Application of stem cell composition in endometrial repair

Abstract

The invention provides a stem cell composition for treating endometrial repair. Specifically, the invention also provides an oligopeptide ZGC-5 which can significantly inhibit ectopic endometrial cell proliferation and has therapeutic activity, and the oligopeptide has the effects of inhibiting ectopic endometrial cell proliferation and promoting apoptosis. After the oligopeptide and the stem cells are prepared into the medicine box, the medicine box has a good effect of treating endometriosis.

Description

Technical Field

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

[0002] The uterus is composed of the inner endometrium, the outer myometrium, and the exometrium. Endometrial repair is an important physiological function of the uterus, which occurs in the late menstruation, uterine involution after delivery, miscarriage, and other types of endometrial damage. A woman's reproductive period can go through about 400 menstrual cycles. In each menstrual cycle, the endometrium undergoes severe inflammatory damage and exfoliates (producing menstruation), and is repaired and reconstructed in the late menstruation. During pregnancy, the uterus needs to increase rapidly and synergistically to accommodate the growth of the embryo. During the entire pregnancy process, the human uterus increases in volume by about 500-1000 times and in weight by about 24 times. The uterine smooth muscle layer and myometrium are reconstructed, and both have obvious hypertrophy and hyperplasia. During delivery, due to the exfoliation of the placenta, the endometrium disappears completely, but it can still be regenerated and reconstructed during the uterine involution process. If the endometrial repair is abnormal, it will lead to a variety of serious diseases and even infertility.

[0003] Stem cell research began in the 1960s, and tissue stem cells have been found in most organs of the body. Studies have shown that the repair and regeneration of tissue and cell damage is generally closely related to stem cells. During the menstrual cycle, pregnancy and after childbirth, a woman's uterus can regenerate and reconstruct rapidly, which requires the uterus to have a strong regenerative ability. Recent studies have shown that there may be a group of uterine stem / progenitor cells with proliferation and differentiation potential in the uterus, which play a decisive role in these processes. Due to the unique biological characteristics of stem cells, they have great application prospects in the fields of regenerative medicine and immunotherapy. Based on various animal experimental models and clinical experiments, it has been found that stem cells play an important role in the repair of endometrial damage.

[0004] Stem cells have high self-renewal and multidirectional differentiation potentials. They are widely present in various tissues throughout the body. They can be expanded under in vitro culture conditions and have the ability of "horizontal differentiation" or "cross-lineage differentiation". Under specific conditions, they can differentiate into neurons, osteoblasts, chondrocytes, adipocytes, etc. Stem cells have become the preferred seed cells for tissue engineering research because of this characteristic. For patients with endometrial damage, domestic and foreign scholars have done a lot of research on whether the differentiation potential of stem cells can regenerate the damaged endometrium and replenish the missing endometrium. The study found that by preparing endometrial conditioned culture medium to simulate the microenvironment of endometrial growth and adding estrogen in an in vitro environment, it was found that rabbit bone marrow mesenchymal stem cells (BMSCs) differentiated into endometrial epithelial cells. Human embryonic stem cells were co-cultured with endometrial stromal cells, estrogen and cytokines, and the results showed that human embryonic stem cells differentiated into endometrial-like cells. There are also studies that endometrial stromal stem cells differentiate into endometrial epithelial cells. Mouse BMSCs were co-cultured with endometrial stromal cells, and BMSCs could differentiate into endometrial epithelial cells. Co-culture of rat 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. The above studies demonstrate the feasibility of stem cells differentiating into endometrial-like cells, providing experimental evidence for the application of stem cell therapy after endometrial injury. Angiogenesis is an essential part of the repair process after endometrial injury. The application of cytokines, cells or other substances to stimulate angiogenesis in ischemic tissues, improve the blood supply and oxygen supply to the tissues, and thus promote tissue repair, is called therapeutic angiogenesis. Stem cell-based therapeutic angiogenesis first requires that the transplanted stem cells have chemotaxis and migration capabilities, also known as homing capabilities, that is, they migrate to specific areas of injury and settle there, thereby exerting various tissue repair functions. A large number of experiments have shown that stem cells have the ability to chemotaxis and migrate to damaged tissues. Direct differentiation into vascular cells is a mechanism for stem cells to promote angiogenesis. When tissues are damaged, especially ischemic damage, the vascular structure is destroyed, ischemia and hypoxia lead to tissue cell necrosis, and the body's own mechanism initiates angiogenesis. This process requires the participation of a large number of endothelial cells, pericytes and vascular smooth muscle cells in the repair. After receiving stem cell transplantation, the stem cells can differentiate into vascular cells, provide a source of vascular cells for angiogenesis, promote and accelerate the formation of new blood vessels in the damaged area, and thus promote tissue repair.

