A preparation method of ovarian stem cells and its application in anti-ovarian aging

Abstract

The present invention provides a method for preparing ovarian stem cells and their use in treating ovarian aging. More specifically, the present invention screens and prepares ovarian stem cells from ovaries and isolates LRP-C5, a peptide with promising therapeutic effects on ovarian aging, from deer antler antler polypeptides. Both, when used alone or in combination, are effective in treating ovarian aging in rats and have promising application prospects.

Description

Technical Field

[0001] The present application relates to the biological field, and specifically to a method for preparing ovarian stem cells and their application in anti-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 factors. The incidence rate is 1% to 3% in the general population and 2% to 10% in women with amenorrhea. It is a common disease in the field of gynecological endocrinology, and the incidence rate has been increasing 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 research indicates that premature ovarian failure is primarily caused by the following factors. First, genetic factors. 10% of POF patients have a family history, and the condition can be vertically transmitted. POF is a group of X-linked genetic disorders. It is often accompanied by chromosomal rearrangements, translocations, or haploidy. X-chromosome cytogenetic abnormalities, such as XTurner syndrome and its variants, are the most common causes of POF. Other X-chromosome abnormalities include trisomy X, isochromosomes, and partial deletions of the short and long arms of the X chromosome. Balanced translocations involving the X chromosome can also lead to POF. The presence of mosaicism is a significant pathogenic factor for POF. Second, gonadotropin dysfunction. Defects in the transmission of the gonadotropins FSH and LH and their receptors can cause POF. Early studies have found that some patients with POF do not experience complete follicular failure but lack a response to endogenous gonadotropins. Furthermore, external factors, such as radiotherapy and chemotherapy, also play a significant role. 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, the ovaries of almost all women of childbearing age will suffer irreversible damage.

[0004] Treatment for premature ovarian failure primarily involves hormone replacement therapy for unmarried women or those who do not desire childbearing. However, the side effects of hormone therapy make long-term treatment difficult for patients to tolerate. Treatment for those who desire childbearing is more complex, but all aim to induce fertility. Currently, the primary approach for POF patients who desire childbearing is to receive donated eggs to achieve conception. Studies have suggested that continuous estrogen-progestin therapy for POF patients who do not desire childbearing, by establishing regular withdrawal bleeding and "menstruation," can improve perimenopausal and postmenopausal symptoms and reduce the incidence of cardiovascular disease and osteoporosis. For POF patients who desire childbearing, ovarian stimulation therapy is performed. Approximately 50% of these patients have some degree of ovarian follicle development, and 5%-10% can resume ovulation and achieve natural pregnancy. In 1997, Hiroyuki Takeuchi recommended estrogen and progesterone replacement therapy, often using the Kaufmann method: starting on the fifth day of withdrawal bleeding, administer 1.25 mg of Premarin daily for 14 consecutive days, followed by 6 mg of medroxyprogesterone daily for another 7 days. This cycle is repeated for three to four cycles, and follicular development is observed after one to two months of discontinuation. Estrogen promotes granulosa cell proliferation, induces the production of FSH receptors on these cells, and inhibits atresia of remaining ovarian follicles. It also improves perimenopausal and postmenopausal symptoms, making it the preferred method for ovulation induction in women with POF. However, the clinical success rate of ovulation induction is low, with a 42.9% ovulation recovery rate for women with amenorrhea of less than one year and less than 10% for those with amenorrhea of more than one year. Patients with POF have no ovarian follicles or, if present, are unresponsive to exogenous gonadotropins. Their uterine morphology is normal, and their endometrium responds well to estrogen and progesterone. Financially feasible, patients can undergo artificial insemination with donated eggs and embryo transfer, resulting in pregnancy, with a clinical pregnancy success rate of approximately 25% to 35%. Another study showed that 31 patients with POF initially received continuous estrogen-progesterone therapy to establish menstrual cycles, followed by ovulation induction after 3 to 6 cycles. Among 19 patients with POF, 8 experienced relief or resolution of hot flashes, sweating, and irritability, increased vaginal discharge, and significant improvement in sexual quality. All 19 patients experienced menstrual-like bleeding 3 to 6 days after stopping hormone therapy, but most were unable to menstruate naturally after stopping hormone therapy. The overall efficacy rate for treating POF with artificial cycles is 88%. Clinical observations of POF patients who have taken hormones for 2 to 4 years have shown that hormone therapy carries certain risks. Patients may experience breast tenderness, which may be caused by the large amount of progesterone entering the bloodstream, increasing breast tissue volume, increasing intracellular water content, and stimulating breast nerve endings. Long-term hormone therapy can induce cystic changes in the mammary ducts or poor mammary duct function and increase the mitotic rate of breast cells.

