Application of amniotic mesenchymal stem cells in preparation of medicine for improving and / or treating premature ovarian failure

Abstract

The invention discloses application of amniotic mesenchymal stem cells in preparation of a medicine for improving and / or treating premature ovarian failure. The invention finds that the amniotic mesenchymal stem cells can significantly improve the granular cell proliferation ability and hormone secretion ability, and the premature ovarian failure animal model proves that the amniotic mesenchymal stem cells have leading treatment effects in the aspects of restoring the ovarian functions (hormone secretion, ovary volume and follicle number of each level) of POF mice and improving fertility again. And a foundation is laid for optimizing a treatment strategy of premature ovarian failure and promoting clinical application of mesenchymal stem cell treatment.

Description

Technical Field

[0001] The present invention relates to the field of cell therapy and regenerative medicine, and in particular to the use of amniotic mesenchymal stem cells in the preparation of drugs for improving and / or treating premature ovarian failure. Background Art

[0002] Premature ovarian failure, also known as ovarian dysfunction, refers to a weakened ovarian capacity to produce eggs and decreased follicle quality, leading to decreased fertility and sex hormone deficiency. The average age of natural menopause is 50 to 52 years old, but over 1% of women experience premature ovarian failure (POF), which is amenorrhea before the age of 40 due to ovarian failure. Primary or secondary amenorrhea is accompanied by elevated blood gonadotropin levels and decreased estrogen levels, along with varying degrees of hypoestrogenic symptoms. POF can progress to ovarian failure, making it impossible for women to conceive naturally. In recent years, the incidence of premature ovarian failure has been increasing, affecting approximately 3% of the general population, due to factors such as lifestyle and stress. This condition severely impairs women's fertility, jeopardizing their physical and mental health and family stability. The pathogenesis of premature ovarian failure is unclear, with numerous influencing factors, and a stable, effective treatment strategy remains lacking. Therefore, it is crucial to find a safe and effective treatment for premature ovarian failure to meet women's reproductive needs.

[0003] Mesenchymal stem cells (MSCs) are a type of adult stem cell with the potential for self-renewal and multidirectional differentiation. The rapid development of stem cell therapy has provided new treatments for a variety of difficult diseases. In recent years, more and more studies have found that various MSCs can effectively improve the function of aging ovaries and restore the reproductive capacity of individual animals. Both umbilical cord mesenchymal stem cells (UMSCs) and adipose-derived mesenchymal stem cells (ADMSCs) have the advantages of being easy to obtain and having no ethical controversy, and have greater application prospects in clinical treatment. However, providing a type of mesenchymal stem cell that is more suitable for the treatment of POF and has better therapeutic effects is of great significance for optimizing the treatment strategy of POF and promoting the clinical application of MSCs therapy. Summary of the Invention

[0004] The purpose of the present invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide an application of amniotic mesenchymal stem cells (AMSCs) in the preparation of a drug for improving and / or treating premature ovarian failure.

[0005] The second object of the present invention is to provide an application of an amniotic mesenchymal stem cell conditioned medium in the preparation of a drug for improving and / or treating premature ovarian failure.

[0006] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0007] The present invention obtained mesenchymal stem cells from three different sources (umbilical cord mesenchymal stem cells (UMSCs), adipose-derived mesenchymal stem cells (ADMSCs), and amniotic membrane mesenchymal stem cells (AMSCs)) through in vitro culture and found that P6 AMSCs had the fastest migration ability. The three different mesenchymal stem cell conditioned medium (CM) was mixed at a 30% ratio with 60% high-glucose DMEM and 10% serum to prepare different granulosa cell culture media. It was found that the granulosa cells cultured with AMSCs-CM had a significantly higher proliferation rate than those cultured with the other two CMs. Similarly, the levels of estradiol and anti-Müllerian hormone detected in the culture medium were also the highest, suggesting that AMSCs can better improve the function of aging granulosa cells. The results of in vivo experiments in mice also proved that the transplantation of three types of mesenchymal stem cells can restore the serum hormone levels, ovarian volume, and the number of growing and mature follicles in POF mice to a certain extent. The therapeutic effect on premature ovarian failure is close to that of normal control mice. Among them, P6 generation AMSCs have the best therapeutic effect, and even indicators such as the number of offspring are significantly higher than those of normal control mice. It can effectively promote the repair and regeneration of ovarian tissue, thereby improving ovarian function and restoring fertility.

