sound hedgehog factor, or small nucleic acid factor. The induction temperature was 35-40℃, 7.5% carbon dioxide, and the induction time was 7-15 days. Then, 2×10... 3 Endometrial stem cell suspensions were aliquoted into 96-well plates at a density of 150 μL per well. The culture plates were aseptically treated and placed in a CO2 incubator at 37°C and 5% CO2 concentration. Functional peptides prepared in Example 1 were added to the endometrial stem cell suspensions at concentrations 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. After 4 days of culture, cells from each well were collected into 4 mL LEP tubes. 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 of testing the relative expression levels of Laminin and HOXA1 in Schwann cells at different concentrations of functional peptides are shown.

[0067] Table 3

[0068]

[0069] As shown in Table 3, similar to Example 3, this example achieves the effect of activating Laminin transcription and inhibiting HOXA1 expression through the synergistic cooperation of functional peptides and Schwann cells. Schwann cells are the core cells of the basement membrane and can secrete Laminin to participate in the construction of the nerve perineurium basement membrane. In this example, the expression level of Laminin gradually increases with the increase of functional peptide concentration. When the concentration of functional peptide increases to a certain level, the expression level of Laminin shows a gradual and stable trend. Compared with the combination of endometrial stem cells and functional peptides in Example 3, the synergistic cooperation of functional peptides and Schwann cells has a more efficient response and expression level. Regarding the HOXA1 test, the expression level of HOXA1 gradually decreases with the increase of functional peptide concentration. In this embodiment, the functional peptide can block the positive regulation of inflammatory factors by inhibiting HOXA1 expression, and the oxidative activity of the functional peptide can reduce the damage of oxidative stress to Schwann cells, thereby ensuring the anti-inflammatory function of Schwann cells. Furthermore, the functional peptide can promote the secretion of matrix metalloproteinases by Schwann cells, thereby degrading collagen fibers in scar tissue, promoting ECM remodeling, and inhibiting excessive proliferation of fibroblasts.

[0070] Example 5

[0071] Safety testing of functional peptides: In this example, 120 female SD rats were randomly divided into 6 groups of 20 each. The six groups of SD rats were further divided into high-, medium-, and low-dose functional peptide groups, and three control groups were administered via PBS solvent. Abnormal toxic reactions (body weight, general condition) were observed in each group. 24 hours after administration, skin pathology, blood biochemical indicators, and toxic effects on major organs such as the liver, kidneys, and spleen were examined. Results showed that with increasing treatment time, the high-dose peptide group exhibited better therapeutic effects, significantly superior to the other groups. These results indicate that the functional peptides have a strong ability to promote the expression of Laminin and inhibit the expression of HOXA1 in endometrial stem cells. Simultaneously:

[0072] The rat skin pathology examination was normal: no pathological conditions such as edema or erythema were found in the skin, and the skin pathology examination showed that the squamous endothelial cell layer was intact, the cell layers were clear and neatly arranged, the hair follicle structure and gland morphology were clear, and no abnormal reactions such as basal cell changes, hyperplasia and hemorrhage were observed, indicating that the functional polypeptide has no obvious irritation and toxicity to skin tissue.

[0073] Normal blood biochemistry indicators: Liver and kidney function (ALT, AST, BUN, CRE), electrolytes (Na) are normal. + K +The normal levels of blood glucose (GLU) and blood lipids indicate that the use of functional peptides will not have an adverse effect on the body's metabolism and physiological functions, nor will it cause systemic toxicity.

[0074] Major organs were normal: The liver, kidneys, spleen, and other major organs were all normal. Specifically, liver tissue HE staining showed intact lobular structure in all groups, regular hepatocyte arrangement, and no obvious ballooning degeneration, necrosis, or inflammatory cell infiltration. Kidney tissue HE staining showed clear glomerular and tubular structures, with no tubular necrosis, protein casts, or inflammatory cell infiltration. Spleen tissue HE staining showed intact splenic corpuscle structure, evenly distributed lymphocytes, and no necrosis or fibrosis.