[0005] Many researchers emphasize that stem cells have rich secretory activity, and their role 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, anti-apoptotic factors, etc. Stem cells cultured in vitro can secrete many cytokines related to angiogenesis, such as vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), placental growth factor (PIGF), platelet-derived factor (PDGF), etc. Animal experiments based on stem cell transplantation have shown that stem cells can also secrete and increase the level of vascular growth factors in tissues in vivo, which is beneficial to angiogenesis, thereby promoting the repair of ischemic tissues. After entering the injured site, transplanted stem cells can produce cytokines through autocrine and paracrine effects. Studies have found that exosomes secreted by stem cells have an up-regulatory effect on anti-apoptotic genes BCI2L1, BCI2 and BIRC8, and a down-regulatory effect on pro-apoptotic genes CASP1, CASP8 and LTA, thereby inhibiting the apoptosis of damaged cells and playing an important role in tissue repair and regeneration. When tissues are damaged and the vascular system is destroyed, stem cells immediately respond to signals secreted by damaged tissues, such as inflammatory factors, by secreting vasoactive factors, stimulating the proliferation and differentiation of endothelial cells, and recruiting perivascular cells and (and) vascular smooth muscle cells. When tissue damage is severe, endogenous stem cells are not enough to maintain the homeostasis of the internal environment, and exogenous stem cells need to be transplanted to play the role of angiogenesis and promote tissue repair.

[0006] Patents CN201910692005.8 and CN201910925566.8, both inventions are to obtain endometrial stem cells by isolating them from human endometrial tissue, concentrating the culture supernatant, freeze-drying it, and mixing it with a solvent to finally obtain a human endometrial stem cell composite repair factor / preparation. However, the seed cells used in the above inventions are endometrial stem cells, which are difficult to obtain, and industrialization is restrictive. In addition, the composite repair factor finally prepared is in liquid form, and the effective utilization of the product during the application process is poor. Therefore, developing an effective stem cell composition for the treatment of endometrial diseases is an important research direction. Summary of the invention

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

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

[0009] Specifically, the present invention also provides a stem cell composition, characterized in that it consists of a ZGC-5 polypeptide and a pharmaceutically acceptable carrier.

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

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

[0012] Further, substitution variants generally comprise an exchange of one amino acid with another amino acid at one or more sites of a polypeptide, and can be designed to have a corresponding activity. The substitution can be conservative, i.e., 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 changes: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine ​​to serine; glutamine to asparagine; glutamic acid 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, the substitution can be non-conservative, so that the function or activity of the polypeptide is affected. Non-conservative changes generally involve replacing a residue with a chemically dissimilar residue, such as replacing a non-polar or uncharged amino acid with a polar or charged amino acid, or vice versa.Furthermore, the polypeptides of the present invention are in a therapeutically effective amount for therapeutic use.

[0013] As used herein, an "effective amount" or "therapeutically effective amount" refers to administering a sufficient amount of a polypeptide disclosed herein that will alleviate one or more symptoms of the disease or condition being treated to some extent. In some embodiments, the result is a reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired change in a biological system. For example, an "effective amount" for therapeutic use is the amount of a composition comprising a polypeptide disclosed herein required to provide a clinically significant reduction in disease symptoms. Specifically, examples of therapeutically effective amounts of polypeptides 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 an excipient. An "excipient" refers to a substance that is not a therapeutic agent in itself but is used as a diluent, adjuvant, binder and / or vehicle for addition to a pharmaceutical composition to improve its handling or storage properties or to allow or facilitate the formation of a compound or pharmaceutical composition into a unit dosage form for administration. As known to those skilled in the art, pharmaceutical excipients can provide various 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 carboxymethylcellulose, ethylcellulose, cellulose acetate, hydroxypropyl methylcellulose, hydroxypropyl cellulose, microcrystalline cellulose, and cross-linked carboxymethylcellulose (e.g., cross-linked sodium carboxymethylcellulose); (4) tragacanth powder; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter. and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, 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) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution

