CN113633754B

CN113633754B - Application of ovarian stem cells in preparation of ovarian anti-aging drugs

Info

Publication number: CN113633754B
Application number: CN202111139153.0A
Authority: CN
Inventors: 张冬久; 钟春秀
Original assignee: Shanghai Lanweisaier Biotechnology Co ltd
Current assignee: Shanghai Lanweisaier Biotechnology Co ltd
Priority date: 2021-09-28
Filing date: 2021-09-28
Publication date: 2022-08-30
Anticipated expiration: 2041-09-28
Also published as: CN113633754A

Abstract

The invention relates to application of ovarian stem cells in preparation of an ovarian anti-aging medicine. According to the invention, the ovarian stem cells are obtained by separation and preparation, meanwhile, the active peptide with an anti-aging effect is obtained by separation from polygonum multiflorum, and the stem cells and the active peptide are used together for treating an ovarian aging model, so that the ovarian aging model can effectively promote follicular secretion and E2 secretion, and has a good anti-ovarian aging effect.

Description

Application of ovarian stem cells in preparation of ovarian anti-aging medicines

Technical Field

The invention relates to the field of biology, in particular to application of ovarian stem cells in preparation of an ovarian anti-aging medicine.

Background

Women are a progressive physiological process from fetus to the elderly, and the ovary is used as a gonadal organ and performs important physiological functions of periodic ovulation and sex hormone secretion. With age, ovarian function gradually declines, the reproductive organs also begin to atrophy, age, and affect the function of other systems of the body. Ovarian aging is a complex biological process of multifactorial interactions, progressive accumulation. As the ovary grows older, follicles are continuously consumed, and the decrease of the number of the follicles is the main cause of the ovarian senescence; accumulation of metabolites in the body, change of ovarian microenvironment and decrease of the quality of the remaining follicles are also closely related to aging. Ovarian aging is marked by menopause, which is known as premature ovarian failure by age 40, i.e., pathological ovarian aging.

Stem cells are a class of early undifferentiated cells, have the characteristics of self-renewal, self-replication, unlimited proliferation, multi-directional differentiation potential and the like, and can be divided into adult stem cells and embryonic stem cells from the source. The adult stem cells are widely present in human body tissues, are easy to separate, culture and amplify, have low immunogenicity and strong migration and immunoregulation capabilities, and have unique advantages in repairing tissue defects, serving as seed cells in tissue engineering and the like. Therefore, MSCs have been widely studied and applied in the fields of cell therapy, gene therapy, tissue engineering, and the like.

The MSCs not only have a certain repairing effect on the immune injury type premature ovarian failure, but also have a certain repairing effect on the chemically injury type premature ovarian failure. A researcher uses 10% hydrogen peroxide to damage ovaries to establish an ovary premature senility mouse model, carries out abdominal cavity transplantation on model mice and control mice respectively and infuses phosphate buffer solution with the same quantity, researches whether the MSCs have a repairing effect on chemically damaged ovaries by observing appearance and pathological sections, finds that the function of transplanted MSCs is recovered faster than that of ovaries of a control group, and meanwhile, the number of follicles growing at each stage is obviously more than that of the control group. Researchers also use a local injection method to inject the MSCs suspension into bilateral ovaries of an ovarian premature senility model mouse caused by the deoxyvinylcyclohexene, observe the distribution of the MSCs and discuss the feasibility of treatment of the ovarian premature senility; the research observes the change of the estrus cycle of the mouse, the ratio level of FSH and luteinizing hormone, the distribution of the transplanted MSCs and the like, and the result shows that the MSCs injected into the ovary are mainly distributed in the ovarian interstitial region and a small amount of the MSCs are distributed in the ovarian cyst membrane cell region; the estrus cycle of the MSCs group is shortened compared with that of the control group, the FSH/luteinizing hormone level is lower than that of the control group, and the difference has statistical significance; namely, the MSCs can improve the endocrine function of mouse ovary and exist in ovarian tissues for a long time, on one hand, cytokines secreted by the MSCs can inhibit the apoptosis of ovarian cells and promote the secretion of steroid hormone cells, and on the other hand, partial MSCs can be differentiated into the ovarian cells to replace damaged cells and promote tissue repair. In conclusion, the MSCs not only have a repairing effect on artificially-established premature ovarian failure, but also can repair damage of the ovary caused by environmental toxins.