[0008] The amniotic membrane is the innermost layer of the placenta. Amniotic mesenchymal stem cells are stem cells isolated from the amniotic tissue on the side of the placenta close to the fetus. They have the advantages of multipotency, unlimited proliferation and anti-inflammatory properties, non-tumorigenicity and immunogenicity, and are easy to obtain and have no ethical disputes. They can repair the function of ovarian granulosa cells, promote oocyte regeneration, and greatly avoid the occurrence of immune rejection reactions. They have high safety and good utilization prospects.

[0009] Therefore, the present invention provides the use of amniotic mesenchymal stem cells in the preparation of a medicament for improving and / or treating premature ovarian failure.

[0010] The present invention also provides an application of an amniotic mesenchymal stem cell conditioned medium in the preparation of a drug for improving and / or treating premature ovarian failure. The preparation method of the amniotic mesenchymal stem cell conditioned medium comprises culturing the amniotic mesenchymal stem cells, collecting the cell culture medium, and filtering the conditioned medium.

[0011] Furthermore, the amniotic membrane mesenchymal stem cells are cultured for 48 hours.

[0012] Furthermore, the drug improves and / or treats premature ovarian failure by restoring ovarian tissue function and fertility.

[0013] Furthermore, the restoration of ovarian tissue function is restoration of one or more of hormone levels, ovarian volume, follicle number or granulosa cell function.

[0014] Furthermore, the concentration of the amniotic membrane mesenchymal stem cells is 1×10 6 ~2×10 7 MSCs / mL.

[0015] Furthermore, the concentration of the amniotic membrane mesenchymal stem cells is 1×10 7 MSCs / mL.

[0016] Furthermore, the amniotic membrane mesenchymal stem cells are cultured in a DMEM / F12 culture medium containing 8-12% fetal bovine serum.

[0017] Preferably, the amniotic mesenchymal stem cells are cultured in a DMEM / F12 medium containing 10% fetal bovine serum.

[0018] Furthermore, the culture time is to replace the entire medium every 23 to 25 hours and continue the culture.

[0019] Preferably, the culture time is to perform a full volume of medium replacement every 24 hours and continue the culture.

[0020] Furthermore, the culture medium also contains antibiotics.

[0021] Preferably, the antibiotic is penicillin and / or streptomycin, and the added concentration is 1%.

[0022] Furthermore, the amniotic mesenchymal stem cells are derived from humans.

[0023] Furthermore, the amniotic mesenchymal stem cells are P5 to P7 amniotic mesenchymal stem cells.

[0024] Preferably, the amniotic mesenchymal stem cells are P6 amniotic mesenchymal stem cells.

[0025] Furthermore, the drug includes a pharmaceutically acceptable carrier.

[0026] The purpose of this invention is to identify a more effective mesenchymal stem cell for improving granulosa cell and ovarian reproductive function, providing a novel therapeutic approach for delaying premature ovarian failure and enhancing fertility. In summary, the present invention has discovered that AMSCs are ideal for restoring the function of senescent granulosa cells and treating premature ovarian failure, bringing a new breakthrough in the field of cell therapy. Advantages include significantly enhancing the proliferation and hormone secretion capacity of senescent granulosa cells, demonstrating significant efficacy in restoring ovarian dysfunction, enabling the full utilization of discarded medical resources, and exhibiting excellent safety. These advantages will promote the application and development of AMSCs in the treatment of aging diseases and regenerative medicine.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention provides the use of amniotic mesenchymal stem cells (AMSCs) in the preparation of a drug for ameliorating and / or treating premature ovarian failure. The present invention discovered that AMSCs can significantly enhance the proliferation and hormone secretion capacity of granulosa cells. Using an animal model of premature ovarian failure, the present invention further confirmed that AMSCs have a leading therapeutic effect in restoring ovarian function (hormone secretion, ovarian volume, and number of follicles at various stages) and improving fertility in POF mice. This research lays the foundation for optimizing treatment strategies for premature ovarian failure and promoting the clinical application of mesenchymal stem cell therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The morphology of mesenchymal stem cells from three different sources. Note: Scale bar: 100 μm.

