Preparation method of ovarian stem cells and application of ovarian stem cells in ovarian aging resistance

Abstract

The invention provides a preparation method of ovarian stem cells and application of the ovarian stem cells in ovarian aging resistance. More specifically, ovarian stem cells and polypeptide LRP-C5 which is separated from pilose antler polypeptide and has a good ovarian aging treatment effect are prepared and screened from ovaries, ovarian aging of rats can be well treated after the ovarian stem cells and the polypeptide LRP-C5 are used independently or jointly, and the application prospect is good.

Description

Technical Field

[0001] The present application relates to the biological field, and specifically to a method for preparing ovarian stem cells and an application thereof in resisting ovarian aging. 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] Current studies have shown that premature ovarian failure is mainly caused by the following factors. The first is genetic factors. 10% of POF patients have a family history and can be vertically transmitted. POF is a group of X-linked genetic diseases. It is often accompanied by chromosome recombination, translocation or haploid changes. X chromosome cytogenetics abnormalities such as XTurner syndrome and its variants are the most common causes of POF. Some other X chromosome abnormalities such as X trisomy, isochromosome, partial deletion of the short arm and long arm of X. Balanced translocation involving X chromosomes can lead to the occurrence of POF. The presence of mosaicism is an important pathogenic factor of POF. The second is gonadotropin action disorder. The conduction defects of gonadotropins FSH, LH and their receptors can cause POF. Early studies have found that some POF patients do not have complete follicle failure, but lack response to endogenous gonadotropins. In addition, external factors such as radiotherapy and chemotherapy are also important influences. When the ovaries receive a direct radiation dose of 1.5 to 8.0 Gy, about 50% to 70% of women aged 15 to 40 will experience ovarian failure. When the dose is greater than 8 Gy, almost all women of childbearing age will suffer irreversible damage to their ovaries.

[0004] The treatment of premature ovarian failure is mainly hormone replacement therapy for unmarried girls or those who do not want to have children, but due to the side effects of hormone therapy, patients find it difficult to tolerate long-term treatment. The treatment for those who want to have children is more complicated, but the purpose is to induce fertility. In the near future, the main way for POF patients who want to have children is to receive donated eggs to make them pregnant. Some studies have shown that the use of continuous estrogen and progesterone therapy to treat POF patients who do not have fertility requirements, in order to establish regular withdrawal bleeding to form "menstruation", can improve perimenopausal and postmenopausal symptoms and reduce the incidence of cardiovascular disease and osteoporosis. For POF patients who have fertility requirements, ovulation induction treatment is performed. About 50% of POF patients have a certain degree of development of follicles in their ovaries, and 5% to 10% can resume ovulation and become pregnant naturally. The methods recommended by Takeuchi Hiroyuki in 1997 include: estrogen and progesterone replacement therapy, and the commonly used Kaufmann therapy: starting from the 5th day of withdrawal bleeding, give 1.25 mg of Premarin daily, and add 6 mg / d of medroxyprogesterone after 14 consecutive days, and continue to use it for 7 days. This is done for 3 to 4 cycles, and observe whether there is follicular development after 1 to 2 months of discontinuation. Estrogen can promote granulosa cell proliferation, induce the formation of FSH receptors on granulosa cells, inhibit the atresia of residual follicles in the ovaries, and improve perimenopausal or postmenopausal symptoms. It is the preferred method for POF ovulation induction. However, the success rate of ovulation induction in clinical treatment is low. The ovulation recovery rate after treatment within 1 year of amenorrhea is 42.9%, and the ovulation recovery rate after treatment for amenorrhea for more than 1 year is less than 10%. POF patients have no follicles in both ovaries or have follicles but lack response to exogenous gonadotropin. The uterus of the patients is normal in morphology, and the endometrium responds well to estrogen and progesterone. If the economic conditions permit, artificial insemination and embryo transfer of donated eggs can be performed to achieve fertility, and the clinical pregnancy success rate is about 25% to 35%. Another study showed that 31 POF patients were first treated with estrogen and progesterone continuous therapy to establish a menstrual cycle, and ovulation induction treatment was used after 3 to 6 cycles. As a result, 8 of the 19 POF patients with hot flashes, sweating and irritability had their symptoms relieved or disappeared, vaginal secretions increased compared to before, and the quality of sexual life was significantly improved. All 19 cases had menstrual bleeding on the 3rd to 6th day after stopping the drug, but most of them could not have natural menstruation after stopping hormone treatment. The use of artificial cycles to treat POF has a total effective rate of 88%. Clinical observations of POF patients who have used hormones for 2 to 4 years have shown that hormone therapy has certain risks. Patients may experience breast pain, which may be caused by the large amount of progesterone entering the blood, increasing the volume of breast tissue, increasing the water content in cells, and stimulating breast nerve endings. Long-term use of sex hormone therapy can induce cystic changes or poor results in the breast ducts and increase the mitotic rate of breast cells.