[0015] ssolution); (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, buccal and sublingual administration, acceptable solid dosage forms include: powders, suspensions, granules, tablets, pills, capsules, soft capsules, and capsules. The preparation method is, for example, mixing one or more compounds of the present invention, or pharmaceutically acceptable salts or tautomers thereof, adding at least one additive, such as starch or other additives. Suitable additives include: sucrose, lactose, cellulose sugar, mannitol, maltitol, dextran, starch, agar, alginate, chitin, chitosan, pectin, tragacanth, gum arabic, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymers or glycerides. Alternatively, the oral dosage form may contain other components to assist 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, flavoring agents or aromatics. Tablets and pills may be further treated with suitable coating materials known in the art.

[0017] Furthermore, the endometrial stem cells of the present invention are used at a concentration of 1×10 5 -1×10 9 / kg administration, more preferably, 1×10 6 -1×10 9 / kg administration; more preferably, 1×10 7 -1×10 9 / kg administration; more preferably, 1×10 8 -1×10 9 / kg administration; more preferably, 5×10 8 -1×10 9 / kg administration.

[0018] Specifically, the endometrial stem cells of the present invention can be cultured using a serum-free culture medium conventional in the art.

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

[0020] In some embodiments, the culture medium comprises one or more of the following: Supplements, Xeno-Free Supplements, GS21 TMSupplement or its combination. In some embodiments, the culture medium comprises or further comprises amino acids, monosaccharides, inorganic ions. In some embodiments, the amino acids include arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine or valine or its combination. In some embodiments, the inorganic ions include sodium, potassium, calcium, magnesium, nitrogen or phosphorus, or its combination or salt. In some embodiments, the medium also comprises one or more of the following: molybdenum, vanadium, iron, zinc, selenium, copper or manganese, or its combination. In certain embodiments, the culture medium comprises or consists essentially 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-1-thyronine, B-27 supplement, xeno-free B-27 supplement, GS21 TM 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] The present invention provides a stem cell composition for treating endometrial repair. Specifically, a short peptide ZGC-5 with therapeutic activity that can significantly inhibit the proliferation of ectopic endometrial cells is provided, and the short peptide has the effects of inhibiting the proliferation of ectopic endometrial cells and promoting apoptosis. The short peptide and stem cells are prepared into a drug kit, which has a good effect of treating endometriosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 OD value of each group at 570nm wavelength DETAILED DESCRIPTION

[0024] The specific embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although the specific embodiments of the present invention are shown in the accompanying drawings, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present invention and to enable the scope of the present invention to be fully communicated to those skilled in the art.

[0025] When implementing or testing the embodiments of the present invention, optional methods and materials similar or equivalent to the methods and materials described in this specification may be used, and preferred methods, devices, 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 size, shape, dimensions, materials, methods, means, etc. described in this specification may be changed according to conventional experimental methods and optimization purposes, and therefore the present invention is not limited thereto. It should also be understood that the technical terms used in this specification are only used to illustrate specific types or embodiments and are not used 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 sterile conditions, the ectopic endometrial tissue of the patient was fully minced into small particles of about 1 mm in size, and then 0.1% type W collagenase was added and digested for 3.0-4.0 hours. The cells were resuspended and counted. 9 The cells were inoculated at a cell concentration of 25 cm 2 In the culture bottle, add 4.0-5.0mL of high-glucose DMEM culture medium containing 10% FBS, 10g / L insulin and working concentration of double antibodies. Culture in a 37℃, 5% CO2 incubator, change the medium after 2 days, remove non-adherent cells, change the medium intermittently for two or three days, and observe under an inverted microscope that the cells are arranged in a vortex-like cluster, with large, round nuclei and filamentous connections between cells, indicating that ectopic endometrial cells have been successfully isolated.

