Use of a stem cell composition in endometrial repair

Abstract

This invention provides a stem cell composition for treating endometrial repair. Specifically, it also provides a short peptide ZGC-5 with therapeutic activity that can significantly inhibit the proliferation of ectopic endometrial cells. This short peptide has the effects of inhibiting the proliferation of ectopic endometrial cells and promoting apoptosis. When the short peptide and stem cells are prepared into a kit, it has a good therapeutic effect on endometriosis.

Description

Technical Field

[0001] This application relates to the biological field, specifically to the therapeutic field, and to the use of a stem cell composition in endometrial repair. Background Technology

[0002] The uterus consists of the endometrium on the inner side and the myometrium and adventitia on the outer side. Endometrial repair is a vital physiological function of the uterus, occurring after menstruation, postpartum uterine involution, miscarriage, and other types of endometrial damage. A woman's reproductive lifespan consists of approximately 400 menstrual cycles. During each menstrual cycle, the endometrium undergoes severe inflammatory damage and shedding (resulting in menstruation), followed by repair and reconstruction in the later stages of menstruation. During pregnancy, the uterus needs to rapidly and collaboratively enlarge to accommodate the growth of the embryo. Throughout pregnancy, the human uterus increases in volume by approximately 500-1000 times and in weight by approximately 24 times. The smooth muscle layer and myometrium undergo reconstruction, exhibiting significant hypertrophy and hyperplasia. During delivery, the endometrium disappears almost entirely due to placental shedding, but it still regenerates and reconstructs during uterine involution. Abnormal endometrial repair can lead to various serious diseases and even infertility.

[0003] Stem cell research began in the 1960s, and tissue stem cells have now been discovered in most organs of the body. Existing research shows that the repair and regeneration of damaged tissues and cells are generally closely related to stem cells. During the menstrual cycle, pregnancy, and postpartum, a woman's uterus can rapidly regenerate and remodel, requiring the uterus to have extremely strong regenerative capabilities. Recent studies suggest that a group of uterine stem / progenitor cells with proliferative and differentiation potential may exist within the uterus, playing a decisive role in these processes. Due to the unique biological characteristics of stem cells, they have great application prospects in regenerative medicine and immunotherapy. Studies based on various animal models and clinical trials have found that stem cells play an important role in the repair of endometrial damage.

[0004] Stem cells possess high self-renewal and multi-lineage differentiation potential, are widely distributed in various tissues throughout the body, can be expanded under in vitro culture conditions, and have the ability to differentiate into "transdifferentiation" or "interlineal differentiation." Under specific conditions, they can differentiate into nerve cells, osteoblasts, chondrocytes, adipocytes, etc. Due to this characteristic, stem cells have become the preferred seed cells for tissue engineering research. For patients with endometrial damage, the differentiation potential of stem cells has been used to regenerate the damaged endometrium, thereby replenishing the missing endometrium. Numerous studies have been conducted by scholars both domestically and internationally. Research has found that by preparing endometrial conditioned culture media to simulate the microenvironment of endometrial growth and adding estrogen in vitro, rabbit bone marrow mesenchymal stem cells (BMSCs) differentiated into endometrial epithelial cells. Co-culturing human embryonic stem cells with endometrial stromal cells, estrogen, and cytokines showed that human embryonic stem cells differentiated into endometrial-like cells. Other studies have also found that endometrial stromal stem cells differentiate into endometrial epithelial cells. Co-culturing mouse BMSCs with endometrial stromal cells showed that BMSCs differentiated into endometrial epithelial cells. Co-culturing rat bone marrow mesenchymal stem cells (BMSCs) with endometrial stromal cells revealed that BMSCs can differentiate into endometrial stromal cells. BMSCs can differentiate into endometrial stromal cells and endometrial epithelial cells. These studies demonstrate the feasibility of stem cell differentiation into endometrial-like cells, providing experimental evidence for stem cell therapy after endometrial injury. Angiogenesis is an essential step in the repair process after endometrial injury. Therapeutic angiogenesis involves stimulating angiogenesis in ischemic tissues with cytokines, cells, or other substances to improve blood and oxygen supply, thereby promoting tissue repair. Stem cell-based therapeutic angiogenesis first requires that the transplanted stem cells possess chemotaxis and migration abilities, also known as homing ability—that is, migrating to the specific area of ​​injury and settling there to perform various tissue repair functions. Numerous experiments have demonstrated that stem cells have the ability to chemotaxis and migrate to damaged tissues. Direct differentiation into vascular cells is one mechanism by which stem cells promote angiogenesis. When tissues are damaged, especially ischemic injuries, vascular structures are destroyed, leading to ischemia and hypoxia, resulting in tissue cell necrosis, and the body's own mechanisms initiate angiogenesis. This process requires the participation of a large number of endothelial cells, pericytes, and vascular smooth muscle cells for repair. When stem cells are transplanted, they can differentiate into vascular cells, providing a source of vascular cells for angiogenesis, promoting and accelerating the formation of new blood vessels in the damaged area, and thus promoting tissue repair.