In addition, free radicals existing in the organism and an antioxidant system are balanced mutually in a normal physiological state, and once the balance is broken, the organism reduces the scavenging capacity of the free radicals, so that excessive accumulation is caused, and then oxidative stress reaction can occur. Recent studies have shown that ovarian OS increases and the expression level of anti-OS related genes decreases with age. And the oocyte maturation and ovulation, ovarian hormone secretion, corpus luteum formation and dissolution process in the ovary are all regulated and influenced by free radicals. During the normal female menstrual cycle, there are various classes of biomarkers for OS; while the expression of antioxidant superoxide dismutase (SOD) was detected at various stages of follicular development. And studies have shown that the expression profile of SOD family varies in antral follicles at different developmental stages. The study of domestic scholars shows that the traditional Chinese medicine can obviously improve SOD in the ovary tissues of aged rats and further play the anti-aging role of the aged rats after being drunk everyday. Meanwhile, the damage effect of ROS on oocytes cannot be ignored, the quality of the oocytes is directly influenced, and the rate of producing congenital anomaly infants is increased for women with age.

However, the number of drugs for treating premature ovarian failure by using stem cells and antioxidant drugs is few at present, and the market has urgent needs.

Disclosure of Invention

The present invention provides a kit for preventing and or treating premature ovarian failure according to the needs of the prior art.

In one aspect, the invention provides a kit for preventing and or treating premature ovarian failure, wherein the kit comprises ovarian stem cells and LC-2 polypeptide.

Further, the ovarian stem cells are prepared by adopting the following method for isolated culture: excised ovarian tissue was washed 3 times with PBS, then cut into approximately 0.1 x 0.1cm3 tissue pieces, collagenase added to 3 times the tissue volume to the dish, blown and transferred to centrifuge tubes, and placed in a 37 ℃ incubator. Digesting for about 30min, and thoroughly blowing every 10min until the tissue mass is dissolved and the digestive juice becomes turbid. Adding DMEM, 10% fetal calf serum, 1% penicillin and streptomycin, mixing, centrifuging at 1500r/min for 5min, discarding supernatant, adding appropriate amount of PBS to resuspend cells for 3 times, filtering with 200 mesh filter screen, centrifuging, adding 5ml of dry cell culture solution into culture dish, 37 deg.C, O.5% CO ₂ And culturing in an incubator, wherein the stem cell culture solution comprises 1000 mL: 150mL KnockOut serum replacement +10mL HyClone non-essential amino acids +7g glutamine +1mL beta-mercaptoethanol +4mg beta-FGF +10mL penicillin-streptomycin solution 100X +2mg coenzyme Q10, DMEM make up to 1000 mL; changing the liquid every other day; digestion passage was performed when the cells in the dish grew to 80% confluence. Discarding the culture solutionAdding PBS for washing for 3 times, adding O.25% pancreatin to cover the bottom of the dish, placing the dish in an incubator at 37 ℃ until most adherent cells become round and bright, adding equivalent DMEM containing 10% fetal calf serum, 1% penicillin and streptomycin to stop digestion, and repeatedly and gently blowing the bottom of the dish by using a pipette to form a single cell suspension. Centrifuging at 1500r/min for 5min, discarding the supernatant, adding appropriate amount of PBS to resuspend cells for 3 times, adding the above stem cell culture solution, and mixing at a ratio of 1: 3, the obtained colony cells are the ovary stem cells after being passaged to P3.

Furthermore, the sequence of the LC-2 polypeptide is FQGIVQLPYWPSFAMW, and the polypeptide is prepared by extracting polygonum multiflorum with water, performing enzymolysis, and performing chromatographic separation.

According to all the preceding aspects of the invention, the LC-2 polypeptides share an amino acid sequence of FQGIVQLPYWPSFAMW of at least 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, or 74%, preferably at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83% or 84%, even more preferably have a sequence identity with the peptide of (i) of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% or 94%, most preferably at least 95%, 96%, 97%, 98% or 99% over its entire sequence.

The polypeptides of the invention may also include structural modifications that may, for example, enhance the therapeutic efficacy of the polypeptide or assist a clinician in monitoring a course of treatment with the polypeptide. Structural modifications may be made during or after translation or chemical synthesis of the polypeptide. For example, the polypeptide may be amidated. Alternatively, or in addition, the polypeptide may comprise a detectable label. The form and location of the detectable label may vary so long as the polypeptide retains sufficient biological activity to remain useful. The label may be, for example, a photoaffinity ligand, a radioisotope, or a fluorescent or chemiluminescent compound.

