Application of endometrial stem cells in ovarian repair

Abstract

The present invention provides an application of endometrial stem cells in ovarian repair. Specifically, the endometrial stem cells are used in combination with PDPA-N-5 polypeptide to treat premature ovarian failure caused by chemotherapy, which has a significant effect of promoting the recovery of ovarian function and has good application prospects.

Description

Technical Field

[0001] The present application relates to the biological field, and specifically to an application of endometrial stem cells in ovarian repair. Background Art

[0002] Premature ovarian failure (POF) refers to a disease characterized by amenorrhea, infertility, estrogen deficiency and elevated gonadotropin levels in women before the age of 40. It is an ovarian failure caused by multiple causes. The incidence rate is 1% to 3% in the general population and 2% to 10% in amenorrhea. It is a common disease in the field of gynecological endocrinology. The incidence rate has been on the rise in recent years. POF patients face a series of symptoms such as infertility and menopause, which endangers women's physical and mental health.

[0003] The main causes of premature ovarian failure include genetic factors, immune factors, environmental factors and lifestyle. In terms of genetic factors, studies have found that multiple genes are associated with premature ovarian failure. FOXL2 gene mutations may lead to abnormal ovarian development and follicular atresia; PRDM1 gene mutations may affect ovarian function and lead to premature menopause. Androgen receptor genes, bone morphogenetic protein 15 genes, etc. are also related to premature ovarian failure. Immune factors also play a certain role in the onset of premature ovarian failure. Autoimmune diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) can cause ovarian inflammatory reactions, thereby affecting ovarian function. Changes in the immune microenvironment of the ovary itself may also lead to premature ovarian failure. In terms of environmental factors, long-term exposure to certain chemicals such as plasticizers and heavy metals may induce premature ovarian failure by damaging ovarian tissue. Lifestyle habits such as excessive weight loss, long-term mental stress, and smoking may also have adverse effects on ovarian function. Lifestyle is also an important factor affecting premature ovarian failure. Bad lifestyle habits such as staying up late for a long time, lack of exercise, and bad eating habits may lead to decreased immunity and affect ovarian function.

[0004] Treatments for POF mainly involve hormone replacement, gene therapy, immunotherapy, and lifestyle adjustments. Hormone replacement therapy is one of the commonly used treatments. Since the estrogen level in POF patients is reduced, exogenous hormone replacement therapy can effectively relieve symptoms, prevent and improve symptoms and complications caused by premature ovarian failure. Estrogen can enter the body through oral or injection administration, which helps regulate the menstrual cycle, protect the endometrium and prevent osteoporosis. However, long-term use of hormone replacement therapy also requires vigilance against potential side effects, such as breast cancer, endometrial hyperplasia and even cancer. In addition to hormone replacement therapy, research on premature ovarian failure in the field of gene therapy has also made significant progress in recent years. Through in-depth exploration of genetic factors, scientists have found that some cases of premature ovarian failure are related to gene defects or abnormal expression. For these patients who may have a genetic predisposition, targeted gene therapy can be tried to restore ovarian function under the premise of risk assessment. Gene editing technology is used to repair specific gene mutations in ovarian tissue in order to restore ovarian function. Immunotherapy has also shown certain potential in the treatment of premature ovarian failure. Ovarian damage and inflammatory response caused by autoimmune factors are considered to be one of the important mechanisms of POF. Regulating the function of the immune system may be beneficial to POF patients. At present, some experimental immunotherapies have shown the effect of restoring ovarian function in clinical trials. The application of drug therapy in premature ovarian failure is showing a diversified trend. Doctors will choose the most appropriate treatment method for different patients. Whether it is hormone supplementation or gene or immunotherapy, a lot of clinical research is still needed to verify its effectiveness and safety. On the basis of individualized treatment, lifestyle adjustments are also indispensable, especially maintaining a healthy lifestyle, a reasonable diet and moderate exercise, which can help alleviate the symptoms of premature ovarian failure and improve the quality of life.