[0005] Selecting alternative treatments without side effects is a key research direction. Proliferation is a vital biological characteristic. Promoting the proliferation of various ovarian cells, particularly granulosa cells, and activating follicular function are also important approaches for treating 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. An international animal study demonstrated that umbilical cord blood-derived mesenchymal stem cells (MSCs) directly restore the function of existing oocytes and thus treat premature ovarian failure by promoting the proliferation of ovarian surface epithelial cells and repairing the tunica albuginea. They also indirectly repair impaired ovarian function by inhibiting caspase-3 and activating cytokeratin 8 / 18, transforming growth factor-β, and homologous loss-of-phosphatase / tensin genes. Another study using human menstrual blood-derived MSCs to treat mice with premature ovarian failure showed an increase in the ovarian tissue proliferation marker Ki67, suggesting that the therapeutic mechanism may be through cell proliferation. The study also demonstrated that human menstrual blood-derived MSCs can be induced to differentiate into ovarian tissue-like cells, particularly granulosa-like cells. Microarray analysis of cDNA expression profiles in this study showed that the expression profiles of human menstrual blood-derived mesenchymal stem cells differed significantly from those of human ovarian tissue before transplantation. However, after surviving for several days in premature ovarian failure, their gene expression profiles became similar to those of human ovarian tissue. This result strongly demonstrates that human menstrual blood-derived mesenchymal stem cells can be 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 deer antler polypeptides can have a good therapeutic effect on premature ovarian failure, the inventors of the present invention used neutral protease to hydrolyze the deer antler polypeptides, and then used ultrafiltration, column separation and HPLC separation and identification, and then mass spectrometry identification to obtain the polypeptide LRP-C5 with good therapeutic effect on ovarian aging, whose amino acid sequence is shown in SEQ ID NO: 1.

[0009] Furthermore, the polypeptides 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 the composition comprises ovarian stem cells and anti-ovarian aging polypeptide LRP-C5 as 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 lauryl sulfate (sodium lauryl sulfate), dodecyldimethylamine oxide, sodium docusate, cetyltrimethylammonium bromide (CTAB), polyethoxylated alcohols, polyoxyethylene sorbitan, octoxynol, N,N-dimethyldodecylamine-N-oxide, cetyltrimethylammonium bromide, polyoxyethylene (10) lauryl ether, surfactants (polyoxyethylene fatty alcohol polyoxyethylene ethers of plant oils derived from lauryl alcohol, cetyl alcohol, stearyl alcohol and oleyl alcohol), bile salts (e.g., sodium deoxycholate and sodium cholate), polyoxyethylene castor oil, nonylphenol ethoxylates, cyclodextrins, 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, carboxylic acid esters, 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-imidazoline, 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-cocoamidoethyl-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] The example of pharmaceutically acceptable preservative that is suitable for includes but is 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 their mixture. Preferred pharmaceutically acceptable preservatives include disodium EDTA (disodium ethylenediaminetetraacetic acid) and benzalkonium chloride or their mixture. In one embodiment, the presence of the pharmaceutically acceptable preservative is about 0.01% to about 2.0% (weight / volume), preferably about 0.05% to about 1% (weight / volume). The example of pharmaceutically acceptable buffer that is suitable for includes but is not limited to sodium chloride, glucose, lactose and phosphate buffered saline (PBS) or their arbitrary combination. Other suitable pharmaceutically acceptable buffers include, but are not limited to, disodium succinate hexahydrate, borates, citrates, phosphates, acetates, saline, tris-HCl (tris(hydroxymethyl)aminomethane hydrochloride), HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), sodium phosphate, sodium borate, saline, citrates, carbonates, phosphates, 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 the 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 preparing a pharmaceutical composition for treating ovarian aging.