[0005] Choosing other treatments without side effects is an important research direction. Proliferation is the most important life characteristic of an organism. Promoting the proliferation of various ovarian cells, especially ovarian granulosa cells, and activating follicular function are also important ways to treat premature ovarian failure. Only when the number of granulosa cells increases and the number of follicles at all levels recovers, can ovarian function gradually recover. A foreign animal study confirmed that umbilical cord blood-derived mesenchymal stem cells directly restore the function of existing oocytes by promoting the proliferation of ovarian surface epithelial cells and re-repairing the white membrane to treat premature ovarian failure, and indirectly repair damaged ovarian function by inhibiting cystine protease 3 and activating cytokeratin 8 / 18, transforming growth factor β, and homologous lost phosphatase tensin gene. Another study used human menstrual blood-derived mesenchymal stem cells to treat mice with premature ovarian failure, and the ovarian tissue proliferation marker Ki67 increased, proving that its treatment mechanism may be to promote cell proliferation. The study also proved that human menstrual blood-derived mesenchymal stem cells can also be induced to differentiate into ovarian tissue-like cells, especially ovarian granulosa-like cells. The results of the microarray analysis of cDNA expression profiles in this study showed that the expression profiles of human menstrual blood-derived mesenchymal stem cells were significantly different from those of human ovarian tissue before transplantation, but after surviving for several days in premature ovarian failure, their gene expression profiles were similar to those of human ovarian tissue. This result strongly proves that human menstrual blood-derived mesenchymal stem cells are induced to differentiate into ovarian tissue-like cells in the ovaries. Therefore, mesenchymal stem cells can treat premature ovarian failure by promoting cell proliferation.

[0006] Based on the good application prospects of mesenchymal stem cells, developing better stem cell treatments for ovarian-related diseases is a key research direction. Summary of the invention

[0007] The present invention provides a method for preparing ovarian stem cells and ovarian stem cells prepared based on the method.

[0008] In addition, based on previous research that velvet antler polypeptides can have a good therapeutic effect on premature ovarian failure, the inventors of the present invention used neutral protease hydrolysis of the velvet antler polypeptides, and then used ultrafiltration, column separation and HPLC separation and identification, and then mass spectrometry identification to obtain a polypeptide LRP-C5 that has a good therapeutic effect on ovarian aging, whose amino acid sequence is shown in SEQ ID NO: 1.

[0009] Further, the polypeptide can also be provided with conservative substitutions of amino acids with similar functions, which are well known to those skilled in the art. For example, the characteristics of the amino acid side chains are hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), and side chains with the following common functional groups or characteristics: aliphatic side chains (G, A, V, L, I, P); hydroxyl groups containing side chains (S, T, Y); sulfur atoms containing side chains (C, M); carboxylic acids and amino compounds containing side chains (D, N, E, Q); bases containing side chains (R, K, H); and aromatic hydrocarbons containing side chains (H, F, Y, W).

[0010] The present invention also provides a pharmaceutical composition for treating ovarian aging, characterized in that ovarian stem cells are the main active ingredients.

[0011] The present invention also provides a pharmaceutical composition for treating ovarian aging, characterized in that the anti-ovarian aging polypeptide LRP-C5 is a main active ingredient.

[0012] The present invention also provides a pharmaceutical composition for treating ovarian aging, characterized in that ovarian stem cells and anti-ovarian aging polypeptide LRP-C5 are main active ingredients.

[0013] The pharmaceutical composition further contains a carrier well known in the art.

[0014] Specifically, the carrier includes a wetting agent or surfactant, an excipient, a pH buffer, a stabilizer, a preservative, and the like.