[0030] Figure 2 Comparison of the migration ability of mesenchymal stem cells from three different sources.

[0031] Figure 3 Comparison of autophagy levels in mesenchymal stem cells from three different sources. Figure 3 A and B are the expressions of autophagy substrate protein SQSTM1 / p62; C and D are the expressions of autophagy marker protein LC3B.

[0032] Figure 4 Identification of ovarian granulosa cells by immunofluorescence staining.

[0033] Figure 5 The effects of co-incubation with different concentrations of mesenchymal stem cell conditioned medium on the proliferation and vitality of granulosa cells.

[0034] Figure 6 This is the effect of co-incubation with different concentrations of mesenchymal stem cell conditioned medium on the hormone secretion level of granulosa cells.

[0035] Figure 7 The effect of co-incubation with conditioned medium of mesenchymal stem cells from three different sources on the proliferation of granulosa cells.

[0036] Figure 8 The effect of co-incubation of mesenchymal stem cell conditioned medium from three different sources on the hormone secretion level of granulosa cells. Figure 8 A in the figure represents the secretion level of E2; B represents the secretion level of AMH.

[0037] Figure 9 The changes in the estrous cycle of mice after modeling. Figure 9 A is a smear of vaginal exfoliated cells during the estrous cycle of mice after modeling (scale: 200 μm); B is the statistics of the estrous cycle of mice after modeling (3 mice were randomly selected from each group for plotting). Note: P: proestrus; E: estrus; M: metestrus; D: diestrus.

[0038] Figure 10 Comparison of serum hormone levels in mice after modeling. Note: “**” indicates P < 0.01; “***” indicates P < 0.001; “****” indicates P < 0.0001.

[0039] Figure 11 The changes in mouse body weight after transplantation of mesenchymal stem cells from three different sources. Figure 11 A shows the weight changes of mice during the treatment period; B shows the weight comparison of mice in each group at the end of treatment. Note: "ns" indicates P>0.05.

[0040] Figure 12 The estrous cycle of mice was monitored after transplantation of mesenchymal stem cells from three different sources. Note: P: proestrus; E: estrus; M: metestrus; D: diestrus.

[0041] Figure 13 This study compares the serum hormone levels of mice at different days (7, 14 and 21 days) after transplantation of mesenchymal stem cells from three different sources.

[0042] Figure 14 Comparison of ovarian size and structure in mice after UCMSCs transplantation. Figure 14 A represents the size of the ovary; B represents the structure of the ovary.

[0043] Figure 15 This study compares the number of mouse follicles after transplantation of mesenchymal stem cells from three different sources. Figure 15 A is the comparison of total follicle numbers; B is the comparison of follicle numbers at each level.

[0044] Figure 16 Comparison of the number of pups born after transplantation of mesenchymal stem cells from three different sources. Note: n = 5. DETAILED DESCRIPTION

[0045] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0046] Unless otherwise specified, all reagents and materials used in the following examples were commercially available.

[0047] 1. Source of Reagents

[0048] (1) The reagents required for the present invention are as follows: DMEM / F12 medium, high-glucose DMEM medium, fetal bovine serum, penicillin G, and streptomycin were from Gibco, USA; DMSO and trypsin were from Sigma, Germany.

[0049] 2. Culture Medium Preparation

[0050] (1) Preparation of MSCs cell culture medium: The concentration of DMEM / F12 basal culture medium is 89%, the concentration of fetal bovine serum is 10%, and the concentration of penicillin and streptomycin is 1%.

[0051] (2) Preparation method of granulosa cell culture medium: the concentration of high-glucose DMEM basal culture medium is 89%, the concentration of fetal bovine serum is 10%, and the concentrations of penicillin and streptomycin are both 1%.

[0052] (3) Preparation of MSCs cryopreservation medium: DMEM / F12 basal culture medium: fetal bovine serum (FBS): DMSO = 1:8:1, prepared and used immediately.

