Application of endometrial stem cells in fascial repair

Abstract

This invention discloses the application of endometrial stem cells in fascial repair, belonging to the field of biomedical technology. This invention obtains a combined polypeptide that promotes the co-expression of KGF and LOX through screening. When used separately or in combination with endometrial stem cells and endometrial endothelial progenitor cells, the combined polypeptide effectively promotes the co-expression of KGF and LOX, thereby effectively repairing uterine fascial damage. The combined polypeptide's regulation of the synergistic expression of KGF and LOX in endometrial stem cells and endometrial endothelial progenitor cells improves the efficiency and quality of tissue repair, enabling damaged uterine fascia to recover its normal structure and function more quickly.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of endometrial stem cells in fascial repair. Background Technology

[0002] In the fields of tissue engineering and regenerative medicine, fascial repair has always been a crucial and challenging issue. As an important connective tissue structure in the human body, fascia is widely distributed throughout the body, playing a vital role in supporting, connecting, protecting, and maintaining organ position. However, fascial injuries are common clinically and often have serious consequences due to trauma, surgery, infection, or disease. Once injured, if not effectively repaired, it can lead not only to structural damage and functional impairment of local tissues but also to a series of complications such as chronic pain, limited joint mobility, and muscle atrophy, severely impacting the patient's quality of life.

[0003] In recent years, with the deepening of stem cell research, the potential of stem cells in tissue repair and regeneration has received widespread attention. Endometrial stem cells (EnSCs), as adult stem cells with strong self-renewal capacity and multi-lineage differentiation potential, are relatively abundant and possess immunomodulatory properties, making them a promising candidate for tissue engineering. However, using endometrial stem cells solely for fascial repair presents challenges such as low cell survival rates after transplantation, short retention time in the target tissue, and difficulty in effectively controlling differentiation direction, thus affecting their repair efficacy and clinical application value.

[0004] As bioactive molecules, peptides play an important signaling regulatory role in physiological processes such as cell proliferation, differentiation, migration, and tissue remodeling. Designing specific peptide sequences that can interact with endometrial stem cells and guide their directed migration, proliferation, and differentiation at fascial injury sites is of great significance for improving the efficiency of stem cell application in fascial repair. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing the application of endometrial stem cells in fascial repair.

[0006] In one aspect of the present invention, endometrial stem cells are provided for the repair of uterine fascia injuries.

[0007] Current research has found that endometrial stem cells possess characteristics such as multi-directional differentiation potential, self-renewal capacity, abundant source, and low immunogenicity, making them widely applicable in aging and tissue damage repair. Some studies have demonstrated the potential of endometrial stem cells to differentiate into endometrial endothelial progenitor cells through in vitro experiments. KGF, by activating the FGFR2b receptor, promotes DNA synthesis and division of endometrial endothelial cells. Simultaneously, high KGF expression can synergistically upregulate VEGF expression, promoting vascular endothelial cell migration and lumen formation, thereby shortening the repair cycle. LOX catalyzes the lysine oxidation of collagen I / III, forming covalent cross-links, which enhances the mechanical strength and tensile strength of collagen fibers. LOX expression can improve tissue relaxation caused by insufficient collagen cross-linking in the fascia of patients with post-inflammatory hyperplasia (POP), improving the fascia's resistance to pressure. Therefore, co-expression of KGF and LOX by endometrial stem cells and endometrial endothelial progenitor cells can effectively achieve fascial repair.

[0008] Furthermore, the present invention provides a combined polypeptide that can promote the co-expression of KGF and LOX, the amino acid sequence of which is shown in SEQ ID NO.1, and the combined polypeptide is obtained by library construction and screening.

[0009] The combined 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 combined peptides or give them better anti-digestion properties.

[0010] To achieve this goal, we can generate diverse libraries with desired properties by altering one or more specific amino acids at certain positions within the conjugate 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 conjugate peptide are excluded from modification to ensure the core function of the peptide remains unaffected.

[0011] 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.

[0012] In this invention, the method for preparing the combined polypeptide includes:

[0013] Obtain the amino acid sequence number of the combined peptide and determine the load plasmid. Select the promoter and terminator, and insert the gene of the combined peptide into the load plasmid.

[0014] The loaded plasmid was transferred into host cells using a heat shock method to obtain recombinant cells. PCR was used to identify recombinant cells that successfully expressed the combined polypeptide.

[0015] The combined peptide was expressed in recombinant cells by inducing expression of the combined peptide with the inducer IPTG, and then the combined peptide was isolated and purified from the recombinant cells.