[0028] Example 2 Preparation of endometrial stem cells

[0029] Obtaining materials: Collect human menstrual blood and dilute it with an equal volume of sterile phosphate buffered saline (PBS) and mix it. Use 1.077g / mL lymphocyte stratification fluid and density gradient centrifugation (2000r / min, 20min) to separate menstrual blood mononuclear cells, carefully aspirate the middle white film layer, add 2 times the volume of PBS buffer, 1500r / min, 10min wash twice, completely discard the supernatant, add 2.0mL complete culture medium to blow, mix, count, and adjust to the appropriate cell density for cell culture. The cells were inoculated in a 75mL culture bottle and placed in an incubator with a volume fraction of 5% CO2 saturated humidity at 37℃. After 72h, the culture medium was replaced and the non-adherent cells were discarded. According to the cell growth, the full amount of medium was replaced once every three or four days. When the primary cells grow to 80% to 90%, they are subcultured: the culture medium is removed, digestion solution (0.25% trypsin + 0.02% EDTA solution) is added, digestion is terminated with an equal amount of serum, centrifuged at 1300r / min for 10min, the supernatant is removed, and DMEM high-glucose culture solution containing 20% ​​fetal bovine serum, 1% amphotericin, and 1% penicillin / streptomycin is added, the cells are blown and dispersed, and subculture is performed. P3 cells are identified by flow cytometry for stem cell surface markers. Specifically, P3 cells are prepared into 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 respectively, and the same amount of mouse anti-human FITC-IgG1, PerCP-IgG1, and PE-IgG1 were added to the control tubes, respectively, and incubated at room temperature in the dark for 20 minutes. The cells were washed twice with PBS, centrifuged at 1500 r / min for 5 minutes, and re-made into cell suspension. The results of flow cytometry showed that P3 cells highly expressed CD44, CD29, and HLA-ABC, and basically did not express CD34 and CD45, indicating that endometrial stem cells were prepared.

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

[0031] Experiment 1: The ectopic endometrial cells and endometrial stem cells prepared in Example 1 were divided into 1×10 5 / 1×10 5 The upper and lower chambers of the Transwell co-culture plate were inoculated in a proportional manner to establish a non-contact co-culture system of upper and lower double-layer cells. Ectopic endometrial cells (1×10 5 ) were cultured alone as the control group.

[0032] Experiment 2: The ectopic endometrial cells prepared in Example 1 were divided into 1×10 5The cells were inoculated in the upper chamber of a Transwell co-culture plate in a certain proportion and cultured separately. Three groups, AC, with different final concentrations of ZGC-5 peptide (10 μg / mL, 50 μg / mL, and 100 μg / mL) were set up respectively.

[0033] Experiment 3: The ectopic endometrial cells and endometrial stem cells prepared in Example 1 were added at 1×10 5 / 1×10 5 The cells were inoculated in a proportional manner in the upper and lower chambers of a Transwell co-culture plate to establish an upper and lower double-layer cell non-contact co-culture system, wherein the final concentration of the ZGC-5 peptide in the system was 100 μg / mL.

[0034] Experiment 4: The ectopic endometrial cells prepared in Example 1 were cultured at 1×10 5 The cells were inoculated in the middle chamber of a Transwell co-culture plate and cultured separately, wherein the final concentration of gestrinone in the liquid was 100 μg / mL.

[0035] The MTT method was used to detect cell proliferation. After culturing the cells in each experimental group and the control group for 72 hours, the ectopic endometrial cells were digested and the cells were concentrated to 1×10 8 / L cell concentration was inoculated into 96-well culture plates, 20μL of 5g / L MTT was added to each well, and after culturing in the incubator for 4h, the culture medium was aspirated and 150μL of DMSO was added to each well, and the crystals were fully vortexed to dissolve; the number of proliferating cells was detected by enzyme-linked detector at a wavelength of 570nm, 3 replicates were set for each well, and a blank well was set as a control adjustment. All experiments were repeated 3 times and the average value was taken. The results are shown in Figure 1 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). As the concentration of the peptide group increased, the OD570 value also gradually decreased, indicating that the peptide was dose-dependent. After using the peptide and stem cells together, the OD570 was only (0.39±0.03), which was significantly lower than that of the control group (1.82±0.13), and the cell proliferation was better inhibited.

[0037] Flow cytometry was used to detect cell cycle and cell apoptosis rate: cells co-cultured for 72 hours were taken to digest ectopic endometrial cells, washed 3 times with PBS, and then suspended and fixed with 70% pre-cooled ethanol. After overnight at 4°C, an equal amount of PBS was added to wash 2 more times, 100 μL RNaseA was added to the cells and the cells were bathed at 37°C for 30 minutes, then the cells were colorized at 4°C in the dark for 30 minutes, and the percentage of apoptotic cells was detected by flow cytometry. The results are shown in Table 1.