[0005] Many researchers emphasize that stem cells possess abundant secretory activity, and their effects may be due to paracrine factors. These factors include the secretion of different types of cytokines, such as growth factors, anti-inflammatory and pro-inflammatory factors, and anti-apoptotic factors. Cultured stem cells in vitro can secrete numerous angiogenesis-related cytokines, including vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), placental growth factor (PIGF), and platelet-derived factor (PDGF). Animal experiments based on stem cell transplantation have demonstrated that stem cells can also promote angiogenesis and thus the repair of ischemic tissues by secreting and increasing the level of vascular growth factors in tissues. After transplanted stem cells enter the site of injury, they can produce cytokines through autocrine and paracrine mechanisms. Studies have found that exosomes secreted by stem cells upregulate anti-apoptotic genes BCI2L1, BCI2, and BIRC8, and downregulate pro-apoptotic genes CASP1, CASP8, and LTA, thereby inhibiting apoptosis of damaged cells and playing an important role in tissue repair and regeneration. When tissue damage occurs and the vascular system is disrupted, stem cells respond immediately to signals secreted by the damaged tissue, such as inflammatory factors. They stimulate the proliferation and differentiation of endothelial cells by secreting vasoactive factors, and recruit perivascular cells and / or vascular smooth muscle cells. In cases of severe tissue damage, endogenous stem cells are insufficient to maintain homeostasis, necessitating the transplantation of exogenous stem cells to promote angiogenesis and tissue repair.

[0006] Patents CN201910692005.8 and CN201910925566.8 describe inventions that involve isolating endometrial stem cells from human endometrial tissue, concentrating and freeze-drying the culture supernatant, and then mixing it with a solvent to obtain a human endometrial stem cell composite repair factor / preparation. However, the seed cells used in these inventions are endometrial stem cells, which present challenges in obtaining and limit industrialization. Furthermore, the final composite repair factor is in liquid form, resulting in poor utilization during application. Therefore, developing an effective stem cell composition for the treatment of endometrial diseases is an important research direction. Summary of the Invention

[0007] Through high-throughput screening of peptide libraries, the inventors obtained a short peptide compound with therapeutic activity that can significantly inhibit the proliferation of ectopic endometrial cells. This compound is named ZGC-5, and its amino acid sequence is shown in SEQ ID NO: 1.

[0008] Furthermore, the present invention also provides the use of a stem cell composition in endometrial repair.

[0009] Specifically, the present invention also provides a stem cell composition characterized by comprising a ZGC-5 peptide and a pharmaceutically acceptable carrier.

[0010] Furthermore, the present invention also provides a medicine box for treating endometriosis, the medicine box being composed of ZGC-5 polypeptide and endometrial stem cells.

[0011] More specifically, the ZGC-5 polypeptide of the present invention can be modified or substituted, but still retains the corresponding activity.