[0016] Furthermore, the present invention provides a pharmaceutical composition for fascia repair, the pharmaceutical composition comprising endometrial stem cells, endometrial endothelial progenitor cells and a combined polypeptide, the amino acid sequence of the combined polypeptide being shown in SEQ ID NO.1.

[0017] Furthermore, the present invention provides the use of endometrial stem cells, endometrial endothelial progenitor cells and a combined polypeptide in the preparation of a pharmaceutical composition for treating fascial injuries, wherein the amino acid sequence of the combined polypeptide is shown in SEQ ID NO.1.

[0018] Furthermore, the present invention also provides the use of endometrial stem cells and combined polypeptides in the preparation of a pharmaceutical composition for treating fascial injuries, wherein the amino acid sequence of the combined polypeptide is shown in SEQ ID NO.1.

[0019] Furthermore, the present invention also provides the use of endometrial endothelial progenitor cells and a combined polypeptide in the preparation of a pharmaceutical composition for treating fascial injuries, wherein the amino acid sequence of the combined polypeptide is shown in SEQ ID NO.1.

[0020] Furthermore, the present invention provides a pharmaceutical composition that further contains a pharmaceutically acceptable carrier.

[0021] 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 combined polypeptides 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 use in impregnated bandages and other dressings, or sprayed onto the infected area using a pump or aerosol sprayer.

[0022] Furthermore, the present invention provides pharmaceutical compositions in the form of tablets, pills, granules, capsules, etc.

[0023] The tablets, pills, capsules, and granules 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 combined polypeptide 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 combined polypeptide of the present invention can be incorporated into sustained-release formulations and devices.

[0024] 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.

[0025] Those skilled in the art can determine the appropriate dosage and range of the combined peptides 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.

[0026] Compared with the prior art, the embodiments of this application have the following main advantages:

[0027] This invention provides the application of endometrial stem cells in fascial repair. By combining peptides to regulate the expression of KGF and LOX in endometrial stem cells and endometrial endothelial progenitor cells, it can effectively repair uterine fascial damage. Furthermore, the combined peptides, by promoting the co-expression of KGF and LOX, can accelerate cell proliferation, differentiation, and angiogenesis, shortening the repair cycle of uterine fascial damage and reducing the risk of fibrosis during fascial repair. High KGF expression promotes cell proliferation and growth, providing a greater source of cells for tissue repair. Appropriate amounts of LOX ensure the cross-linking and stability of the extracellular matrix, creating favorable conditions for cell attachment and growth. The synergistic effect of the combined peptides in regulating the KGF and LOX expression in endometrial stem cells and endometrial endothelial progenitor cells improves the efficiency and quality of tissue repair, enabling damaged uterine fascia to recover its normal structure and function more quickly. Attached Figure Description

[0028] Figure 1 The results of assays showing the promotion of KGF expression in endometrial endothelial progenitor cells by different types of peptides are presented.

[0029] Figure 2 The results of LOX immunohistochemical scoring assay in endometrial endothelial progenitor cells at different concentrations of combined peptides are shown.

[0030] Figure 3 The results of testing the expression level of KGF in endometrial stem cells by different concentrations of combined peptides are shown.

[0031] Figure 4 The results of the test on the number of fascial glands were shown when evaluating the effect of combined peptide repair on uterine fascial injury. Detailed Implementation

[0032] 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 be limiting of the application; the terms "comprising" and "having," and any variations thereof, in the specification, claims, and foregoing drawings of this application are intended to cover non-exclusive inclusion. The terms "first," "second," etc., in the specification, claims, or foregoing drawings of this application are used to distinguish different objects, not to describe a particular order.

[0033] Example 1: Preparation of Combined Peptides

[0034] Obtain the amino acid sequence number of the combined peptide and determine the load plasmid. Select the promoter and terminator, and insert the gene of the combined peptide into the load plasmid.

[0035] The load plasmid can be pcDNA3.1 plasmid or pCMV-Myc vector plasmid.

[0036] The plasmid-loaded cells were transferred into host cells using a heat shock method to obtain recombinant cells. PCR was used to identify recombinant cells successfully expressing the combined polypeptide. The host cells could be Pichia pastoris MG1003 or Pichia pastoris KM71. After obtaining recombinant cells by heat shock, they could be cultured in seed culture medium, such as LB medium, SOC medium, or YPD medium. When using YPD medium, it contained 10 g / L glucose, 15 g / L peptone, 5 g / L yeast extract, 0.2 g / L potassium chloride, and 0.5 g / L manganese chloride.