[0005] Stem cells can be directed to differentiate into functional cells of different germ layers, and autologous transplantation can repair damaged tissues and improve tissue and organ functions. At present, they are highly valued in the treatment of various systemic diseases, especially degenerative diseases and organ failure, and are considered to be a new hope for solving such diseases. Reproductive system diseases are mostly caused by dysfunction or structural damage, resulting in weakened reproductive ability. Stem cells from different sources can improve reproductive organ function and restore fertility through various mechanisms, which has become a current research hotspot. Studies have found that stem cells from various sources, including human umbilical cord MSCs, adipose-derived stem cells, ovarian granulosa-like cells derived from human induced pluripotent stem cells (PSCs), human amniotic epithelial cells, human umbilical cord blood mononuclear cells, human endometrial MSCs, bone marrow-derived MSCs, skin-derived MSCs, and human induced PSCs transfected with microRNA-17-3p, can increase the ovarian weight of POF model animals, balance serum hormone levels, increase the number of pregnancies, and restore fertility, significantly improving the ovarian function of POF model animals, proving the potential of stem cell transplantation for the treatment of POF. However, at present, there are not enough studies on the use of endometrial stem cells alone or in combination with other drugs to treat premature ovarian failure. Summary of the invention

[0006] In one aspect, the present invention provides a use of endometrial stem cells alone or in combination with other drugs in a medicine kit for treating premature ovarian failure.

[0007] The endometrial stem cells are separated from endometrial tissue or from menstrual blood. The separation of endometrial stem cells is well known in the art and is also a well-known method.

[0008] Specifically, the present invention modifies the amino acids of the polypeptide PFPA1 for activating primordial follicles to treat or assist in the treatment of premature ovarian failure in the prior art to obtain a new polypeptide PDPA-N-5 with improved functions, the amino acid sequence of which is shown in SEQ ID NO.1.

[0009] Furthermore, the peptide may be in the form of a salt, specifically an acid addition salt.

[0010] Non-limiting examples of pharmaceutically acceptable acid addition salts include: salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid or perchloric acid; salts formed with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid; and salts formed by using other methods used in the art such as ion exchange. Non-limiting examples of pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, and valerate. Pharmaceutically acceptable salts derived from appropriate bases include alkali metals, alkaline earth metals, ammonium and N+(C1-4 alkyl)4 salts. The present disclosure also envisions the quaternization of any basic nitrogen-containing groups of the polypeptides disclosed herein. Suitable non-limiting examples of alkali metal salts and alkaline earth metal salts include sodium salts, lithium salts, potassium salts, calcium salts and magnesium salts. Other non-limiting examples of pharmaceutically acceptable salts include ammonium, quaternary ammonium and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates and aryl sulfonates. Other suitable non-limiting examples of pharmaceutically acceptable salts include benzenesulfonates and glucosamine salts.

[0011] The polypeptide PDPA-N-5 has been tested on mice at a large dose and found to have no obvious animal toxicity and good safety.

[0012] On this basis, another aspect of the present invention provides a use of endometrial stem cells in combination with a novel polypeptide PDPA-N-5 with improved functions in a medicine kit for treating premature ovarian failure.

[0013] On this basis, another aspect of the present invention provides a use of endometrial stem cells in combination with a novel polypeptide PDPA-N-5 with improved functions in a drug for treating estradiol hormone recovery.

[0014] On this basis, another aspect of the present invention provides a use of endometrial stem cells in combination with a novel polypeptide PDPA-N-5 with improved functions in a drug for treating and promoting oocyte development.

[0015] Furthermore, the dosage of endometrial stem cells in the kit or drug of the present invention is 1×10 9 / kg, or about 5x10 8 / kg, or about 4x10 8 / kg, or about 3x10 8 / kg, or about 2x10 8 / kg, or about 1x10 8 / kg, or about 5x10 7 / kg, or about 4x10 7 / kg, or about 3x10 7 / kg, or about 2x10 7 / kg, or about 1x10 7 Pieces / kg.

[0016] Further, the dosage of the polypeptide in the medicine kit or drug is 10 mg / kg, preferably 9 mg / kg, preferably 8 mg / kg, preferably 7 mg / kg, preferably 6 mg / kg, preferably 5 mg / kg, preferably 4 mg / kg, preferably 3 mg / kg, preferably 2 mg / kg, and preferably 1 mg / kg.

[0017] Furthermore, the medicine kit or drug of the present invention further contains a pharmaceutically acceptable carrier.