[0015] Examples of suitable pharmaceutically acceptable wetting agents or surfactants include, but are not limited to, amphoteric, nonionic, cationic or anionic molecules. Suitable surfactants include, but are not limited to, polysorbates, sodium dodecyl sulfate (sodium lauryl sulfate), dodecyl dimethylamine oxide, sodium docusate, cetyl trimethyl ammonium bromide (CTAB), polyethoxylated alcohols, polyoxyethylene sorbitan, octoxynol, N,N-dimethyldodecylamine-N-oxide, cetyl trimethyl ammonium bromide, polyoxyethylene (10) lauryl ether, surfactants (polyoxyethylene fatty alcohol polyoxyethylene ethers derived from lauryl alcohol, cetyl alcohol, stearyl alcohol and oleyl alcohol), bile salts (e.g., sodium deoxycholate and sodium cholate), polyoxyethylene castor oil, nonylphenol ethoxylate, cyclodextrin, lecithin, methylbenzethonium chloride, carboxylates, sulfonates, petroleum sulfonates, alkylbenzene sulfonates, naphthalene sulfonates, olefin sulfonates, alkyl sulfates, sulfates, sulfated natural oils and fats, sulfated esters, sulfated alkanolamides, alkylphenols (ethoxylated and sulfated), ethoxylated fatty alcohols, polyoxyethylene surfactants, carboxylates, polyethylene glycol esters, sorbitan esters and ethoxylated derivatives thereof, fatty acid glycol esters, carboxamides, monoalkanolamine condensates, polyoxyethylene fatty acid amides, quaternary ammonium salts, amines having amide bonds, polyoxyethylene alkylamines and polyoxyethylene alicyclic amines, N,N,N,N-tetrasubstituted ethylenediamines, 2-alkyl-1-hydroxyethyl-2-imidazolines, N-cocoyl-3-aminopropionic acid / sodium salt, N-tallow-3-iminodipropionic acid disodium salt, N-carboxymethyl-N,N-dimethyl-N-9-octadecenyl ammonium hydroxide, N-cocoylamideethyl-N-hydroxyethylglycine sodium salt, etc., polyoxyethylene, sorbitan monolaurate and stearate, (polyethoxylated castor oil), (ethylene oxide / 12-hydroxystearic acid), polysorbate, tyloxapol, and any combination thereof. Preferred pharmaceutically acceptable surfactants include tyloxapol and (sorbitan monooleate) or mixtures thereof.

[0016] Examples of suitable pharmaceutically acceptable preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride and cetylpyridinium chloride, benzyl bromide, benzyl alcohol, disodium EDTA, phenylmercuric nitrate, phenylmercuric acetate, ethylmercuric sodium thiosalicylate, thimerosal, acetate and phenylmercuric borate, polymyxin B sulfate, chlorhexidine, methylparaben and propylparaben, phenylethyl alcohol, quaternary ammonium chloride, sodium benzoate, sodium propionate, stable chlorine oxygen complex, sorbic acid or a mixture thereof. Preferred pharmaceutically acceptable preservatives include disodium EDTA (disodium ethylenediaminetetraacetic acid) and benzalkonium chloride or a mixture thereof. In one embodiment, the pharmaceutically acceptable preservative is present in an amount of about 0.01% to about 2.0% (weight / volume), preferably about 0.05% to about 1% (weight / volume). Examples of suitable pharmaceutically acceptable buffers include, but are not limited to, sodium chloride, glucose, lactose and phosphate buffered saline (PBS) or any combination thereof. Other suitable pharmaceutically acceptable buffers include, but are not limited to, disodium succinate hexahydrate, borate, citrate, phosphate, acetate, saline, tris-HCl (tris(hydroxymethyl)aminomethane hydrochloride), HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), sodium phosphate, sodium borate, saline, citrate, carbonate, phosphate and / or mixtures thereof. In one embodiment, the pharmaceutically acceptable buffer is present in an amount of about 0.01% to about 2.0% (weight / volume), preferably about 0.05% to about 1% (weight / volume).

[0017] Furthermore, the present invention also provides the use of ovarian stem cells as a main active ingredient in preparing a pharmaceutical composition for treating ovarian aging.

[0018] Furthermore, the present invention also provides the use of the anti-ovarian aging polypeptide LRP-C5 as a main active ingredient in the preparation of a pharmaceutical composition for treating ovarian aging.

[0019] Furthermore, the present invention also provides the use of the anti-ovarian aging polypeptide LRP-C5 and ovarian stem cells as main active ingredients in the preparation of a pharmaceutical composition for treating ovarian aging.

[0020] Furthermore, the ovarian stem cells may be mature cell lines purchased commercially, or may be directly isolated and prepared from ovaries.