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

[0021] Specifically, the ovarian stem cells of the present invention are used at a concentration of 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 use in treating ovarian aging. More specifically, the present invention screens and prepares ovarian stem cells from ovaries and isolates LRP-C5, a peptide with promising therapeutic effects on ovarian aging, from deer antler antler polypeptides. Both, when used alone or in combination, are effective in treating ovarian aging in rats and have promising 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 the apoptosis rate of ovarian granulosa cells DETAILED DESCRIPTION

[0027] Specific embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although 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. Rather, these embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0028] Example 1 Preparation of Mouse Ovarian Stem Cells

[0029] Take the ovarian tissue of a female newly adult C57BL / 6 mouse, take a tissue block of about 1 cm3, place it in a sterile vial containing 1 ml of normal saline and rinse the tissue 3 times with PBS to remove red blood cells as much as possible. Use ophthalmic scissors to fully chop the tissue into a paste. Add 3 times the volume of collagenase IV to the dish, pipette and transfer to a centrifuge tube, and place it in a 37°C incubator. Digest for about 30 minutes, pipetting 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 1300 rpm for 8 minutes, discard the supernatant, resuspend the cells three times in an appropriate amount of PBS, filter through a 200-mesh filter, and add 5 ml of culture medium II to the culture dish. Culture medium II consists of 80% knockout DMEM (Gibco) + 20% knockout Serum Replacement (Gibco) + 1% non-essential amino acids (Hyclone) + 1% glutamine (Gibco) + 1% β-dimercaptoethanol (Gibco) + 4 ng / ml β-FGF (Gibco) + 1% penicillin and streptomycin (Sigma). Incubate at 37°C in a 0.5% CO2 incubator. Change the medium every other day, and passage the cells when they reach 80% confluence. Discard the culture medium, wash twice with PBS, cover the bottom of the dish with 0.25% trypsin, and incubate at 37°C in a 37°C incubator. Observe the digestion progress under a microscope every 2 minutes. When most adherent cells have become round and bright, add an equal volume of medium I containing 10% fetal bovine serum to terminate digestion. Gently pipette repeatedly to the bottom of the dish to obtain a single-cell suspension. Centrifuge at 1500 rpm for 5 minutes, discard the supernatant, and resuspend the cells twice in an appropriate amount of PBS. Add an appropriate amount of medium II and passage at a 1:2 ratio. Incubate at 37°C, 0.5% CO2 incubator. Change the medium every other day, select MVH+ positive cells by flow cytometry, and continue culturing to obtain mouse ovarian stem cells.

[0030] Identification of Mouse Ovarian Stem Cells: Microscopic examination of cultured mouse ovarian stem cells revealed round cells with scant cytoplasm, spherical nuclei, a large nucleocytoplasm ratio, and nuclear diameters of 15-20 μm. AKP and Oct-4 staining for pluripotency assessment revealed positive results for both, confirming their identification as ovarian stem cells. MVH staining yielded 99.87% positive results, a marker expressed exclusively in germ cells, confirming that the isolated cells were ovarian stem cells.

[0031] Example 2: Verification of the efficacy of polypeptide LRP-C5

[0032] Female, newly adult C57BL / 6 mice were oocyte-collected. The follicular fluid after oocyte retrieval was stored on ice and quickly transferred to the laboratory for granulosa cell extraction. After low-temperature centrifugation, the cell pellet was placed in 50% (volume fraction) Percoll cell separation solution. After centrifugation, the cell layer was aspirated and washed with PBS to remove the Percoll solution. Three volumes of red blood cell lysis solution were then taken to remove a small amount of residual red blood cells. After centrifugation, the pellet was washed with PBS to remove the red blood cell lysis solution and centrifuged again to obtain a cell pellet. The cells were cultured for 24 hours using DMEM / F12 {15% (volume fraction) fetal bovine serum, 1% double antibody, and a final concentration of 1 μmol / L androstenedione solution}. Under an inverted microscope, the cultured cells were observed to be uniform, round mononuclear cells. The results of cell slide staining showed that positive staining was localized to the cell membrane and cytoplasm, showing brown staining, and the cell nucleus was blue. After identification, it was found that more than 96.3% were granulosa cells, indicating that ovarian granulosa cells were isolated and prepared.