[0012] Furthermore, substitution variants typically involve the exchange of one amino acid for another at one or more sites on the polypeptide and can be engineered to have the corresponding activity. Substitution can be conservative, meaning one amino acid is replaced by one having a similar shape and charge. Conservative substitutions are well known in the art and include, for example, the following variations: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartic acid to glutamate; cysteine ​​to serine; glutamine to asparagine; glutamate to aspartic acid; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine, or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Alternatively, substitution can be non-conservative, affecting the function or activity of the polypeptide. Non-conservative changes typically involve substituting chemically dissimilar residues, such as replacing nonpolar or uncharged amino acids with polar or charged amino acids, and vice versa. Furthermore, the polypeptides of the present invention are therapeutically based in a therapeutically effective amount.

[0013] The term "effective amount" or "therapeutic effective amount" as used in this application refers to the administration of a sufficient amount of the polypeptide disclosed in this application, which will alleviate one or more symptoms of the treated disease or condition to some extent. In some embodiments, the result is a reduction and / or mitigation of the signs, symptoms, or causes of the disease, or any other desired alteration of the biological system. For example, an "effective amount" for therapeutic use is the amount of a composition comprising the polypeptide disclosed in this application required to provide a clinically significant reduction in disease symptoms. Specifically, examples of therapeutically effective doses of peptides include, but are not limited to, 1-1500 mg, 1-1400 mg, 1-1300 mg, 1-1200 mg, 1-1000 mg, 1-900 mg, 1-800 mg, 1-700 mg, 1-600 mg, 1-500 mg, 1-400 mg, 1-300 mg, 1-250 mg, 1-200 mg, 1-150 mg, 1-125 mg, 1-100 mg, 1-80 mg, 1-60 mg, 1-50 mg, 1-40 mg, 1-2 5mg, 1-20mg, 5-1500mg, 5-1000mg, 5-900mg, 5-800mg, 5-700mg, 5-600mg, 5-500mg, 5-400mg, 5-300mg, 5-250mg, 5-20 0mg, 5-150mg, 5-125mg, 5-100mg, 5-90mg, 5-70mg, 5-80mg, 5-60mg, 5-50mg, 5-40mg, 5-30mg, 5-25mg, 5-20mg, 10-150 0mg, 10-1000mg, 10-900mg, 10-800mg, 10-700mg, 10-600mg, 10-500mg, 10-450mg, 10-400mg, 10-300mg, 10-250mg, 10 -200mg, 10-150mg, 10-125mg, 10-100mg, 10-90mg, 10-80mg, 10-70mg, 10-60mg, 10-50mg, 10-40mg, 10-30mg, 10-20mg ;20-1500mg, 20-1000mg, 20-900mg, 20-800mg, 20-700mg, 20-600mg, 20-500mg, 20-400mg, 20-350mg, 20-300mg, 20-2 50mg, 20-200mg, 20-150mg, 20-125mg, 20-100mg, 20-90mg, 20-80mg, 20-70mg, 20-60mg, 20-50mg, 20-40mg, 20-30mg;50-1500mg, 50-1000mg, 50-900mg, 50-800mg, 50-700mg, 50-600mg, 50-500mg, 50-400mg, 50-300mg, 50-250mg, 50-200mg, 50-150mg, 50-125mg, 5 0-100mg; 100-1500mg, 100-1000mg, 100-900mg, 100-800mg, 100-700mg, 100-600mg, 100-500mg, 100-400mg, 100-300mg, 100-250mg, 100-200mg. ;

[0014] Furthermore, the carrier of the present invention may include excipients. "Excipient" refers to an agent that is not itself a therapeutic agent but serves as a diluent, excipient, binder, and / or medium for addition to a pharmaceutical composition to improve its disposal or storage properties or to allow or promote the formation of a unit dosage form of the compound or pharmaceutical composition for administration. As known to those skilled in the art, pharmaceutical excipients can provide a variety of functions and can be described as wetting agents, buffers, suspending agents, lubricants, emulsifiers, disintegrants, absorbents, preservatives, surfactants, colorants, flavoring agents, and sweeteners. Examples of pharmaceutical excipients include, but are not limited to: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, cellulose acetate, hydroxypropyl methyl cellulose, hydroxypropyl cellulose, microcrystalline cellulose and croscarmellose (e.g. croscarmellose sodium); (4) tragacanth gum powder; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter. (9) Oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) Diols, such as propylene glycol; (11) Polyols, such as glycerol, sorbitol, mannitol, and polyethylene glycol; (12) Esters, such as ethyl oleate and ethyl laurate; (13) Agar; (14) Buffers, such as magnesium hydroxide and aluminum hydroxide; (15) Alginate; (16) Atherless water; (17) Isotonic saline; (18) Ringer's solution

[0015] (19) ethanol; (20) pH buffer solution; (21) polyester, polycarbonate and / or polyanhydride; and (22) other non-toxic compatible substances used in pharmaceutical preparations.