[0021] Specifically, the concentration of ovarian stem cells used in the present invention is 1×10 7 / kg, 2×10 7 / kg, 3×10 7 / kg, 4×10 7 / kg, 5×10 7 / kg, 6×107 / kg, 7×10 7 / kg, 8×10 7 / kg, 9×10 7 Pieces / kg.

[0022] Specifically, the ovarian stem cells of the present invention are used at a concentration of 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1 mg / kg, 1.5 mg / kg, or 2 mg / kg.

[0023] Beneficial Effects

[0024] The present invention provides a method for preparing ovarian stem cells and their application in anti-ovarian aging. More specifically, the present invention prepares and screens ovarian stem cells from ovaries and separates LRP-C5, a polypeptide with good ovarian aging treatment effect from deer antler polypeptides. The two can effectively treat rat ovarian aging when used alone or in combination, and have good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Effect of each experimental group on the E2 concentration in the culture medium

[0026] Figure 2 Effects of each group on apoptosis rate of ovarian granulosa cells DETAILED DESCRIPTION

[0027] 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.

[0028] Example 1 Preparation of mouse ovarian stem cells

[0029] Take the ovarian tissue of a female C57BL / 6 mouse that has just reached adulthood, take a tissue block of about 1 cm3, place it in a sterile vial containing 1 ml of normal saline, rinse the tissue 3 times with PBS, and try to remove red blood cells. Use ophthalmic scissors to fully chop the tissue into a paste. Add 3 times the volume of collagenase IV to the dish, blow and transfer to a centrifuge tube, and place it in a 37°C incubator. Digest for about 30 minutes, and blow thoroughly every 10 minutes until the tissue block dissolves and the digestion fluid becomes turbid. Add an equal amount of culture medium I and mix thoroughly. The culture medium I is DMEM (Hyclone) + 10% fetal bovine serum (Hyclone) + 1% penicillin and streptomycin (Sigma). Centrifuge at 1300r / min for 8min, discard the supernatant, add appropriate amount of PBS to resuspend the cells 3 times, filter with a 200-mesh filter, add 5ml culture solution II to the culture dish after centrifugation, the culture solution II is 80% Knockout DMEM (Gibco) + 20% Knockout Serum Replacement (Gibco) + 1% non-essential amino acids (Hyclone) + 1% glutamine (Gibco) + 1% β-dimercaptoethanol (Gibco) + 4ng / ml β-FGF (Gibco) + 1% penicillin, streptomycin (Sigma), and culture in a 37°C, 0.5% CO2 incubator. Change the medium every other day, and digest and passage the cells in the dish when they grow to 80% confluence. Discard the culture solution, add PBS to wash twice, add 0.25% trypsin to cover the bottom of the dish, and place it in a 37°C incubator. Observe the digestion status under the microscope every 2 minutes. When most of the adherent cells become round and bright, add an equal amount of culture medium I containing 10% fetal bovine serum to stop digestion. Use a pipette to repeatedly and gently blow the bottom of the dish to make a single cell suspension. Centrifuge at 1500r / min for 5 minutes, discard the supernatant, add an appropriate amount of PBS to resuspend the cells twice, add an appropriate amount of culture medium II, pass the cells at a ratio of 1:2, and culture in a 37℃, 0.5% CO2 incubator. Change the medium every other day, screen MVH+ positive cells using flow cytometry, and continue to culture to prepare mouse ovarian stem cells.

[0030] Identification of mouse ovarian stem cells: Under the microscope, the cultured mouse ovarian stem cells were round cells with little cytoplasm, spherical nuclei, a large nuclear-cytoplasmic ratio, and a nuclear diameter of 15-20μm. The cells were stained with AKP and Oct-4 for pluripotency identification, and the results showed that both were positive, so they were identified as ovarian stem cells. MVH was used for identification, and 99.87% were positive. This marker is only expressed in germ cells, which also shows that the isolated cells are ovarian stem cells.