[0075] Example 6

[0076] Verification of the repair effect of functional peptides in a rat model of clitoral dorsal nerve transection: Unmated SD female rats were housed for 2 weeks to acclimatize to the environment. They were fasted for 10 hours before treatment. The rats were anesthetized by injecting 0.8 mL / 100 g of chloral hydrate. After shaving the hair on the abdomen and perineum, the area was disinfected three times alternately with povidone-iodine (1:20 dilution) and 75% ethanol. A sterile drape was used to expose the surgical area. A longitudinal incision (2-3 cm in length) was made along the midline of the lower abdomen of the rat. The skin, subcutaneous fat and anterior rectus abdominis sheath were cut layer by layer to expose the extraperitoneal fat. The rats were then separated laterally along the vaginal wall to expose the clitoral suspensory ligament. The clitoral body was pulled cephalad to expose the neurovascular bundle on the dorsal surface of the clitoris. The dorsal nerve of the clitoris was then cut with microscissors. The clitoral body was rinsed with physiological saline. After hemostasis, the anterior rectus abdominis sheath, subcutaneous tissue and skin were sutured layer by layer. Immediately after surgery, 7 mg / kg tramadol was injected intraperitoneally to relieve pain. After confirming successful model establishment through behavioral and histological examinations, the rats with successful model establishment were divided into the following groups for testing:

[0077] Test 1: Starting on the seventh day after surgery, 0.4 mL of endometrial stem cells were injected into the clitoral region of rats, once every 3 days for a total of six weeks;

[0078] Test 2: Endometrial stem cells were modified using functional peptides. The functional peptides were introduced into the endometrial stem cells via electroporation for stable expression. Then, the endometrial stem cells with introduced functional peptides were dissolved in 120 mg of physiological saline with 25 mg of sodium alginate. 20 mg of calcium chloride aqueous solution was added to the physiological saline and mixed thoroughly to prepare a combined gel. Starting on the seventh day after the operation, 0.4 mL of the combined gel was injected into the clitoral region of rats, once every 3 days for a total of 6 weeks.

[0079] Test 3: Starting on the seventh day after surgery, 0.4 mL of prepared Schwann cells were injected into the clitoral region of the rats, once every 3 days, for a total of six weeks;

[0080] Test 4: Schwann cells were modified with functional peptides. The functional peptides were introduced into the Schwann cells by electroporation for stable expression. Then, the Schwann cells with functional peptides were dissolved in 120 mg of physiological saline with 25 mg of sodium alginate. 20 mg of calcium chloride aqueous solution was added to the physiological saline and mixed thoroughly to prepare a combined gel. Starting on the seventh day after the operation, 0.4 mL of the combined gel was injected into the clitoral region of the rats, once every 3 days for a total of 6 weeks.

[0081] Test 5: Starting on the seventh day after surgery, 0.4 mL of endometrial stem cells and Schwann cells were injected into the clitoral region of the rats, once every 3 days for a total of six weeks;

[0082] Test 6: Endometrial stem cells and Schwann cells were modified with functional peptides. The functional peptides were introduced into the endometrial stem cells and Schwann cells by electroporation for stable expression. Then, the endometrial stem cells and Schwann cells with the introduced functional peptides were dissolved with 25 mg of sodium alginate in 120 mg of physiological saline. 20 mg of calcium chloride aqueous solution was added to the physiological saline and mixed thoroughly to prepare a combined gel. Starting on the seventh day after the operation, 0.4 mL of the combined gel was injected into the clitoral region of rats, once every 3 days for a total of 6 weeks.

[0083] Positive control group: Starting on the seventh day after surgery, 0.4 mL of nerve growth factor (NGF) was injected into the clitoral region of the damaged rats, once every 3 days for a total of six weeks.

[0084] Model group: Starting on the seventh day after surgery, 0.4 mL of PBS solution was injected into the damaged clitoral area of ​​the rats, once every 3 days for a total of six weeks.