[0016] For oral, sublingual, and sublingual administration, acceptable solid dosage forms include: powders, suspensions, granules, tablets, pills, capsules, soft capsules, and microcapsules. Preparation methods include, for example, mixing one or more compounds of the present invention, or pharmaceutically acceptable salts or their tautomers, with at least one additive, such as starch or other additives. Suitable additives include: sucrose, lactose, cellulose sugars, mannitol, maltitol, dextran, starch, agar, alginate, chitin, chitosan, pectin, tragacanth gum, gum arabic, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymers, or glycerides. Alternatively, oral dosage forms may contain other components to aid administration, such as inert diluents, or lubricants such as magnesium stearate, or preservatives such as parabens or sorbic acid, or antioxidants such as ascorbic acid, vitamin E, or cysteine, disintegrants, binders, thickeners, buffers, sweeteners, flavorings, or aromatizers. Tablets and pills can also be further processed with suitable coating materials known in the art.

[0017] Furthermore, the concentration of endometrial stem cells used in this invention is 1×10⁻⁶. 5 -1×10 9 Dosage: / kg, more preferably, 1×10 6 -1×10 9 / kg dosage; more preferably, 1×10 7 -1×10 9 / kg dosage; more preferably, 1×10 8 -1×10 9 / kg dosage; more preferably, 5×10 8 -1×10 9 / kg dosage.

[0018] Specifically, the endometrial stem cells of the present invention can be cultured using serum-free culture media commonly used in the art.

[0019] The culture medium can be a serum-free medium suitable for cell development. For example, the culture medium may include... Supplements, xeno-free Supplements.

[0020] In some implementations, the culture medium comprises one or more of the following: Supplements, no foreign species Supplements, GS21 TMSupplements or combinations thereof. In some embodiments, the culture medium comprises or further comprises amino acids, monosaccharides, and inorganic ions. In some embodiments, the amino acids include arginine, cysteine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine, or combinations thereof. In some embodiments, the inorganic ions include sodium, potassium, calcium, magnesium, nitrogen, or phosphorus, or combinations thereof, or salts thereof. In some embodiments, the medium further comprises one or more of the following: molybdenum, vanadium, iron, zinc, selenium, copper, or manganese, or combinations thereof. In some embodiments, the culture medium comprises or is substantially composed of: one or more vitamins discussed herein and / or one or more proteins discussed herein and / or one or more of the following: corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite or triiodo-I-thyroxine, B-27 supplement, xenobiotic-free B-27 supplement, GS21™ supplement, amino acids (such as arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine or valine), monosaccharides, inorganic ions (such as sodium, potassium, calcium, magnesium, nitrogen and / or phosphorus) or salts thereof and / or molybdenum, zinc, selenium, copper or manganese.

[0021] Beneficial effects

[0022] This invention provides a stem cell composition for treating endometrial repair. Specifically, it also provides a short peptide ZGC-5 with therapeutic activity that can significantly inhibit the proliferation of ectopic endometrial cells. This short peptide has the effects of inhibiting the proliferation of ectopic endometrial cells and promoting apoptosis. When the short peptide and stem cells are prepared into a kit, it has a good therapeutic effect on endometriosis. Attached Figure Description

[0023] Figure 1 OD values ​​at 570nm wavelength for each group Detailed Implementation

[0024] Specific embodiments of the invention will now be described in more detail with reference to the accompanying drawings. While specific embodiments of the invention are shown in the drawings, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the invention and to fully convey the scope of the invention to those skilled in the art.