[0053] 3. Preparation of Reagents for Establishing the Mouse POF Model

[0054] (1) Cyclophosphamide injection: Weigh 480 mg of cyclophosphamide powder using an analytical balance and dissolve it in a centrifuge tube containing 40 mL of 0.9% sodium chloride solution. Mix thoroughly by pipetting and dispense into 1.5 mL EP tubes. Prepare the solution immediately before use.

[0055] (2) Busulfan injection: Weigh 48 mg of busulfan powder using an analytical balance, dissolve it in a centrifuge tube containing 40 mL of DMSO solution, pipette thoroughly to mix, and dispense into 1.5 mL EP tubes for immediate use.

[0056] Example 1 Comparative study of biological characteristics of mesenchymal stem cells from three different sources (fat, umbilical cord, and amniotic membrane)

[0057] 1. Detection of basic features

[0058] P6 UCMSCs, P6 AMSCs and P6 ADMSCs were cultured to 90% and subjected to scratch test to detect the migration ability of cells after 6h, 12h and 24h of culture.

[0059] The morphology of mesenchymal stem cells from three different sources is shown in the figure Figure 1 shown.

[0060] The results of the migration ability of mesenchymal stem cells from three different sources are as follows Figure 2 As shown in the results, the migration ability of P6 AMSCs was the fastest, followed by P6 UCMSCs and P6 ADMSCs (P>0.05).

[0061] 2. Detection of autophagy levels

[0062] After P6 UCMSCs, P6 AMSCs and P6 ADMSCs were cultured to 80%, the expression levels of autophagy-related proteins (LC3B, SQSTM1 / p62) were detected by immunofluorescence technique.

[0063] The results are as follows Figure 3 As shown, all three cell lines highly expressed the autophagy marker protein LC3B and lowly expressed the autophagy substrate protein SQSTM1 / p62, and there was no significant difference among the groups (P>0.05).

[0064] Example 2 Effects of three different sources (adipose, umbilical cord, and amniotic membrane) of mesenchymal stem cell conditioned medium on bovine granulosa cell function

[0065] 1. Effects of different ratios of umbilical cord mesenchymal stem cell conditioned medium on the function of bovine ovarian granulosa cells

[0066] (1) Preparation of Mesenchymal Stem Cell Conditioning Medium (CM)

[0067] After umbilical cord mesenchymal stem cells reached 80% confluence at passage 6, the culture medium was discarded, the cells were washed three times with PBS, and serum-free medium (50% F12 + 50% high-glucose DMEM) was added. After 48 hours, the conditioned medium in the culture dish was collected. Cell debris was pelleted by centrifugation at 3000 × g for 20 minutes at room temperature. The supernatant was collected, filtered through a 0.22 μm filter, and aliquoted into sterile 15 mL centrifuge tubes. Store at -80°C until needed.

[0068] (2) Cell culture and identification

[0069] Granulosa cells (GCs) were collected and cultured to adhere to the wall. Immunofluorescence was used to detect the expression of FSHR (follicle-stimulating hormone receptor), AMH (anti-Müllerian hormone, secreted by granulosa cells of small antral follicles of the ovary, whose level directly reflects the number of remaining follicles in the ovary), and WT1 protein (a granulosa cell-specific marker protein).

[0070] The results are as follows Figure 4 As shown, granulosa cells expressed FSHR, AMH, and WT1.

[0071] (3) Determination of cell co-incubation concentration

[0072] Granulocytes were collected and cultured in granulosa cell culture medium (high glucose DMEM, 10% FBS medium). After the cells adhered to the wall and the confluence reached 90%, they were digested and inoculated into 24-well plates. The collected conditioned medium was added at a ratio of 10%, 20%, and 30% and co-cultured with the granulosa cells for 48 hours. EDU was used to detect cell proliferation ability and CCK8 was used to detect cell activity.

[0073] The results are as follows Figure 5 As shown in the figure, compared with the control group, the addition of different concentrations (10%, 20%, 30%) of umbilical cord MSCs-CM can increase the proliferation rate and cell viability of granulosa cells, among which the granulosa cells cultured with 30% umbilical cord MSCs-CM have the fastest proliferation rate.

[0074] (4) Detection of granulosa cell hormone secretion ability

[0075] The supernatant of granulosa cell culture was collected, and the levels of E2 (estradiol) and AMH (anti-Müllerian hormone) in the supernatant were detected by ELISA.