In addition to the polypeptides described herein, the invention features pharmaceutical formulations comprising these polypeptides, nucleic acids encoding them, host cells expressing them, and kits comprising one or more of these compositions. The nucleic acids, vectors and host cells containing them may also be formulated as pharmaceutical preparations.

Methods for constructing nucleic acids encoding a given polypeptide are well known in the art. Nucleic acids encoding fragments or biologically active variants thereof include those that are codon optimized. For expression, the nucleic acid can be readily incorporated into vectors (e.g., plasmids or viral vectors), and such vectors are included in the present invention. The nucleic acids are operably linked to regulatory regions suitable for use in prokaryotic or eukaryotic systems, many of which are known in the art and can be used to produce the polypeptides described herein. In particular embodiments, the regulatory region may be, for example, a promoter or enhancer. Useful promoters include cell type specific promoters, tissue specific promoters, constitutively active promoters and widely expressed promoters. As mentioned above, the invention also includes host cells comprising a vector expressing a polypeptide of the invention, and these cells may be prokaryotic (e.g., bacteria) or eukaryotic (e.g., mammals).

In some aspects, the kits of the invention exhibit a characteristic following oral, inhalation, intranasal, topical, intravenous, subcutaneous, intra-articular, intramuscular, intraperitoneal, intrasynovial, vaginal, rectal, pulmonary, ocular, buccal, sublingual, intrathecal administration or any combination thereof to a subject.

The polypeptides, nucleic acids, vectors and host cells of the invention may be formulated with stem cells into pharmaceutical compositions. For example, these compositions may be formulated as non-toxic formulations for parenteral (e.g., intravenous) administration. The pharmaceutical composition may comprise more than one sequence of polypeptides. As noted above, the polypeptides of the invention include polypeptides within a longer polypeptide, and such multimers, whether assembled as fusion proteins or conjugates, may also be formulated for administration to a patient. Carriers and stabilizers may be added to facilitate drug delivery and ensure shelf life. For example, encapsulation of the polypeptide in a suitable delivery vehicle (e.g., a polymeric microparticle, an implantable device, or any structure for timed, delayed, or controlled release) can increase delivery efficiency.

Furthermore, the medicine box or the medicine composition of the invention also adds a pharmaceutically acceptable carrier.

Advantageous effects

The ovarian stem cells are obtained by separation and preparation, the active peptides with the anti-aging effect are separated from the polygonum multiflorum, and the stem cells and the active peptides are used together for treating an ovarian aging model to find that the ovarian stem cells and the active peptides can effectively promote the secretion of follicles and the secretion of E2, so that the ovarian aging resistant effect is better.

Drawings

FIG. 1 is a graph showing the results of cell viability

FIG. 2 is a graph showing the results of the expression levels of p53 and p16 proteins

Detailed Description

The following detailed description refers, by way of illustration, to specific details and embodiments in which the invention may be practiced. These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention. Other embodiments may be utilized and structural and logical changes may be made without departing from the scope of the present invention. Various embodiments are not necessarily mutually exclusive, as some embodiments may be combined with one or more other embodiments to form new embodiments.

EXAMPLE 1 isolation preparation of ovarian Stem cells

Isolated ovarian tissue was harvested, washed 3 times with PBS, and then cut to approximately 0.1 x 0.1cm ³ The tissue mass of (1) was prepared by adding collagenase in a volume 3 times the tissue volume to a dish, pipetting and transferring to a centrifuge tube, and placing into a 37 ℃ incubator. Digesting for about 30min, and thoroughly blowing every 10min until the tissue mass is dissolved and the digestive juice becomes turbid. Adding DMEM, 10% fetal calf serum, 1% penicillin and streptomycin, mixing, centrifuging at 1500r/min for 5min, discarding supernatant, adding appropriate amount of PBS to resuspend cells for 3 times, filtering with 200 mesh filter screen, centrifuging, adding 5ml of dry cell culture solution into culture dish, 37 deg.C, O.5% CO ₂ Culturing in an incubator, wherein the composition of the stem cell culture solution is (1000 mL): 150mL KnockOut serum replacement +10mL HyClone nonThe essential amino acids (cat # SH30238.01) +7g glutamine +1mL beta-mercaptoethanol +4mg beta-FGF +10mL penicillin-streptomycin solution (100X) +2mg coenzyme Q10, DMEM make-up to 1000 mL. The liquid is changed every other day.