[0037] The expression of a combined polypeptide was induced in recombinant cells using the inducer IPTG, and the polypeptide was then isolated and purified from the recombinant cells. It should be noted that IPTG is a lactose analogue, structurally similar to lactose but chemically more stable. It consists of a glucose molecule and a thiogalactose molecule linked by a glycosidic bond. Because IPTG is readily soluble in water, with a solubility of approximately 196 g / L at room temperature, and its aqueous solution is usually colorless and transparent, it exhibits good solubility and stability in experimental procedures.

[0038] After cleaning and removing fat from pig skin and beef Achilles tendon, the meat is chopped and degreased with 35% sodium hydroxide solution for 10-15 hours. It is then washed with ethanol and rinsed twice with deionized water. The cleaned pig skin and beef Achilles tendon are placed in a water bath and subjected to trypsin hydrolysis at 40℃ for 30 hours. Non-collagenous proteins are removed using Triton X-100 buffer to obtain collagen. 4g of gelatin is dispersed in 55mL of PBS solution at 55℃. 0.5g of collagen is added to the PBS solution and stirred at 100rpm for 10 minutes. The mixture is dialyzed using a dialysis bag to obtain collagen-gelatin. The collagen-gelatin is then freeze-dried and pulverized to obtain collagen-gelatin powder for later use.

[0039] Dissolve 5g of collagen-gelatin powder in 40mL of PBS solution at 60℃, then add 0.5g of hyaluronic acid, stir well, add the combined peptide, stir at 100rpm for 10min, add 0.02g of EDC crosslinking agent to the mixture, stir at 50rpm for 3min, transfer the mixture into a mold with a 3mm hole, irradiate with ultraviolet light for 1min to solidify the mixture, and freeze for later use.

[0040] Example 2: Culture and Identification of Endometrial Endothelial Progenitor Cells

[0041] Sixty SD magnetic rats were randomly divided into 6 groups of 10 each. The rats were acclimatized to the environment for one week and fasted for 8 hours before treatment. Before harvesting fascia tissue, the rats were anesthetized by injecting 0.8 mL / 100 g of chloral hydrate. Abdominal hair was removed using a scalpel, and the area was disinfected twice with povidone-iodine. Under aseptic conditions, a longitudinal incision was made in the midline of the lower abdomen to expose the uterus. The endometrium from the cervix was harvested, washed with PBS solution, and placed in a culture dish. The surrounding adipose tissue was removed, and the endometrium was minced and digested with 250 U / mL collagenase and DNase for 45 min. The progenitor cells were separated and pipetted to obtain a suspension. Culture medium was added to terminate the digestion. The progenitor cell suspension was centrifuged at 1200 rpm, the supernatant was discarded, and the progenitor cells were counted at 4 × 10⁻⁶. 3 Cells were aliquoted into 96-well plates at a density of 150 μL per well. The plates were aseptically incubated at 37°C with 5% CO2. Pre-prepared conjugate peptide gels were added to each well at a concentration of 15 μg / mL. Four conjugate peptide gels (G1, G2, G3, and G4 peptides) and an ineffective peptide gel served as a co-culture control. After 3 days of culture, cells were collected from each well into 4 mL EP tubes, centrifuged at 4500 rpm for 5 min, and the supernatant was discarded. 100 μL of cell lysis buffer was added, and the cells were then transferred to 96-well white ELISA plates. The bioluminescence intensity was detected using a chemiluminescence analyzer. KGF expression and LOX immunohistochemical scores in endometrial endothelial progenitor cells were also measured. The results of different peptides promoting KGF expression and LOX immunohistochemical scores in endometrial endothelial progenitor cells are shown in Table 1. Figure 1 The results of assays showing the promotion of KGF expression in endometrial endothelial progenitor cells by different types of peptides are presented.