[0018] The kit or medicine disclosed herein may optionally include a surfactant. A surfactant or surfactant mixture will typically reduce the interfacial tension between the solid dispersion and the aqueous medium. An appropriate surfactant or surfactant mixture may also enhance the water solubility and bioavailability of the polypeptide. Surfactants useful in conjunction with the present disclosure include, but are not limited to, sorbitan fatty acid esters (e.g., sorbitan monostearate), polyoxyethylene sorbitan fatty acid esters (e.g., sodium lauryl sulfate (SLS), sodium dodecylbenzene sulfonate (SDBS), sodium octyl sulfosuccinate (docusate sodium), deoxycholic acid sodium salt (DOSS), sorbitan monostearate, sorbitan tristearate, cetyltrimethylammonium bromide (HTAB), sodium N-lauroyl sarcosinate, sodium oleate, sodium myristate, sodium stearate, sodium palmitate, Gelucire 44 / 14, ethylenediaminetetraacetic acid (EDTA), vitamin E d-alpha tocopheryl polyethylene glycol 1000 succinate (TPGS), lecithin, monosodium glutamate monohydrate, Labrasol, PEG 8 caprylic / capric glycerides, Transcutol, diethylene glycol monoethyl ether, Solutol HS-15, polyethylene glycol / hydroxystearate, taurocholic acid, Pluronic F68, Pluronic F108 and Pluronic F127 (or any other polyoxyethylene-polyoxypropylene copolymer or saturated polyglycolized glyceride). Specific examples of such surfactants that can be used in conjunction with the present disclosure include, but are not limited to, Span 65, Span 25, Tween 20, Capryol 90, Pluronic F108, sodium lauryl sulfate (SLS), Vitamin E TPGS, pluronic and copolymers.

[0019] Beneficial Effects

[0020] The present invention provides an application of endometrial stem cells in ovarian repair. Specifically, the endometrial stem cells are used in combination with PDPA-N-5 polypeptide to treat premature ovarian failure caused by chemotherapy, which has a significant effect of promoting ovarian function recovery and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Effects of each group on the estradiol hormone level in mice

[0022] Figure 2 Results of superovulated eggs in each group of mice DETAILED DESCRIPTION

[0023] Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described by preferred embodiments, and relevant personnel can obviously change or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and apply the technology of the present invention. The methods, equipment, and materials in the following implementation cases, if not specifically stated, are all conventional methods, equipment, and materials in the art, and can be purchased from the market.

[0024] Example 1 Preparation and functional identification of PFPA1 modified peptide

[0025] Using the original peptide PFPA1---SGLIYEETRGVLKV as a template, the peptide was modified and restructured. Through computer modeling analysis, 8 candidate peptides with good expected activities were screened, as shown in Table 1.

[0026] Table 1 List of candidate peptides

[0027] Candidate peptide name Amino acid sequence PDPA-N-2 SGLIYERTRGVLKV PDPA-N-5 SGLIYEESRGVLKV PDPA-N-9 SGLIYERTRVVLKV PDPA-N-14 SGLIYEESRVVLKV PDPA-N-20 SGLIYEQTRGVLKV PDPA-N-29 SGLIYRETRGVLKV PDPA-N-33 SGLIYEEVRGVLKV

[0028] The candidate peptides and the original polypeptide PFPA1---SGLIYEETRGVLKV were synthesized by Hongpeptide Biotechnology and dissolved in water at appropriate concentrations.

[0029] Healthy C57BL / 6 female mice purchased from Beijing Biolab Technology Co., Ltd. were cultured in the SPF experimental animal room. After sexual maturity, the mouse ovaries were collected under a dissecting microscope, transferred to a culture dish, and cultured in a conventional incubator at 37°C and 5% CO2 for 24 hours; the ovarian tissue in vitro culture medium contained 50μM concentration of peptide, 1% penicillin / streptomycin, 1% ITS, 0.05mg / ml vitamin C, and 3mg / ml BSA in DMEM / F12 medium. An equal volume of water was added as the control group. Ovarian tissue was taken after 24 hours. The treated ovarian tissues of each group were fixed in 4% paraformaldehyde, dehydrated with various concentrations of alcohol the next day, embedded in paraffin, and continuously sectioned at a thickness of 5μm / sheet. The sections were stained with hematoxylin. When counting follicles, the first of every 5 consecutive sections was taken to count follicles at all levels, and the follicle typing was based on the recognized standards established by Pedersen and Peters. The percentage of follicles at each level in each group was statistically analyzed and the results are shown in Table 2.