[0025] When implementing or testing embodiments of the present invention, optional methods and materials similar to or equivalent to those described in this specification may be used, although preferred methods, apparatus, and materials are described in this specification. However, before describing the materials and methods of the present invention, it should be understood that the specific sizes, shapes, dimensions, materials, methods, means, etc., described in this specification can be modified according to conventional experimental methods and for optimization purposes; therefore, the present invention is not limited to these. Furthermore, it should be understood that the technical terms used in this specification are only used to describe specific types or embodiments and are not intended to limit the scope of the present invention, which is limited only by the scope of the appended claims.

[0026] Example 1: Preparation and Identification of Ectopic Endometrial Cells

[0027] Under aseptic conditions, the patient's ectopic endometrial tissue was thoroughly minced into tiny particles approximately 1 mm in size. Then, 0.1% W-type collagenase was added, and digestion was carried out for 3.0-4.0 hours. Cells were resuspended and counted at 10⁻⁶ cells per cell. 9 Cells were seeded at a concentration of / L at 25cm 2 Add 4.0–5.0 mL of high-glucose DMEM medium containing 10% FBS, 10 g / L insulin, and working concentration of penicillin antibiotics to the culture flask. Incubate at 37°C in a 5% CO2 incubator. Change the medium after 2 days to remove non-adherent cells. Change the medium intermittently every two to three days. Under an inverted microscope, observe that the cells are arranged in swirling clusters with large, round nuclei and filamentous connections between cells, indicating successful isolation of ectopic endometrial cells.

[0028] Example 2: Preparation of endometrial stem cells

[0029] Materials Acquisition: Collect human menstrual blood and dilute it with sterile phosphate-buffered saline (PBS) by an equal volume and mix well. Separate mononuclear cells from the menstrual blood using density gradient centrifugation (2000 rpm, 20 min) with 1.077 g / mL lymphocyte separation medium. Carefully aspirate the middle white membrane layer, add 2 volumes of PBS buffer, wash twice at 1500 rpm for 10 min, discard the supernatant completely, add 2.0 mL of complete culture medium, mix well, count the cells, and adjust to an appropriate cell density for cell culture. Seed the cells in 75 mL culture flasks and incubate at 37°C in a 5% CO2 incubator with saturated humidity. Change the culture medium after 72 h, discarding any non-adherent cells. Replace the entire medium every three to four days depending on cell growth. When primary cells reach 80%–90% confluence, they are passaged: the culture medium is removed, and digestion is performed with a digestion solution (0.25% trypsin + 0.02% EDTA solution). Digestion is terminated with an equal volume of serum. The cells are centrifuged at 1300 rpm for 10 minutes, the supernatant is discarded, and DMEM high-glucose culture medium containing 20% ​​fetal bovine serum, 1% amphotericin B, and 1% penicillin / streptomycin is added. The cells are then dispersed by pipetting and passaged. P3 cells are used to identify stem cell surface markers using flow cytometry. Specifically, P3 cells are prepared as a cell suspension. Using the direct method, 20 μL of mouse anti-human CD29-PE, CD44-FITC, HLA-ABC-FITC, CD34-PE, CD45-PerCP, and HLA-DR-FITC were added to the control tubes, respectively. The control tubes were incubated at room temperature in the dark for 20 min, washed twice with PBS, centrifuged at 1500 r / min for 5 min, and the cells were resuspended. Flow cytometry results showed that P3 cells highly expressed CD44, CD29, and HLA-ABC, and barely expressed CD34 and CD45, indicating that endometrial stem cells were successfully prepared.

[0030] Example 3: Effects of ZGC-5 peptide and endometrial stem cells on endometrial cells

[0031] Experiment 1: Ectopic endometrial cells and endometrial stem cells prepared in Example 1 were mixed at a ratio of 1×10⁻⁶. 5 / 1×10 5 Cells were seeded proportionally into the upper and lower chambers of a Transwell co-culture plate to establish a non-contact co-culture system with two layers of cells. Ectopic endometrial cells (1×10⁻⁶) were also included. 5 They were cultured separately as a control group.