[0042] Table 1

[0043]

[0044] As shown in Table 1, compared to the four associated peptide gels (G1 peptide, G2 peptide, G3 peptide, G4 peptide) and the ineffective peptide gel, the combined peptide gel in this invention exhibits a stronger ability to promote the co-expression of KGF and LOX. While increasing KGF expression, it dynamically balances LOX expression, ensuring that LOX promotes extracellular matrix (ECM) cross-linking and structural stability while avoiding over-activation leading to fibrosis. Therefore, the combined peptide gel can effectively enhance KGF expression. KGF, as an important growth factor, stimulates cell proliferation and differentiation, playing a crucial role, especially in endothelial cell repair and regeneration. Its high expression helps accelerate the repair and regeneration of damaged tissues. While promoting KGF expression, the combined peptide gel can also dynamically regulate LOX expression. LOX plays an important role in extracellular matrix cross-linking and stability, but its over-activation may trigger fibrosis. This gel maintains LOX expression at an appropriate level, ensuring normal extracellular matrix cross-linking and structural stability while avoiding fibrosis caused by excessive LOX activation.

[0045] Example 3: Effects of different concentrations of combined peptides on the co-expression of KGF and LOX in endometrial endothelial progenitor cells

[0046] Endometrial endothelial progenitor cells prepared in Example 2 were digested with 250 U / mL collagenase and DNase for 45 min. The cells were then separated and pipetted to obtain a progenitor cell suspension. The progenitor cell suspension was counted at a ratio of 4 × 10⁻⁶ cells / mL. 3 Progenitor 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. Pre-prepared combined peptide gels were added to the wells at concentrations of 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, and 500 μg / mL per well. KGF expression and LOX immunohistochemical scores were measured. The results of different concentrations of combined peptides promoting KGF expression and LOX immunohistochemical scores in endometrial endothelial progenitor cells are shown in Table 2. Figure 2 The results of LOX immunohistochemical scoring assay in endometrial endothelial progenitor cells at different concentrations of combined peptides are shown.

[0047] Table 2

[0048] Peptide concentration (μg / mL) 100 200 300 400 500 KGF expression level 1.42 1.65 1.67 1.78 1.92 LOX immunohistochemical score (%) 41.3 45.7 45.8 45.8 45.7

[0049] As shown in Table 2, the combined peptide exhibits a stronger effect on promoting the co-expression of KGF and LOX in endometrial endothelial progenitor cells with increasing concentration. This indicates that the combined peptide of the present invention can effectively promote the co-expression of KGF and LOX in endometrial endothelial progenitor cells. However, when the concentration of the combined peptide is too high, the expression level of LOX remains stable without overexpression, thus avoiding the problems of excessive cross-linking of uterine fascia collagen leading to scar formation, inhibition of angiogenesis, and endothelial regeneration caused by LOX overexpression. Therefore, the combined peptide may promote the co-expression of KGF and LOX by activating or regulating specific intracellular signaling pathways. For example, after the combined peptide binds to receptors on the surface of endometrial endothelial progenitor cells, it activates downstream signal transduction pathways, such as the MAPK and PI3K / Akt pathways, thereby promoting the expression of KGF and LOX genes. The rational regulation of LOX by the combined peptide helps maintain the dynamic balance of the extracellular matrix. Appropriate amounts of LOX can catalyze the cross-linking of extracellular matrix components such as collagen, enhancing their stability and mechanical strength, while avoiding excessive cross-linking. This balanced extracellular matrix environment is conducive to cell attachment, migration and proliferation, and promotes tissue repair.

[0050] Example 4: Effects of different concentrations of combined peptides on the co-expression of KGF and LOX in endometrial stem cells

[0051] Commercially available human endometrial stem cells were placed in a cell laboratory. The liquid in a 20mL centrifuge tube was discarded in a clean bench. The cells were digested with a pre-prepared 0.02% EDTA solution and 0.5% trypsin for 3 hours. The resulting suspension of endometrial stem cells was separated and pipetted. The endometrial stem cell suspension was counted at a ratio of 4 × 10⁻⁶. 3 Endometrial stem cell suspension was 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. Pre-prepared combined peptide gels were added to the wells at concentrations of 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, and 500 μg / mL per well. KGF expression and LOX immunohistochemical scores were measured. The results of different concentrations of combined peptides promoting KGF expression and LOX immunohistochemical scores in endometrial stem cells are shown in Table 3. Figure 3 The results of testing the expression level of KGF in endometrial stem cells by different concentrations of combined peptides are shown.

[0052] Table 3

[0053]

[0054] As shown in Table 3, the combined peptide exhibits a stronger effect in promoting the co-expression of KGF and LOX in endometrial stem cells with increasing concentration. This indicates that the combined peptide of the present invention can effectively promote the co-expression of KGF and LOX in endometrial stem cells. However, when the concentration of the combined peptide is too high, the expression level of LOX remains stable without overexpression, thus avoiding the problems of excessive cross-linking of uterine fascia collagen leading to scar formation, inhibition of angiogenesis, and endothelial regeneration caused by LOX overexpression.