[0085] Functional and histological tests were performed on tests 1-6, the positive control group, and the model group in this embodiment. Functional tests included stimulating the clitoral tissue of rats at 8 and 10 weeks post-surgery and recording the frequency of hip lift and urination reflexes. Each rat was tested three times, and the behavioral reflex rate was calculated. The results of the rat behavioral reflex rate tests are shown in Table 4 and... Figure 4 As shown in Table 4. During histological examination of rats, the maximum thickness of the scar at the site of clitoral nerve injury was recorded using HE staining. Figure 5 As shown in Table 4. Simultaneously, the integrity of the basement membrane recovery was verified by testing the proportion of Laminin-positive areas at the clitoral sensitive nerve injury site in rats at week 8 post-surgery. The test results are shown in Table 4. Figure 6 As shown. Axon density was calculated for tests 1-6, the positive control group, and the model group (NF-200). + Area ratio (%), test results are shown in Table 4 and Figure 7 As shown.

[0086] Table 4

[0087]

[0088] As shown in Table 4, compared with the model group and the positive control group, tests 1-6 in this embodiment showed significant advantages in various indicators, including promoting the recovery of behavioral reflexes after clitoral nerve injury in rats, reducing the maximum scar thickness at the injury site, promoting the recovery of the proportion of Laminin-positive area (integrity) of the basement membrane, and increasing axon density. This indicates that functional peptides, endometrial stem cells, Schwann cells, and their combinations help reduce scar formation and promote tissue repair. Furthermore, the test results show that functional peptides relieve the inhibition of axon growth by inhibiting HOXA1 expression. At the same time, functional peptides can synergistically enhance the axonal microtubule polymerization capacity with neurotrophic factors.

[0089] In summary, this invention provides a functional polypeptide and its application for promoting the repair of clitoral sensitive nerves by endometrial stem cells. The repair of clitoral sensitive nerves is achieved through the modification of endometrial stem cells and Schwann cells with the functional polypeptide and delivery via local sustained-release gel. Furthermore, the functional polypeptide can activate laminin expression and regulate ECM degradation balance, 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. Simultaneously, the functional polypeptide can effectively inhibit HOXA1 expression, enhancing axonal elongation capacity, and strengthening the axonal microtubule polymerization capacity at the nerve injury site. This systematically solves the problems of inflammation, oxidative stress, and scar inhibition in the microenvironment after clitoral nerve fiber injury, significantly improving nerve repair efficiency and functional recovery quality.

[0090] It should be noted that, for the sake of simplicity, the foregoing embodiments are all described as a series of actions. However, those skilled in the art should understand that the present invention is not limited to the described order of actions, as some steps may be performed in other orders or simultaneously according to the present invention. Furthermore, those skilled in the art should also understand that the embodiments described in the specification are preferred embodiments, and the actions and modules involved are not necessarily essential to the present invention.

[0091] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on these embodiments, all other embodiments obtained by those skilled in the art without creative effort 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, those skilled in the art can still combine, add, delete, or otherwise adjust the features of the various embodiments of the present invention according to the circumstances without conflict or creative effort, thereby obtaining different technical solutions that do not fundamentally depart from the concept of the present invention. 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 mimic peptide and an antioxidant peptide, the NGF mimic peptide sequence being shown in SEQ ID NO: 1 and the antioxidant peptide sequence being shown in SEQ ID NO:

2.

2. A pharmaceutical composition for repairing clitoral sensitive nerves, comprising the functional polypeptide as described in claim 1, characterized in that: The pharmaceutical composition contains endometrial stem cells, Schwann cells, and functional peptides, the functional peptides including NGF mimic peptides and antioxidant peptides, the NGF mimic peptide sequence being shown in SEQ ID NO: 1, and the antioxidant peptide sequence being shown in SEQ ID NO:

2.

3. The use of the functional polypeptide as described in claim 1 in the preparation of a pharmaceutical composition for repairing clitoral sensitive nerves, characterized in that: The functional peptide was introduced into endometrial stem cells.

4. The use of the functional polypeptide as described in claim 1 in the preparation of a pharmaceutical composition for repairing clitoral sensitive nerves, characterized in that: Functional peptides were introduced into endometrial stem cells and Schwann cells.

5. The use of the functional polypeptide as described in claim 1 in the preparation of a pharmaceutical composition for repairing clitoral sensitive nerves, characterized in that: Functional peptides were introduced into Schwann cells.

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