[0032] Experiment 2: Ectopic endometrial cells prepared in Example 1 were processed at a ratio of 1×10⁻⁶. 5The ZGC-5 peptide was seeded proportionally in the inner chamber of a Transwell co-culture plate and cultured separately. Three groups (AC) were set up with different final concentrations of ZGC-5 peptide (10 μg / mL, 50 μg / mL, and 100 μg / mL).

[0033] Experiment 3: Ectopic endometrial cells and endometrial stem cells prepared in Example 1 were mixed at a ratio of 1×10⁻⁶. 5 / 1×10 5 The cells were seeded proportionally into the upper and lower chambers of a Transwell co-culture plate to establish a non-contact co-culture system with two layers of cells. The final concentration of ZGC-5 peptide in the system was 100 μg / mL.

[0034] Experiment 4: Ectopic endometrial cells prepared in Example 1 were processed at a ratio of 1×10⁻⁶. 5 The samples were inoculated onto Transwell co-culture plates and cultured separately in a liquid containing a final concentration of 100 μg / mL of gestrinone.

[0035] Cell proliferation was detected using the MTT assay. After culturing cells from each experimental group and the control group for 72 hours, ectopic endometrial cells were digested and precipitated at a ratio of 1 x 10⁻⁶ cells / mL. 8 Cells were seeded at a concentration of 5 g / L in 96-well plates, with 20 μL of 5 g / L MTT added to each well. After 4 hours of incubation, the culture medium was aspirated, and 150 μL of DMSO was added to each well. The plates were then shaken thoroughly to dissolve the crystals. The number of proliferating cells was detected at 570 nm using an ELISA reader. Each well was divided into three replicates, with blank wells used as controls. All experiments were repeated three times, and the average value was taken. The results are shown below. Figure 1 As shown.

[0036] from Figure 1 It can be seen that the OD570 values ​​of each experimental group were significantly lower than those of the control group (P<0.05). The OD570 value of the peptide group also gradually decreased with increasing concentration, indicating that the peptide exhibits dose-dependent effects. When the peptide and stem cells were used together, the OD570 was only (0.39±0.03), significantly lower than that of the control group (1.82±0.13), indicating better inhibition of cell proliferation.

[0037] Cell cycle status and apoptosis rate were detected by flow cytometry: Cells co-cultured for 72 h were used to digest ectopic endometrial cells. After washing three times with PBS, the ectopic endometrial cells were suspended and fixed in 70% pre-chilled ethanol. After incubation overnight at 4°C, the cells were washed twice more with an equal volume of PBS, and then 100 μL of RNase A was added. The cells were incubated at 37°C for 30 min, followed by P1 at 4°C in the dark for 30 min. The percentage of apoptotic cells was then detected by flow cytometry. The results are shown in Table 1.

[0038] Table 1. Results of apoptosis rates in each group

[0039] Group Apoptosis rate (%) Experiment 1 17.94±0.29 control group 0.68±0.12 Experiment 2 (10 μg / mL) 24.61±0.23 Experiment 2 (50 μg / mL) 29.45±0.25 Experiment 2 (100 μg / mL) 33.54±0.32 Experiment 3 45.23±0.42 Experiment 4 15.89±0.21

[0040] As shown in Table 1, the ZGC-5 peptide of the present invention has a dose-dependent effect on promoting apoptosis of ectopic endometrial cells, and the difference is significant compared with the control group (P<0.01). In particular, the combined use of stem cells and peptides has a significant synergistic effect on promoting cell apoptosis. Compared with the use of stem cells or peptides alone, the improvement is beyond expectations, and the effect is significantly better. It also significantly increases the apoptosis rate compared with the positive control.

[0041] Example 3: Animal experiments with ZGC-5 peptide and endometrial stem cells

[0042] Experimental SPF-grade female SD rats were used to establish an EMs (endometriosis) rat model using the conventional autologous endometrial transplantation method in this field; estradiol valerate was administered by gavage once daily for 3 days prior to modeling.