Digestion passage was performed when the cells in the dish grew to 80% confluence. Removing the culture solution, adding PBS, washing for 3 times, adding O.25% pancreatin to cover the bottom of the dish, placing the dish in an incubator at 37 ℃ until most adherent cells become round and bright, adding equivalent DMEM containing 10% fetal calf serum, 1% penicillin and streptomycin to stop digestion, and repeatedly and gently blowing the bottom of the dish by using a pipette to form a single cell suspension. Centrifuging at 1500r/min for 5min, discarding supernatant, adding appropriate amount of PBS to resuspend cells for 3 times, adding the above stem cell culture solution, and mixing at a ratio of 1: 3, the obtained colony cells are the ovary stem cells after being passaged to P3.

And (3) adding alkaline phosphatase into the collected ovarian stem cells, staining the ovarian stem cells for 30min, then starting staining the colonies, discarding culture solution for 35min to stop staining, adding PBS (phosphate buffer solution), and observing cell masses under a microscope to be purple blue, wherein the alkaline phosphatase is used as an identification marker of the stem cells, which indicates that the cells are the stem cells.

In addition, immunofluorescence assay detection of 0ct-4 expression (1) sterile cover slips were placed in a petri dish for cell slide culture, and washed with PBS for 5min until cells accounted for about 80% of the slide. (2) 4% paraformaldehyde was fixed at room temperature for 20min, washed 3 times with PBS, 5min each time. (3) O.5% PBST membrane rupture for 5min, PBS wash 3 times, each time for 5 min. (4) 5% sheep serum was blocked at 37 deg.C for 30min, and washed with PBS for 2 times, each time for 5 min. Rabbit anti-human 0ct-4 antibody IgG and PBS (as blank control) were added separately and incubated overnight in a wet box at 4 ℃. (5) Washing with PBS for 3 times, each for 10min, adding goat anti-rabbit IgG, and incubating at room temperature in dark for 60 min. (6) PBS was washed 3 times for 5min each and mounted with DAPI-containing mounting medium. (7) When the cell nucleus is observed and photographed under a fluorescent microscope, the result is positive when the cell nucleus is red, and the result is negative when the cell nucleus is not colored. The results showed that 0ct-4 stained nuclei of colony cells collected by culture appeared red, which also indicates that the isolated cultured cells were stem cells.

EXAMPLE 2 preparation of anti-ovarian senescence polypeptide

Soaking 5g of Polygoni Multiflori radix in 5 times of distilled water for 2 hr, boiling with slow fire for 30min, and filtering. Decocting the residue with 3 times of water for 20min, ultrasonic pulverizing at 50 w/w power for 10min, and filtering. Mixing the two filtrates, centrifuging at 8000r/min for 10min, and collecting supernatant. Adding 0.5% of compound protease at 37 deg.C for enzymolysis for 1h, and stopping enzymolysis. Sequentially performing CM-Sepharose Fast Flow and Sephadex G-50 chromatography on a column, performing reversed-phase high performance liquid chromatography to obtain different polypeptide components, screening to obtain 6 polypeptides with the property of reducing the expression of P53, Rb and P16 genes according to the property of inhibiting P53, Rb and P16 by the polypeptides, and determining 2 polypeptides with better effect by MALDI-ToFMS, wherein the amino acid sequences of the 2 polypeptides are LC-2: FQGIVQLPYWPSFAMW, LC-5: GLHCESMNEKQAAYKPLPQQ are provided.

Example 3 anti-senescence cell assay for polypeptides

IMR-90 (human embryonic lung fibroblasts) (Cat: CL-0538, Wuhan Punuo Sai Life technologies Co., Ltd.) cells were grown to 90% confluency in DMEM complete medium containing 10% fetal bovine serum, and the ratio of the total amount of IMR-90 (human embryonic lung fibroblasts) (Cat: CL-0538, Wuhan Punuo Sei, Life technologies Co., Ltd.) was adjusted to 1: 4, subculturing. The LC-2 polypeptide-treated group and the control group used IMR-90 cells (senescent cell-mimicking group) from 55 generations.