[0031] Example 2 Efficacy verification of polypeptide LRP-C5

[0032] Female C57BL / 6 mice that have just grown up are used for egg collection. The follicular fluid after egg collection is stored on ice and quickly transferred to the laboratory for granulosa cell extraction. After low-temperature centrifugation, the cell pellet is taken and placed in 50% (volume fraction) percoll cell separation solution. After centrifugation, the cell layer is aspirated and washed in PBS to remove the percoll solution. Then three times the volume of red blood cell lysis solution is taken from the cell pellet to remove a small amount of residual red blood cells. After centrifugation, the pellet is taken in PBS to wash the red blood cell lysis solution and then centrifuged to obtain a cell pellet. DMEM / F12 {15% (volume fraction) fetal bovine serum, 1% double antibody, and a final concentration of 1 μmol / L androstenedione solution} is used to continue cell culture for 24 hours. Under an inverted microscope, the cultured cells can be observed as uniform round mononuclear cells. The results of cell slide staining show that positive staining is located in the cell membrane and cytoplasm, showing brown coloration, and the cell nucleus is blue. It can be seen that more than 96.3% are granulosa cells after identification, indicating that ovarian granulosa cells have been isolated and prepared.

[0033] The isolated ovarian granulosa cells were adjusted to a cell concentration of 10 using DMEM / F12 {15% (volume fraction) fetal bovine serum, 1% double antibody, and a final concentration of 1 μmol / L androstenedione solution} cell culture medium. 5 / ml, take a 6-well cell culture plate, add 2ml of culture system to each well, and culture it in a 37°C, 5% CO2 incubator for 72h. Discard the old culture medium and replace it with culture medium that does not contain androstenedione and contains different concentrations of polypeptide LRP-C5 (10μg / mL (low concentration), 100μg / mL (medium concentration), 200μg / mL (high concentration)). The negative control is a blank control group with only culture medium, and the culture is continued for 48h. The concentration of E2 in the supernatant of each group was detected by ELISA kit. The operation was based on the instructions of the kit. The absorbance value of the enzyme-linked immunosorbent assay was read at 450nm, and the E2 concentration in the culture medium was calculated using the standard curve. The results are as follows Figure 1 shown.

[0034] from Figure 1 The results showed that with the increase of polypeptide concentration, the expression level of E2 showed a dose-dependent increase compared with the control group (P < 0.05). When the concentration was 200 μg / mL, the expression level of estrogen reached (841.5 ± 13.1) pg / mL.

[0035] CCK-8 proliferation assay was used to detect the effects of each group on the proliferation ability of ovarian cells in vitro. The results are shown in Table 1.

[0036] Table 1 OD values ​​of each group at 450nm wavelength

[0037] Group OD value Negative control group 0.50±0.04 Low concentration peptide group 0.61±0.03 Medium concentration peptide group 0.72±0.04 High concentration peptide group 0.81±0.02

[0038] It can be seen from Table 1 that with the increase of polypeptide concentration, it can significantly promote the proliferation ability of ovarian cells in vitro compared with the negative control group.

[0039] Example 3 Animal Experiment

[0040] SPF grade 10-week-old female SD rats, weighing 250-270g, were purchased from Saiye Biotechnology. The model preparation method is as follows: On the first day, the rats were intraperitoneally injected with 60 mg / kg of cyclophosphamide (prepared with saline to a mass concentration of 6 mg / mL, injected at a volume of 10 mL / kg); subsequently, the rats were intraperitoneally injected with 10 mg / kg of cyclophosphamide (prepared with saline to a mass concentration of 1 mg / mL, injected at a volume of 10 mL / kg), once a day for 14 consecutive days. During the modeling period, vaginal secretions of rats were scraped at a fixed time every day to prepare vaginal smears to observe the estrous cycle of rats. The rat estrous cycle is divided into: proestrus (lasting 17-21 hours, all nucleated epithelial cells on the smear may be accompanied by a small number of keratinized cells), estrus (lasting 9-15 hours, all anucleated keratinized cells or a small number of epithelial cells on the smear), metestrus (lasting 10-14 hours, nucleated epithelial cells, keratinized cells, and leukocytes can be seen on the smear), and diestrus (lasting 60-70 hours, a large number of leukocytes and a small number of epithelial cells and mucosa can be seen on the smear). If the rat estrous cycle is disordered or prolonged or stays in a certain period for a long time, it means that the model is successfully established.

[0041] The POF rats with successful modeling were randomly divided into a model group, a low-dose LRP-C5 peptide group, a high-dose LRP-C5 peptide group, a low-dose peptide + ovarian stem cell group, an ovarian stem cell group, and a positive control group, with 10 rats in each group. Another 10 healthy rats were taken as a negative control group.