[0076] The results are as follows Figure 6 As shown, compared with the control group, different concentrations (10%, 20%, 30%) of umbilical cord MSCs-CM can increase the levels of E2 and AMH hormones, among which 30% umbilical cord MSCs-CM has the best effect. Therefore, the present invention finally selected 30% CM for subsequent experiments.

[0077] 2. Effects of conditioned medium derived from mesenchymal stem cells from different sources on the function of bovine ovarian granulosa cells

[0078] (1) Granulocyte proliferation and cell activity detection

[0079] Granulosa cells were collected and digested and seeded into 24-well plates after adherence and confluence reached 90% using granulosa cell culture medium (high-glucose DMEM, 10% FBS medium). 30% HU-CM (UCMSCs-CM), 30% HA-CM (AMSCs-CM), and 30% HAD-CM (ADMSCs-CM) were added to co-culture with the granulosa cells for 48 h. Cell proliferation was detected by EDU, and cell viability was detected by CCK8.

[0080] The results are as follows Figure 7As shown in the figure, compared with the control group, the addition of conditioned medium of mesenchymal stem cells from different sources can increase the cell proliferation rate and cell viability. Among them, the granulosa cells cultured with 30% HA-CM had the fastest cell proliferation rate.

[0081] (2) Detection of granulosa cell hormone secretion ability

[0082] The supernatant of granulosa cell culture was collected and the levels of E2 and AMH in the supernatant were detected by ELISA.

[0083] The results are as follows Figure 8 As shown in the figure, compared with the control group, the cytokine secretion ability of the cells was improved after adding the conditioned medium of mesenchymal stem cells from different sources. Among them, the reproductive hormone secretion level was the highest after adding 30% HA-CM and co-incubating with granulosa cells, indicating that the addition of 30% HA-CM can significantly restore the function of granulosa cells.

[0084] The present invention confirms that mesenchymal stem cells can improve the activity of granulosa cells and restore the function of aging granulosa cells through paracrine function, and the addition of human amniotic mesenchymal stem cell conditioned fluid has the best therapeutic effect.

[0085] Example 3 Evaluation of the effect of mesenchymal stem cell transplantation from three different sources (fat, umbilical cord, and amniotic membrane) on restoring ovarian function in POF mice

[0086] 1. Establishment of POF Mouse Model

[0087] Ninety 7-week-old SPF-grade healthy Kunming mice were randomly divided into two groups: a normal control group (n=18) and a POF model group (n=72); the POF model group received a single subcutaneous injection of 12 mg / kg busulfan and an intraperitoneal injection of 120 mg / kg cyclophosphamide, while the normal control group received an equal volume of intraperitoneal injection of 0.9% sodium chloride solution and an equal volume of subcutaneous injection of DMSO solution.

[0088] Following modeling and drug administration, body weights of mice in each group were measured daily. Starting on day 8 of modeling and drug administration, vaginal acellular smears were performed daily between 8:00 and 9:00 AM for 8 consecutive days to monitor changes in the estrous cycle. Tail vein blood was collected from mice in each group 14 days after modeling and drug administration to measure serum E2, FSH, and LH concentrations.

[0089] The results are as follows Figure 9 As shown, during proestrus, a large number of nucleated epithelial cells appeared, along with a small number of cornified epithelial cells and leukocytes; during estrus, a large number of cornified epithelial cells appeared, along with a small number of nucleated epithelial cells and leukocytes; during metestrus, cornified epithelial cells, nucleated epithelial cells, and leukocytes appeared in equal proportions; and during diestrus, a large number of leukocytes appeared, along with a small number of cornified epithelial cells and nucleated epithelial cells. Statistical results indicate that POF mice developed estrous cycle disruption after injection of cyclophosphamide and busulfan.

[0090] The results of ELISA detection of serum E2, FSH and LH concentrations are as follows Figure 10 As shown in the data, compared with the normal control group, the E2 level in the POF model group mice was significantly decreased (P<0.001), the FSH level was significantly increased (P<0.0001), and the LH level was significantly increased (P<0.01).