[0038] Table 1 Results of cell apoptosis rate in each group

[0039] Group Cell 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 can be seen from Table 1, the ZGC-5 peptide of the present invention has a dose-dependent effect of promoting apoptosis of ectopic endometrial cells, and the difference is very significant compared with the control group (P<0.01); in particular, after the combined use of stem cells and polypeptides, there is a significant synergistic effect of promoting cell apoptosis. Compared with the use of stem cells or polypeptides alone, the improvement is beyond expectations, the effect is very significant, and the apoptosis rate is significantly increased compared with the positive control.

[0041] Example 3 Animal Experiments on ZGC-5 Peptide and Endometrial Stem Cells

[0042] SPF female SD rats were used for the experiment, and the EMs (endometriosis) rat model was prepared by the conventional autologous endometrial transplantation method in the field; estradiol valerate was given by gavage once a day starting 3 days before modeling.

[0043] (0.1mg / kg) to regulate the estrous cycle of rats and make them estrus at the same time: after anesthesia by intraperitoneal injection of 10% chloral hydrate (3.5mL / kg), the abdomen was opened at about 2cm above the urethra to expose the left uterus, the left uterine horn was ligated and the uterus of about 1cm in length was cut, placed in normal saline and cut into 0.5cmx0.5cm endometrial tissue blocks, which were transplanted to the right abdominal wall subcutaneous fascia layer and abdominal wall muscle layer, sutured layer by layer, and raised in a conventional way; the sham operation group underwent the same surgical access as the model group, ligated and removed the left uterine horn, but no autologous transplantation was performed. After 4 weeks, the dark gray hypoechoic area was visible under the abdominal wall by small animal ultrasound, which was the ectopic lesion. The black anechoic area in the lesion indicated that there was fluid in the lesion. The volume of the ectopic lesion measured by image segmentation was ≥50mm, and the model was considered successful.

[0044] Treatment groups and medication were performed as follows:

[0045] The normal group and the model group were intraperitoneally injected with normal saline, the intraperitoneal injection volume was 5 mL / kg, the administration frequency was once every 3 days, and the treatment course was 4 weeks.

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

[0047] Endometrial stem cell treatment group: intraperitoneal injection of endometrial stem cells, volume 5mL / kg, 1×10 7 / mL, the administration frequency was once / 3d, and the course of treatment was 4 weeks;

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

[0049] Positive control group: Gestrinone 10 mg / kg was administered orally 3 days / time for 4 weeks.

[0050] Six hours after the last administration, the laparotomy was performed after anesthesia. The endometrial tissues in situ of the rats in the normal group and the endometriotic lesions of the rats in the other groups were obtained. After being rinsed with normal saline, the length (mm), width (mm), and height (mm) of the ectopic lesions were measured with a caliper. The lesion volume (V) was calculated according to the ellipsoid volume calculation formula. V (mm 3 )=0.52×length×width×height; the pelvic adhesion score of ectopic lesions was performed according to the Haber scoring method. The results are shown in Table 2.

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

[0052] Group Pelvic adhesion 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# Combination therapy group 0.87±0.08## Positive control group 2.86±0.16#

[0053] As can be seen from Table 2, the pelvic adhesion scores of each treatment group were significantly reduced compared with the model group (#P<0.05), and the therapeutic effects of the ZGC-5 peptide treatment group and the endometrial stem cell treatment group were better than those of the positive control group, especially the combined treatment group, which had a significantly reduced effect, and the difference was extremely significant compared with the model group (P<0.01). From this result, it can be seen that the polypeptide of the present invention has a better effect when used together with stem cells.

[0054] It is to be understood that, although the present invention has been disclosed as a preferred embodiment, the above embodiment is not intended to limit the present invention. For any person skilled in the art, without departing from the scope of the technical solution of the present invention, the technical content disclosed above can be used to make many possible changes and modifications to the technical solution of the present invention, or modified into equivalent embodiments of equivalent changes. Therefore, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the scope of protection of the technical solution of the present invention.

Claims

1. A short peptide ZGC-5 with therapeutic activity for inhibiting 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 according to claim 1 in the preparation of a drug for treating endometriosis.

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

4. Use of the ZGC-5 polypeptide and endometrial stem cells described in claim 1 in preparing a drug kit for treating endometriosis.

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