[0033] The isolated ovarian granulosa cells were cultured with DMEM / F12 {15% (volume fraction) fetal bovine serum, 1% double antibody, and a final concentration of 1 μmol / L androstenedione solution} to adjust the cell concentration to 10 5 / ml, take a 6-well cell culture plate, add 2ml culture system to each well, and culture in a 37°C, 5% CO2 incubator for 72h. Discard the old culture medium and replace it with a 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 is detected by ELISA kit, and the operation is based on the instructions of the kit. The absorbance value of the enzyme-linked immunosorbent assay is read at 450nm, and the E2 concentration value in the culture medium is 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). At a concentration of 200 μg / mL, the expression level of estrogen reached (841.5 ± 13.1) pg / mL.

[0035] The 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] As can be seen from Table 1, 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] Ten-week-old, SPF-grade female Sprague-Dawley rats, weighing 250-270 g, were purchased from Saiye Biosciences. The model was established as follows: On day 1, rats were intraperitoneally injected with 60 mg / kg of cyclophosphamide (prepared with saline to a concentration of 6 mg / mL, injected at a volume of 10 mL / kg). Subsequently, rats were intraperitoneally injected with 10 mg / kg of cyclophosphamide (prepared with saline to a concentration of 1 mg / mL, injected at a volume of 10 mL / kg), once daily for 14 consecutive days. During the modeling period, vaginal smears were obtained from the rats at fixed times daily to observe the estrous cycle. The rat estrous cycle is divided into: proestrus (lasting 17-21 hours, with all nucleated epithelial cells on the smear and possibly a small number of keratinocytes), estrus (lasting 9-15 hours, with all anucleated keratinocytes or a small number of epithelial cells on the smear), metestrus (lasting 10-14 hours, with nucleated epithelial cells, keratinocytes, and leukocytes visible on the smear), and diestrus (lasting 60-70 hours, with a large number of leukocytes and a small number of epithelial cells and mucosa visible on the smear). If the rat's estrous cycle is disrupted or prolonged, or if it remains in a certain period for a long time, it indicates that the model has been successfully established.

[0041] Successfully established POF rats 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. Ten healthy rats were also selected as a negative control group.

[0042] The rats in the low-dose and high-dose peptide groups were injected with peptide injection 1 and 2 mg / kg, 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 received a tail vein injection of 1 mg / kg polypeptide solution and 2×10 ovarian stem cells from Example 1. 7 pieces / kg;

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

[0046] Rats in the negative control group and model group were injected with 1 mL / kg of normal saline into the tail vein for 4 consecutive weeks.

[0047] Twenty-four hours after the last administration, rats in each group were anesthetized by inhalation of ether, and tail vein blood was collected. The blood was centrifuged at 4400 rpm for 14 minutes at 4°C, and the upper serum layer was collected. Serum levels of E2, AMH, and FSH were measured using a microplate reader according to the instructions of the corresponding ELISA kits. 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] As shown in Table 2, compared with the control group, the serum E2 and AMH levels of the model group rats were significantly decreased, while the FSH level was significantly increased (P < 0.05). Compared with the model group, the serum E2 and AMH levels of the rats in the low-dose and high-dose peptide groups, the ovarian stem cell group, and the combination group were significantly increased, while the FSH level was significantly decreased (P < 0.05). This fully demonstrates that the peptide LRP-C5 and ovarian stem cells are both effective in treating premature ovarian failure.

[0052] After blood was collected, rats in each group were decapitated and the ovaries were removed by laparotomy. Ovarian tissue sections were prepared and TUNEL staining was used to observe the apoptosis of ovarian granulosa cells. 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 As can be seen, compared with the control group, the model group showed increased apoptosis in granulosa cells in the ovarian tissue of rats, and the apoptosis rate of ovarian granulosa cells was significantly increased (P < 0.05). Compared with the model group, apoptosis in ovarian granulosa cells in rats in the low-dose and high-dose polypeptide groups, ovarian stem cell group, and the combination group 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 of the present invention and ovarian stem cells can effectively reduce the apoptosis rate of ovarian granulosa cells.

[0054] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principles of the present invention, and such improvements and modifications should also be considered within the scope of protection of the present invention. Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. Such modifications or replacements do not deviate from the essence of the corresponding technical solutions within 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 treating ovarian aging, wherein the amino acid sequence of the polypeptide is shown in SEQ ID NO:

1.

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 according to claim 4, wherein the ovarian stem cells are prepared by digesting mouse ovarian tissue with collagenase IV, subculturing, screening MVH+ positive cells by flow cytometry, and continuing to culture.

6. The use according to claim 4, wherein the ovarian stem cells are used at a concentration of 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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