[0091] The above results show that the POF mouse model constructed by the combined use of cyclophosphamide and busulfan undergoes significant changes in the estrous cycle and serum hormone levels. These changes are similar to the phenotype of human POF patients, indicating that the POF mouse model has been successfully constructed and can be used for subsequent experiments.

[0092] 2. Transplantation of UMSCs, AMSCs, and ADMSCs into the Mouse Tail Vein

[0093] One week after modeling, the mice with successful modeling were randomly divided into four groups: POF model group (n=18), UMSCs+POF treatment group, AMSCs+POF treatment group, and ADMSCs+POF treatment group.

[0094] Each mouse in the treatment group was injected into the tail vein with 200 μL of 2×10 6 MSCs were injected 3 times in total, with an interval of 1 week between each injection. The same amount of sterile PBS was injected into the tail vein of each mouse in the normal group and POF model group.

[0095] 3. Evaluation of the effect of UMSCs, AMSCs, and ADMSCs transplantation in restoring ovarian function in POF mice

[0096] (1) Analysis of body weight test results after three types of mesenchymal stem cell transplantation

[0097] The mice were weighed every two days.

[0098] The results are as follows Figure 11 As shown in the figure, the body weight of mice in each group increased slightly after treatment, and the difference was not statistically significant; there was no significant difference between the mice in the cell therapy group, indicating that different MSCs treatments had no obvious effect on the body weight of POF mice.

[0099] (2) Analysis of estrous cycle monitoring results after three types of mesenchymal stem cell transplantation

[0100] After the cell transplantation treatment, acellular smears of the mouse vagina were collected by cotton swab to monitor the estrous cycle.

[0101] The results are as follows Figure 12 As shown in the figure, after cell transplantation, the estrous cycles of mice in each cell treatment group returned to normal, similar to that of the normal control group.

[0102] (3) Analysis of the results of serum E2, FSH, and LH levels in each group of mice after three types of mesenchymal stem cell transplantation

[0103] The tail vein blood of mice was collected on the 7th, 14th and 21st day after cell transplantation to detect the changes in serum E2, LH and FSH levels.

[0104] The results are as follows Figure 13 As shown in the results, compared with the POF model group, on day 7 after injection, E2 levels in all treatment groups gradually increased, with extremely significant increases in the AMSCs treatment group (P<0.001) and the UMSCs treatment group (P<0.01). On days 14 and 21 after injection, E2 levels in the AMSCs, UMSCs, and AMSCs treatment groups were extremely significantly increased (P<0.0001). Compared with the POF model group, FSH levels in all treatment groups were extremely significantly decreased after injection (P<0.0001). Compared with the POF model group, on the 7th day after injection, the LH levels of mice in each treatment group gradually decreased, and the LH level in the AMSCs treatment group was significantly decreased (P<0.05); on the 14th day after injection, the LH levels in the AMSCs and UMSCs treatment groups were extremely significantly decreased (P<0.01), and the LH level in the ADMSCs treatment group was significantly decreased (P<0.05); on the 21st day after injection, the LH levels in the AMSCs and UMSCs treatment groups were extremely significantly decreased (P<0.001), and the LH level in the ADMSCs treatment group was significantly decreased (P<0.05).

[0105] Based on the above results, it can be seen that the three types of mesenchymal stem cell transplantation can restore the ovarian hormone levels of POF mice to a certain extent, and the transplantation of AMSCs has the best recovery effect, followed by the UMSCs treatment group.

[0106] (4) Comparative analysis of ovarian size and structure in mice after three types of mesenchymal stem cell transplantation

[0107] One week after the third cell injection, five mice were randomly selected from each group for comparison and analysis of mouse ovarian size. The mouse ovaries were removed, dehydrated, fixed, and paraffin-embedded sections were prepared. The ovarian structure was observed after HE staining.

[0108] The results are as follows Figure 14As shown in the figure, the ovaries of mice in the normal control group were larger, with numerous well-developed growing and mature follicles, closely packed granulosa cells surrounding the oocytes, and no inflammatory cell infiltration observed in the ovarian tissue. Compared with the normal control group, the ovaries of mice in the POF model group were significantly smaller, with fewer growing and mature follicles and loosely packed granulosa cells within mature follicles. Compared with the POF model group, the ovarian volume of mice in the treatment groups recovered, with an increase in the number of growing and mature follicles. The ovarian volume of mice in the AMSCs-treated group was closest to that of the normal control group, followed by the UMSCs-treated group.