[0055] Example 5: Safety Test of Combined Peptides

[0056] Sixty SD magnetic rats were randomly divided into six groups of ten each. These groups were further divided into high-, medium-, and low-dose combined peptide groups, and a PBS solvent control group. Each group was also further divided into an intact fascia group and a damaged fascia group. The rats were administered the drug once daily for six weeks, covered with plastic wrap, and abnormal toxic reactions (body weight, general condition) were observed. Twenty-four hours after administration, skin pathology, blood biochemical indicators, and toxic effects on major organs such as the liver, kidneys, and spleen were examined. In the first three days of treatment, the treatment effects of the intact fascia group and the damaged fascia group were similar. With increasing treatment time, the high-dose peptide group showed significantly better treatment effects than the other groups. These results indicate that the combined peptide has a strong ability to promote the co-expression of KGF and LOX in endometrial stem cells.

[0057] Dermatopathological examination was normal: no pathological conditions such as edema or erythema were found in the skin, and the dermatopathological 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 combined polypeptide has no obvious irritation and toxicity to skin tissue.

[0058] Subcutaneous tissue normal: No edema or inflammatory cell infiltration was observed in the subcutaneous tissue, further proving that the combined polypeptide does not cause inflammatory reactions or tissue damage when applied topically.

[0059] Normal blood biochemistry indicators: Normal blood biochemistry indicators indicate that the use of combined peptides will not have adverse effects on the body's metabolism and physiological functions, and will not cause systemic toxic reactions.

[0060] Major organs were normal: The liver, kidneys, spleen and other major organs were all normal, indicating that the combined peptides will not cause damage to important organs during metabolism and excretion in the body, further verifying its good safety.

[0061] Example 6: Establishment of a rat model of uterine fascia injury and evaluation of the effect of combined peptide repair on uterine fascia injury.

[0062] Unmated SD magnetic rats, six weeks old, were housed for one week to acclimatize. They were fasted for 8 hours prior to treatment. Anesthetized rats were injected with 0.8 mL / 100 g of chloral hydrate. Abdominal hair was removed using a scalpel, and the area was disinfected twice with povidone-iodine. Under aseptic conditions, a longitudinal incision was made in the midline of the lower abdomen to expose the uterus. The uterine fascia was then gently scraped away using a self-made scraper, mimicking clinical uterine fascia curettage to induce pathological uterine fascial damage. After scraping, the surgical site was carefully examined. The uterine fascial endothelium appeared flat or low columnar, the fascia was thinner, the number of stromal glands was reduced, and significant fibrosis was observed, indicating successful model establishment. The rats with successful model establishment were then divided into the following groups for testing:

[0063] Test 1: Starting on the second day after surgery, 0.2 mL of endometrial stem cells were injected into the uterus of rats;

[0064] Test 2: Starting on the second day after surgery, 0.2 mL of endometrial stem cells and 0.4 mg of combined polypeptide gel were injected into the uterus of rats;

[0065] Test 3: Starting on the second day after surgery, 0.2 mL of endometrial endothelial progenitor cells prepared in Example 2 were injected into the uterus of rats;

[0066] Test 4: Starting on the second day after surgery, 0.2 mL of endometrial endothelial progenitor cells and 0.4 mg of combined polypeptide gel prepared in Example 2 were injected into the uterus of rats.

[0067] Test 5: Starting on the second day after surgery, 0.2 mL of endometrial stem cells, 0.2 mL of endometrial endothelial progenitor cells prepared in Example 2, and 0.4 mg of combined polypeptide gel were injected into the uterus of rats.

[0068] Positive control group: Starting on the second day after surgery, 0.2 mL of the gel composition was injected into the damaged uterine fascia. The gel composition included the following ingredients by weight percentage: 50% anti-adhesion gel and 50% 17β-estradiol.

[0069] Model group: Starting on the second day after surgery, 0.2 mL of PBS solution was injected into the damaged uterine fascia.

[0070] Postoperative suturing of the surgical incision was performed. After anesthesia, rats were individually housed in clean cages, with bedding changed every three days and kept dry. They had free access to water and food, and received enhanced nutrition. Tests 1-5 and the positive control group were administered the drug five times, with a three-day interval between each group. Rats were sacrificed on day 17, and the uterus was dissected for HE staining to observe the uterine fascia thickness and count the number of uterine fascial glands. The evaluation results of the combined peptide treatment for repairing uterine fascial damage are shown in Table 4. Figure 4The results of the test on the number of fascial glands were shown when evaluating the effect of combined peptide repair on uterine fascial injury.