[0043] (0.1 mg / kg) to regulate the estrous cycle of rats to induce simultaneous estrus: After anesthesia with intraperitoneal injection of 10% chloral hydrate (3.5 mL / kg), an incision was made approximately 2 cm above the urethra to expose the left uterus. The left uterine horn was ligated, and a 1 cm section of the uterus was harvested, placed in physiological saline, and cut into 0.5 cm x 0.5 cm endometrial tissue blocks. These blocks were then transplanted between the subcutaneous fascia and abdominal muscle layers of the right abdominal wall, and sutured layer by layer. The rats were then fed normally. The sham-operated group underwent the same surgical approach as the model group, with ligation and removal of the left uterine horn, but without autologous transplantation. Four weeks later, small animal ultrasound examination revealed a dark gray hypoechoic area below the abdominal wall, indicating ectopic lesions. A black anechoic area within the lesion indicated the presence of fluid. A ectopic lesion volume ≥50 mm² was considered a successful model establishment.

[0044] The patients were grouped and administered medication according to the following treatment methods:

[0045] Both the normal group and the model group received intraperitoneal injections of normal saline, with an intraperitoneal injection volume of 5 mL / kg, administered once every 3 days, for a course of 4 weeks.

[0046] ZGC-5 peptide treatment group: 10 mg / kg of ZGC-5 peptide was injected intraperitoneally, with a volume of 5 mL / kg, and the administration frequency was once every 3 days, for a course of 4 weeks;

[0047] Endometrial stem cell therapy group: Endometrial stem cells were injected intraperitoneally at a volume of 5 mL / kg, 1×10⁻⁶. 7 The dose was administered once every 3 days, with a dose per mL, for a total of 4 weeks.

[0048] Combined treatment group: Intraperitoneal injection of endometrial stem cells and ZGC-5 peptide, the dosage of which was the same as that of the ZGC-5 peptide treatment group and the endometrial stem cell treatment group, and the frequency of administration was once every 3 days, with a course of treatment of 4 weeks;

[0049] Positive control group: Gestrinone 10mg / kg orally, 3 times a day, for 4 weeks.

[0050] Six hours after the last administration, the abdomen was opened under anesthesia. Endometrial tissue from normal rats and ectopic endometrial lesions from other groups were harvested. After rinsing with physiological saline, the length (mm), width (mm), and height (mm) of the ectopic lesions were measured using calipers. The lesion volume (V) was then calculated using the ellipsoidal volume formula. V (mm²) 3 = 0.52 × length × width × height; pelvic adhesions in ectopic lesions were scored according to the Haber scoring method. The results are shown in Table 2.

[0051] Table 2. Pelvic adhesion scores after treatment in each group

[0052] Group Pelvic adhesions score (points) normal group - Model group 4.57±0.33 ZGC-5 peptide treatment group 1.91±0.12# Endometrial stem cell therapy group 2.68±0.23# Combined treatment group 0.87±0.08## Positive control group 2.86±0.16#

[0053] As shown in Table 2, the pelvic adhesion scores of each treatment group were significantly reduced compared to the model group (#P<0.05). The treatment effects of the ZGC-5 peptide treatment group and the endometrial stem cell treatment group were also better than those of the positive control group. In particular, the combined treatment group showed a significant reduction in efficacy, with an extremely significant difference compared to the model group (P<0.01). This result indicates that the peptides and stem cells of the present invention have good effects when used together.

[0054] It is understood that although the present invention has been disclosed above with reference to preferred embodiments, these embodiments are not intended to limit the present invention. For any person skilled in the art, many possible variations and modifications can be made to the technical solutions of the present invention based on the disclosed technical content, or equivalent embodiments can be modified accordingly, without departing from the scope of the present invention. Therefore, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A short peptide ZGC-5 with therapeutic activity that inhibits the proliferation of ectopic endometrial cells, characterized in that... The amino acid sequence is shown in SEQ ID NO:

1.

2. Use of the short peptide ZGC-5 as described in claim 1 in the preparation of a medicament for treating endometriosis.

3. A medicine box for treating endometriosis, characterized in that... The medicine box is composed of the short peptide ZGC-5 as described in claim 1 and endometrial stem cells.

4. Use of the short peptide ZGC-5 and endometrial stem cells as described in claim 1 in the preparation of a kit for treating endometriosis.

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