[0042] The rats in the low-dose and high-dose groups were injected with 1 and 2 mg / kg of peptide injection solution through the tail vein, respectively (the peptide solution with a mass concentration of 1 and 2 mg / mL was prepared with normal saline and injected at a volume of 1 mL / kg, once a day);

[0043] The ovarian stem cell group was injected with 2×10 ovarian stem cells prepared in Example 1 via the tail vein. 7 Pieces / kg;

[0044] The low-dose polypeptide + ovarian stem cell group was injected with 1 mg / kg polypeptide injection via the tail vein, and 2×10 ovarian stem cells from Example 1 were injected via the tail vein at the same time. 7 Pieces / kg;

[0045] Positive control group: oral administration of estradiol valerate tablets 1 mg / kg;

[0046] The rats in the negative control group and the model group were injected with 1 mL / kg of normal saline via the tail vein. All rats were given the drug for 4 consecutive weeks.

[0047] 24 hours after the last administration, rats in each group were anesthetized by inhalation of ether, and their tail vein blood was collected and centrifuged at 4400r / min for 14 minutes at 4°C. The upper serum was taken and the E2, AMH, and FSH levels in the rat serum were detected by microplate reader according to the instructions of the corresponding ELISA kit. The results are shown in Table 2.

[0048] Table 2 Effects of each group on the levels of E2, AMH and FSH in rat serum

[0049]

[0050]

[0051] From the results in Table 2, it can be seen that compared with the control group, the levels of E2 and AMH in the serum of the rats in the model group were significantly reduced, and the level of FSH was significantly increased (P < 0.05). Compared with the model group, the levels of E2 and AMH in the serum of the rats in the low-dose and high-dose peptide groups, the ovarian stem cell group, and the combination group of the two were significantly increased, and the level of FSH was significantly reduced (P < 0.05). This fully shows that the peptide LRP-C5 and ovarian stem cells can effectively treat premature ovarian failure.

[0052] After blood collection, rats in each group were decapitated and the ovaries were removed by laparotomy. TUNEL staining was used to observe the apoptosis of ovarian granulosa cells, and the apoptosis rate of ovarian granulosa cells was calculated using ImageJ software. Ovarian granulosa cell apoptosis rate (%) = number of apoptotic ovarian granulosa cells / total number of ovarian granulosa cells × 100%. Figure 2 shown.

[0053] from Figure 2 It can be seen that compared with the control group, the apoptosis of granulosa cells in the ovarian tissue of the model group rats increased, and the apoptosis rate of ovarian granulosa cells increased significantly (P < 0.05). Compared with the model group, the apoptosis of ovarian granulosa cells in the low-dose and high-dose groups of the polypeptide, the ovarian stem cell group and the combination group of the two was reduced, and the apoptosis rate of ovarian granulosa cells was significantly reduced, especially the apoptosis rate of ovarian granulosa cells reached (10.82 ± 0.81)%. This shows that the polypeptide LRP-C5 and ovarian stem cells of the present invention can effectively reduce the apoptosis rate of ovarian granulosa cells.

[0054] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention. Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention, rather than to limit it; although the present invention is described in detail with reference to the above embodiments, ordinary technicians in this field should understand that they can still modify the technical solutions recorded in the above embodiments, or replace part or all of the technical features therein with equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An anti-ovarian aging polypeptide LRP-C5, characterized in that The amino acid sequence is shown in SEQ ID NO:

1.

2. A drug for treating ovarian aging, characterized in that The invention comprises the polypeptide LRP-C5 for resisting ovarian aging, and the amino acid sequence thereof is shown in SEQ ID NO:

1.

3. Use of the anti-ovarian aging polypeptide LRP-C5 as the main active ingredient in the preparation of a pharmaceutical composition for the treatment of ovarian aging.

4. Use of the anti-ovarian aging polypeptide LRP-C5 and ovarian stem cells in the preparation of a pharmaceutical composition for treating ovarian aging, wherein the amino acid sequence of the polypeptide is shown in SEQ ID NO:

1.

5. The use as claimed in claim 4, wherein the ovarian stem cells are prepared by digesting mouse ovarian tissue with collagenase IV, subculturing, and then screening MVH+ positive cells using a flow cytometer and continuing to culture.

6. The use according to claim 4, wherein the concentration of ovarian stem cells is 1×10 7 / kg, 2×10 7 / kg, 3×10 7 / kg, 4×10 7 / kg, 5×10 7 / kg, 6×10 7 / kg, 7×10 7 / kg, 8×10 7 / kg or 9×10 7 Pieces / kg.

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