[0109] (5) Comparative analysis of the total number of follicles and the number of follicles at each level after three types of mesenchymal stem cell transplantation

[0110] Five mice were randomly selected from each group. The mouse ovaries were removed, dehydrated and fixed, and paraffin sections were prepared. The follicles were counted after HE staining.

[0111] The results are as follows Figure 15 As shown in A, compared with the POF model group, the number of growing and mature follicles in the ovaries of mice in the three cell treatment groups increased, among which the total number of follicles in the ovaries of mice in the AMSCs treatment group increased extremely significantly (P<0.0001), and the total number of follicles in the ovaries of mice in the ADMSCs treatment group increased significantly (P<0.05). Figure 15 As shown in the results of B, compared with the normal control group mice, the number of primordial follicles, primary follicles, secondary follicles and antral follicles in the ovaries of the POF model group mice was reduced; compared with the POF model group, the growth follicles of all levels in the ovaries of the mice in the three cell treatment groups showed an increasing trend, among which, the growth follicles of all levels in the ovaries of the mice in the AMSCs treatment group were higher than those in the other treatment groups and close to the normal control group.

[0112] (6) Comparison of fertility in mice after three types of mesenchymal stem cell transplantation

[0113] The remaining 5 mice in each group were caged together for fertility test.

[0114] The results are as follows Figure 16 As shown in the data, compared with the normal group, the number of pups born by mice in the POF model group was significantly reduced (P<0.0001); compared with the POF model group, the number of pups born by the three treatment groups was significantly increased (P<0.0001), among which the average number of pups born by the AMSCs treatment group was higher than that of the other two treatment groups.

[0115] In summary, the present invention found that granulosa cells cultured with 30% P6 AMSCs-conditioned medium exhibited the fastest proliferation rate and highest cell viability compared to those cultured with the other two mesenchymal stem cell-conditioned media. Similarly, they secreted the highest levels of estradiol and anti-Müllerian hormone, demonstrating a better ability to improve the function of senescent granulosa cells. In vivo mouse experiments also demonstrated that AMSC transplantation was more effective than the other two mesenchymal stem cell-conditioned media in restoring ovarian hormone levels, ovarian volume, and the number of growing and mature follicles in POF mice, approaching that of normal control mice. Fertility was also significantly restored. Therefore, AMSCs can be used to improve or treat premature ovarian failure.

Claims

1. Application of amniotic mesenchymal stem cells in the preparation of drugs for improving and / or treating premature ovarian failure.

2. Use of an amniotic membrane mesenchymal stem cell conditioned medium in the preparation of a drug for improving and / or treating premature ovarian failure, characterized in that: The preparation method of the amniotic membrane mesenchymal stem cell conditioned medium is as follows: after culturing the amniotic membrane mesenchymal stem cells, collecting the cell culture medium and filtering the medium.

3. The application according to any one of claims 1 or 2, characterized in that: The drug improves and / or treats premature ovarian failure by restoring ovarian tissue function and fertility.

4. The application according to claim 3, characterized in that Restoring ovarian tissue function is restoring one or more of hormone levels, ovarian volume, follicle number or granulosa cell function.

5. The application according to any one of claims 1 or 2, characterized in that: The concentration of the amniotic membrane mesenchymal stem cells was 1×10 6 ~2×10 7 MSCs / mL.

6. The application according to claim 5, characterized in that The concentration of the amniotic membrane mesenchymal stem cells was 1×10 7 MSCs / mL.

7. The use according to any one of claims 1 or 2, characterized in that: The amniotic membrane mesenchymal stem cells are cultured in a DMEM / F12 culture medium containing 8-12% fetal bovine serum.

8. The use according to any one of claims 1 or 2, characterized in that: The amniotic mesenchymal stem cells are derived from humans.

9. The use according to any one of claims 1 or 2, characterized in that: The amniotic mesenchymal stem cells are P5 to P7 amniotic mesenchymal stem cells.

10. The use according to any one of claims 1 or 2, characterized in that: The drug includes a pharmaceutically acceptable carrier.

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