The LC-2 polypeptide treatment group is that the polypeptide acts on cells for 2h and then is normally cultured, the cell acts for 1 time every day and acts for 4 times continuously; the control group of the conventional cultured cells was not subjected to any treatment.

Determination of optimal growth-promoting concentration of polypeptide by MTI method: after 90% confluent monolayer of IMR-90 cells were digested with 0.25% trypsin, the cell suspension was adjusted for density and seeded into 96-well plates at 5 × 10 per well ³ After incubation and culture for 24h, the supernatant was discarded, 100. mu.L of polypeptide solution was added to each well, and the cell viability was measured by the MTT method after 48h of action. The administration concentrations of the polypeptides were 0, 1, 10, 20, 50, 100, 200, 500. mu.g/mL, 6 duplicate wells were provided for each group, and the experiment was repeated three times. The results are shown in FIG. 1.

As can be seen from FIG. 1, the concentration of the polypeptide was plotted on the abscissa and the cell survival rate (absorbance in the administration group/absorbance in the control group x 100%) was plotted on the ordinate, and the survival rate of the cells increased and then decreased as the dose of the polypeptide increased, and the optimal growth-promoting concentration was 200. mu.g/mL.

Western blot is used for detecting the expression conditions of p53 and p16 proteins after the polypeptide acts. Cells treated by the polypeptide treatment group for 48 hours at 500 mu g/mL and cells of a control group are taken, trypsinized and centrifuged to collect the cells. After RIPA lysate, supernatant fluid is collected by centrifugation at 12000r/min at 4 ℃, protein is boiled by adding protein loading buffer solution after BCA protein is quantified, and the supernatant fluid is frozen and stored for later use at 20 ℃ below zero. A12% polyacrylamide gel was prepared and loaded at 20. mu.g for SDS-PAGE. And (3) after electrophoresis, performing wet film transfer (PVDF) and sealing for 2 hours by using skimmed milk powder. The primary antibody is rabbit anti-human p53, p16 (1: 1000) and alpha-actin (1: 800) monoclonal antibody, the secondary antibody is goat anti-rabbit antibody (l: 10000) marked by horseradish peroxidase, and ECL luminescent solution is added for analysis by an imaging system. The results are shown in FIG. 2.

As can be seen from FIG. 2, the aging of the cells significantly enhanced the expression of the P3 and P16 proteins; the polypeptide shows a remarkable effect of down-regulating the expression of p53 and p16 proteins. The results show that the LC-2 polypeptide delays the cell aging, and the inhibition rate of P16 reaches 71.5%.

Example 4 mouse premature ovarian failure model experiment

ICR mice with normal estrus cycle are randomly divided into a model group, a stem cell transplantation group, a polypeptide treatment group, a stem cell combined polypeptide treatment group and a blank control group, and each group contains 30 mice. The model group, the stem cell transplantation group, the polypeptide treatment group and the stem cell and polypeptide combination treatment group are subjected to one-time intraperitoneal injection of 0.1mL of cyclophosphamide solution (120 mg. kg) and busulfan solution (12mg/kg), vaginal secretion smears are taken by cotton swabs each day, the vaginal cast-off cell changes are observed, and when continuous estrus intervals are observed for 10 days continuously, the modeling of premature ovarian failure is considered to be successful. Stem cell transplant group on modeled day 14, 0.1mL of ovarian stem cell suspension of example 1 (1X 10) was injected via tail vein ⁸ one/mL), repeated 1 time every 4d for a total of 5 times. The polypeptide set was injected with 0.1mL of LC-2 polypeptide (10mg/kg) via tail vein on day 14 of modeling, repeated 1 time every 4d for a total of 5 times. Stem cell combination polypeptide therapy group on modeled day 14, 0.1mL of ovarian stem cell suspension of example 1 (1X 10) was injected via tail vein ⁸ one/mL) and 0.1mL of LC-2 polypeptide (10mg/kg), repeated 1 time every 4d for 5 times. The building block and the blank control group are injected with the same amount of physiological saline through tail vein every time.

And (3) reproductive hormone determination: obtaining serum of each group of mice by tail-cutting method, and detecting Follicle Stimulating Hormone (FSH) and estradiol (E) of the mice by chemiluminescence method ₂ ) Levels, all sera were tested with the same batch of reagents. The results are shown in Table 1.