[0030] Table 2 Results of primordial follicle percentage and primary follicle percentage in each group

[0031] Candidate peptide name Primordial follicle percentage (%) Primary follicle percentage (%) PDPA-N-2 89.51±0.23 6.98±0.14 PDPA-N-5 87.26±0.18 9.76±0.25 PDPA-N-9 89.74±0.27 6.84±0.17 PDPA-N-14 88.16±0.30 7.92±0.23 PDPA-N-20 89.07±0.24 7.15±0.18 PDPA-N-29 88.16±0.32 7.99±0.22 PDPA-N-33 91.46±0.18 6.13±0.16 PFPA1 88.52±0.47 7.84±0.33 Control group 91.53±0.38 6.02±0.21

[0032] As can be seen from Table 2, the effect of the modified polypeptide has a certain degree of uncertainty. It is unexpectedly found that only the modified PDPA-N-5 polypeptide has a better effect of increasing the percentage of primary follicles compared with the PFPA1 polypeptide.

[0033] Example 2 Preparation of endometrial stem cells

[0034] The excised endometrial tissue from the ovarian endometriosis cyst surgery (reviewed by the ethics committee and signed the informed consent) was washed with PBS three times, and the eutopic and ectopic endometrium were scraped and cut into 1 mm 3 Add collagenase III (300 μg / ml) + DNase (40 μg / ml) to a small piece of 100 μg / ml volume, place in a 30°C constant temperature water bath shaker (150 rpm / min) for digestion for 45 min, add DMEM / F12 culture medium containing 10% FBS to terminate digestion, filter with a 100-mesh filter, and collect the filtrate. Filter with a 400-mesh filter to separate stromal cells (<400 mesh). Collect the suspension, centrifuge at 1000 rpm / min for 10 min, discard the supernatant, resuspend the precipitate with 1 ml of culture medium, slowly add it dropwise to a centrifuge tube with 8 ml of culture medium, centrifuge at 400 rpm / min for 3 min, and remove the clumped cells. Transfer the supernatant to another centrifuge tube, centrifuge at 1000 rpm / min for 10 min, discard the supernatant, and the bottom precipitate is the purified endometrial stromal cells. The cells were resuspended in DMEM / F12 culture medium containing 20% ​​FBS, cultured in a 37°C, 5% CO2 incubator for 4 hours, and then the medium was changed to remove non-adherent cells. Cell growth and morphological characteristics were continuously observed under an inverted microscope. When the primary cells covered 90% of the flask wall, they were digested with 0.25% trypsin-0.1EDTA digestion solution, and passaged at a ratio of 1:2. The third generation cells were taken for the experiment, which were endometrial stem cells.

[0035] Immunofluorescence was used to detect endometrial stem cell-related markers CD34, CD117, Oct-4, ABCG2, CD90, and CD105. The third generation endometrial stem cell spheres were selected, washed twice with PBS, fixed with 4% paraformaldehyde; washed three times with PBS, 5 minutes each time; blocked with normal goat serum for 20 minutes, incubated with primary antibody (1:100) in a wet box at 4°C overnight; washed three times with PBS, 5 minutes each time; added FITC-labeled goat anti-mouse IgG secondary antibody (1:100), placed in a wet box, and incubated at 37°C for 30-60 minutes; washed twice with PBS, 5 minutes each time, and washed once with distilled water. Observed under a fluorescence microscope. The results showed that the positive rates of CD34, CD117, Oct-4, ABCG2, CD90, and CD105 were 93.6%, 95.7%, 68.5%, 86.5%, 97.5%, and 98.2%, respectively. The above results indicate that endometrial stem cells were isolated.

[0036] Example 4 Ovarian anti-aging experiment of PDPA-N-5 polypeptide combined with endometrial stem cells