[0071] Table 4

[0072]

[0073] Table 4 shows that the number of uterine fascial glands decreased sequentially in Test 5, Test 4, Test 2, Positive Control Group, Test 3, and Test 1. Compared to endometrial endothelial progenitor cells, endometrial stem cells exhibit a certain lag in fascial repair. Both endometrial endothelial progenitor cells and endometrial stem cells, when combined with the combined polypeptide gel, promoted fascial repair. The combination of endometrial endothelial progenitor cells, endometrial stem cells, and the combined polypeptide gel showed the best effect. Furthermore, the number of uterine fascial glands in Test 5, Test 4, Test 2, Positive Control Group, Test 3, and Test 1 was significantly higher than that in the model group, indicating that the combined polypeptide is effective in promoting the co-expression of KGF and LOX in endometrial stem cells. Simultaneously, the thickness of the uterine fascia decreased sequentially in Test 5, Test 4, Test 2, Positive Control Group, Test 3, and Test 1, and the thickness of the uterine fascia in Test 5, Test 4, Test 2, Positive Control Group, Test 3, and Test 1 was significantly higher than that in the model group, further verifying that the combined polypeptide is effective in promoting the co-expression of KGF and LOX in endometrial stem cells.

[0074] Experimental results showed that the combined peptide effectively promoted the co-expression of KGF and LOX in endometrial stem cells. High expression of KGF promoted cell proliferation and regeneration, while reasonable expression of LOX ensured normal cross-linking and structural stability of the extracellular matrix. The results of tests 5 and 4 further verified the mechanism of action of the combined peptide in promoting fascial repair, namely, by regulating the expression of KGF and LOX, it achieves efficient tissue repair and regeneration. By regulating the expression of these two key factors, the combined peptide significantly enhanced the repair capacity of the uterine fascia, providing a new and effective strategy for the treatment of uterine fascial injuries.

[0075] In summary, this invention provides the application of endometrial stem cells in fascial repair. By combining peptides to regulate the expression of KGF and LOX in endometrial stem cells and endometrial endothelial progenitor cells, it can effectively repair uterine fascial damage. Furthermore, the combined peptides, by promoting the co-expression of KGF and LOX, can accelerate cell proliferation, differentiation, and angiogenesis, shortening the repair cycle of uterine fascial damage and reducing the risk of fibrosis during fascial repair. High KGF expression promotes cell proliferation and growth, providing a greater source of cells for tissue repair. Appropriate amounts of LOX ensure the cross-linking and stability of the extracellular matrix, creating favorable conditions for cell attachment and growth. The synergistic effect of the combined peptides in regulating the KGF and LOX expression in endometrial stem cells and endometrial endothelial progenitor cells improves the efficiency and quality of tissue repair, enabling damaged uterine fascia to recover its normal structure and function more quickly.

[0076] 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.

[0077] 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 combination polypeptide capable of promoting fascia repair, characterized in that, The amino acid sequence of the joint polypeptide is shown as SEQ ID NO. 1, which can promote the co-expression of KGF and LOX in endometrial stem cells and endometrial endothelial progenitor cells, and realize fascia repair.

2. A pharmaceutical composition for fascia repair, characterized by: The pharmaceutical composition contains endometrial stem cells, endometrial endothelial progenitor cells and joint polypeptides, the amino acid sequence of the joint polypeptide is shown as SEQ ID NO. 1, and the endometrial stem cells are non-human endometrial stem cells.

3. Use of endometrial stem cells, endometrial endothelial progenitor cells and a combination polypeptide in the preparation of a pharmaceutical composition for the treatment of fascial injury, wherein, The amino acid sequence of the joint polypeptide is shown as SEQ ID NO.

1.

4. Use of endometrial stem cells and a combination polypeptide in the preparation of a pharmaceutical composition for the treatment of fascial injury, wherein, The amino acid sequence of the joint polypeptide is shown as SEQ ID NO.

1.

5. Use of endometrial endothelial progenitor cells and a conjugate polypeptide in the preparation of a pharmaceutical composition for the treatment of fascial injury, wherein, The amino acid sequence of the joint polypeptide is shown as SEQ ID NO.

1. The amino acid sequence of the joint polypeptide is shown as SEQ ID NO. 1.

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