TABLE 1 groups of mice FSH and E ₂ Comparison of

Group of	FSH/(mIU/mL)	E ₂ /(pg/mL)
			Model set	4.75±0.41	33.78±2.58
Blank control group	2.88±0.23	43.85±3.54
			Stem cell transplantation group	3.54±0.29	38.46±2.87
Polypeptide treatment group	3.87±0.19	35.47±1.98
			Stem cell combination polypeptide treatment group	3.01±0.17	42.86±3.45

As can be seen from the results in Table 1, E of the mouse model group relative to the control ₂ Decreased secretion, increased FSH secretion, indicating failure of mouse ovarian function, and mouse E after polypeptide or stem cell therapy ₂ Secretion is increased, FSH secretion is reduced, the secretion level of FSH is approximate to that of normal rat hormone, especially the improvement effect of a stem cell combined polypeptide treatment group is obvious, and the combination of the FSH secretion and the stem cell combined polypeptide treatment group can effectively improve the ovarian function.

All groups of mice which are bled are killed by dislocation of cervical vertebrae, and bilateral ovarian tissues are taken, and the histological change of the ovaries is observed under a frozen section fluorescence microscope (x 400). The results are shown in Table 2.

TABLE 2 comparison of the number of follicles in each group

Group of	Total number of follicles
		Model set	14.87±2.41
Blank control group	41.58±5.03
		Stem cell transplantation group	30.48±2.88
Polypeptide treatment group	28.45±1.97
		Stem cell combination polypeptide treatment group	39.45±3.45

As can be seen from the results in table 2, the total number of follicles in the model group was significantly smaller than that in the placebo group, but the total number of follicles in the model group was significantly increased after each group treatment. After the combination of the stem cells and the polypeptide group is used for treatment, the total number of the follicles reaches 39.45 +/-3.45, is basically similar to that of normal mice, and shows that the ovarian functional recovery effect is good.

It should be understood that the above describes only some embodiments of the present invention and that various other changes and modifications may be affected therein by one of ordinary skill in the related art without departing from the scope or spirit of the invention.

Claims

1. The application of the ovary stem cell and LC-2 polypeptide in preparing a medicine box for treating premature ovarian failure is characterized in that: the sequence of the LC-2 polypeptide is shown as FQGIVQLPYWPSFAMW; the ovarian stem cells are prepared by adopting the following method for isolated culture: isolated ovarian tissue was harvested, washed 3 times with PBS, and then dissected 0.1 x 0.1cm ³ Adding collagenase which is 3 times of the volume of the tissue into a dish, blowing and beating the tissue, transferring the tissue to a centrifuge tube, and putting the centrifuge tube into an incubator at 37 ℃; digesting for 30min, and blowing and beating fully every 10min until the tissue blocks are dissolved and the digestive juice becomes turbid; adding DMEM, 10% fetal calf serum, 1% penicillin and streptomycin, mixing, centrifuging at 1500r/min for 5min, discarding supernatant, adding appropriate amount of PBS to resuspend cells for 3 times, filtering with 200 mesh filter screen, centrifuging, adding 5ml of dry cell culture solution into culture dish, 37 deg.C, and 0.5% CO ₂ The culture box of (2) is used for culturing, wherein the composition of a stem cell culture solution is 1000 mL: 150mL KnockOut serum replacement +10mL HyClone non-essential amino acids +7g glutamine +1mL beta-mercaptoethanol +4mg beta-FGF +10mL penicillin-streptomycin solution 100X +2mg coenzyme Q10, DMEM make up to 1000 mL; changing the liquid every other day; digesting and passaging when the cells in the dish grow to 80% confluence; removing culture solution, adding PBS, washing for 3 times, adding 0.25% pancreatin to cover the bottom of the dish,placing in an incubator at 37 ℃ until most adherent cells become round and bright, adding equivalent DMEM containing 10% fetal calf serum, 1% penicillin and streptomycin to stop digestion, and repeatedly and gently blowing the bottom of the dish by using a suction tube to form a single cell suspension; centrifuging at 1500r/min for 5min, discarding supernatant, adding appropriate amount of PBS to resuspend cells for 3 times, adding the above stem cell culture solution, and mixing at a ratio of 1: 3 to P3 generation, and the collected colony cells are the ovary stem cells.

2. The use of claim 1, wherein said kit further comprises a pharmaceutically acceptable carrier.

3. Use according to claim 2, characterized in that the carrier comprises an excipient.