[0037] The construction of the premature ovarian failure model also adopts the more mature methods in this field. Specifically, C57BL / 6 mice were intraperitoneally injected with cyclophosphamide 120mg / kg + busulfan 30mg / kg once to establish a premature ovarian failure mouse model. The POF mouse model was successfully established by observing the disorder of the estrous cycle. The POF group was enrolled, and the model group was the PBS injected into the tail vein of the mice; the POF mice injected with endometrial stem cells into the tail vein of the mice were the stem cell group; the POF mice injected with endometrial stem cells into the tail vein of the mice were the stem cell group; the POF mice injected with endometrial stem cells into the tail vein of the mice were the stem cell group; the POF mice injected with PDPA-N-5 polypeptide in situ in the ovaries were the polypeptide group; the POF mice injected with PFPA1 polypeptide in situ in the ovaries were the polypeptide control group; the POF mice injected with endometrial stem cells and then injected with PDPA-N-5 polypeptide in situ in the ovaries were the combined treatment group; the normal mice in the same period were injected with PBS into the tail vein as the blank control group. Specifically, the injection of stem cells is the injection of endometrial stem cells isolated in Example 3 into the tail vein of the mice starting 3 days after the injection of chemotherapy drugs. Each mouse is injected with 200μl, containing approximately 2x10 cells 6 The second injection was performed after an interval of 7 days. The in situ ovarian injection of polypeptides was performed by injecting the polypeptides into the left and right ovarian bursae of mice using a capillary, twice a week for a total of 3 weeks, with each dose of 5 mg / kg. The combined treatment group was injected with stem cells and polypeptides at the same time, in the same manner as the single injection, and the dosage was a combination of the two.

[0038] One week after the end of the drug administration in each group, three mice were selected from each group, and their eyeballs were removed to collect blood. After being placed at 4°C for 2 hours, the blood was centrifuged at 5000g for 5 minutes to separate the serum and detect the estradiol hormone level using an Elisa kit. Figure 1 shown.

[0039] Changes of serum estradiol in each group after treatment Figure 1 As shown. Figure 1 It can be seen that compared with the normal control group, the estradiol content in the model group was significantly reduced, and the difference was statistically significant (P<0.01). The model mice were effectively treated with stem cells and peptides or both alone to increase the estradiol content of the model mice. The serum estradiol concentration of the peptide group alone was (185.19±17.25) pg / mL, and the serum estradiol concentration of the peptide combined with stem cells was (231.89±18.97) pg / mL, which was still higher than the estradiol content of the normal control group. This shows that peptides combined with stem cells have a good effect in treating premature ovarian failure.

[0040] The ovaries of the mice in each group were fixed in 4% paraformaldehyde overnight after blood collection, and then embedded in paraffin after dehydration. The ovaries were stained with conventional H and analyzed under an optical microscope. The results showed that the ovaries of the mice in the normal control group were larger and redder in appearance, while the ovaries of the mice in the POF group were significantly smaller and pale in appearance. The ovaries of the mice in the stem cell group or the polypeptide group or the combined treatment group were significantly improved compared with the POF group, with larger volume and redder surface color, indicating that the treatment effect was obvious.

[0041] The remaining mice in each group were subjected to the mouse superovulation experiment. 7 days after the end of the drug administration, 5IU of PMSG was injected intraperitoneally in each group of mice, and 48 hours later, 5IU of HCG was injected intraperitoneally. 15 hours later, the mice were killed by dislocation to obtain bilateral oviducts, and a small hole was torn at the enlarged part of the oviduct ampulla with a 1m injection needle to collect the discharged MI mature eggs. After collagenase digestion, the number of superovulated eggs in each mouse in the three groups was counted.

[0042] from Figure 2 The results show that the number of superovulated ovaries in each group of mice is: normal control group (40.5±2.9), model group (9.8±2.1), stem cell group (24.9±3.0), polypeptide group (27.0±2.1), polypeptide control group (23.4±1.1), combined treatment group (35.4±2.5). From the above results, it can be seen that the number of superovulated ovaries in each treatment group is higher than that in the POF group, and the difference is statistically significant (P<0.05). Each treatment group has a good effect in treating premature ovarian failure.

[0043] Although the embodiments of the present invention are described above in conjunction with the accompanying drawings, the present invention is not limited to the above specific embodiments and application fields, and the above specific embodiments are only illustrative and instructive, rather than restrictive. A person of ordinary skill in the art can also make many forms under the guidance of this specification and without departing from the scope of protection of the claims of the present invention, all of which belong to the protection of the present invention.

Claims

1. A polypeptide PDPA-N-5 for assisting the treatment of premature ovarian failure, characterized in that The amino acid sequence is shown in SEQ ID NO.

1.

2. Use of the polypeptide PDPA-N-5 as claimed in claim 1 in the preparation of a drug for treating premature egg aging.

3. Use of the polypeptide PDPA-N-5 and endometrial stem cells as claimed in claim 1 in the preparation of a pharmaceutical composition for repairing premature ovarian failure.

4. The use according to claim 3, characterized in that The endometrial stem cells are separated from endometrial tissue or from menstrual blood.

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