MSC-sEVs medicine for improving ovarian function as well as preparation method and application of MSC-sEVs medicine

Abstract

The invention discloses an application of MSC-sEVs in treatment of reduction of ovarian reserve. DNA repair genes (such as PARP1) are activated in a targeted manner through MSC-sEVs, and NAD + metabolic regulation is combined, so that the ovarian function and reproductive potential of DOR are remarkably improved. The method has the advantages of high safety and unique advantages, and an innovative treatment scheme is provided for patients with ovarian reserve dysfunction.

Description

Technical Field

[0001] The present invention relates to the technical fields of reproductive medicine and cell therapy, and particularly to the application of mesenchymal stem cell-derived small extracellular vesicles (MSC-sEVs) in the preparation of a drug for improving the ovarian function of a mouse model of cyclophosphamide-induced diminished ovarian reserve (Cy-DOR). Background Art

[0002] Primary ovarian insufficiency (POI) or premature ovarian failure (POF) is a heterogeneous gynecological endocrine disease caused by follicular function failure or dysfunction. It is characterized by elevated serum gonadotropin levels, no residual follicles in the gonads, and is associated with oligomenorrhea, amenorrhea, insufficient fertility or even infertility. According to the progression and severity of the disease, the National Institutes of Health (NIH) and the American Society for Reproductive Medicine (ASRM) have proposed that POI can be divided into three progressive processes: the latent stage, the biochemical abnormal stage (biochemical PO1, bPOI), and the ovarian failure stage. Patients still in the first two stages of POI progression can still conceive offspring with their own oocytes, while patients in the ovarian failure stage often need to choose donor oocyte in vitro fertilization-embryo transfer treatment to solve the fertility problem. Diminished ovarian reserve (DOR) means a reduction in the number of remaining eggs, a decline in quality or growth ability in a woman's ovaries, which may affect fertility. It is usually associated with increasing age, but can also be caused by genetic factors, medical treatments (such as chemotherapy or radiotherapy), surgical removal of ovarian tissue, or other unknown reasons. Diminished ovarian reserve means a reduced chance of natural pregnancy, but it is still possible to achieve.

[0003] There are significant differences between diminished ovarian reserve (DOR) and POI in pathological mechanisms and treatment goals: due to complete ovarian failure in POI, the treatment is mainly hormone replacement or donor oocyte-assisted reproduction; while in DOR, due to the decline in the number and quality of follicles, there is still reproductive potential, and it is necessary to delay the decline by improving the ovarian microenvironment. Hormonal imbalances associated with DOR may have an adverse impact on metabolic health, leading to insulin resistance and metabolic syndrome, which is associated with an increased risk of neurodegenerative diseases such as Alzheimer's disease. Existing POI treatment methods have not effectively restored ovarian function, and treatment strategies for DOR are also limited. Summary of the Invention

[0004] In view of the above background, the present invention provides a new treatment method, that is, by targeting the activation of DNA repair genes (such as PARP1) by MSC-sEVs and combining NAD+ metabolism regulation, aiming to significantly improve ovarian function and reproductive potential in a DOR mouse model. This method has high safety and unique advantages, providing an innovative treatment plan for patients with ovarian reserve dysfunction.

[0005] Our study isolated and characterized mesenchymal stem cell-derived exosomes (MSC-sEVs) by using a scalable isolation method and found their protective effect on the ovaries in a mature cyclophosphamide-induced ovarian reserve reduction (Cy-DOR) mouse model. Specifically, in the Cy-DOR mouse model, MSC-sEVs enhance the DNA repair ability of ovarian cells by upregulating DNA repair genes such as PARP1, thereby protecting them from damage, reducing oxidative stress and enhancing the DNA repair pathway to restore ovarian function.

[0006] On the one hand, the present invention provides the use of MSC-sEVs in the preparation of a drug for treating reduced ovarian reserve.

[0007] Further, the treatment of reduced ovarian reserve is to improve the estrous cycle and / or follicular development.

[0008] Further, the improvement of follicular development is that the number of primordial follicles and the total number of follicles are significantly increased, and the number of atretic follicles is decreased.

[0009] Or the treatment of reduced ovarian reserve is to improve the number of oocytes and / or reproductive outcomes.

[0010] Further, the improvement of the number of oocytes is that the number of oocytes is increased and the number of abnormal oocytes is decreased.

[0011] Further, the improvement of reproductive outcomes is that the number of offspring produced is increased and the weight of the offspring is increased.

[0012] Or the treatment of reduced ovarian reserve is to enhance ovarian function and / or serum hormone levels.

[0013] Further, the enhancement of ovarian function is that the level of AMH is increased and the expression of DDX4 is increased.

[0014] Further, the enhancement of ovarian function is that the expression of cleaved caspase-3 to total caspase-3 decreases.

[0015] Further, the enhancement of ovarian function is that the apoptosis index decreases.

[0016] Further, the enhancement of serum hormone levels is that the serum E2 level is significantly increased and the FSH level is significantly decreased.

[0017] Preferably, the aforementioned treatment for reducing ovarian reserve improves the symptoms of reduced ovarian reserve by maintaining NAD+ levels and DNA.

[0018] On the other hand, the present invention provides the use of MSC-sEVs in the preparation of a drug for improving behavioral and cognitive functions.

[0019] Furthermore, the improvement of behavioral and cognitive functions is the enhancement of learning and memory abilities.

[0020] On the other hand, the present invention provides a method for improving follicular development for non-disease treatment, characterized in that MSC-sEVs or a drug prepared based on MSC-sEVs is used to improve follicular development.

[0021] Furthermore, the improvement of follicular development is that the number of primordial follicles and the total number of follicles are significantly increased, and the number of atretic follicles is decreased.

[0022] On the other hand, the present invention provides a method for improving the ovulation number for non-disease treatment, characterized in that MSC-sEVs or a drug prepared based on MSC-sEVs is used to increase the ovulation number of animals.

[0023] Furthermore, the improvement of the number of oocytes is that the number of oocytes is increased and the number of abnormal oocytes is decreased.

[0024] On the other hand, the present invention provides a method for improving reproductive number for non-disease treatment, characterized in that MSC-sEVs or a drug prepared based on MSC-sEVs is used to improve the reproductive number of animals.

[0025] Furthermore, the improvement of reproductive number is that the number of offspring produced is increased and the weight of the offspring is increased.

[0026] Preferably, the aforementioned method can be applied in scientific research experiments or animal breeding production. For example, it can be used as a positive control for scientific experiments or to increase production in animal breeding.

[0027] Preferably, the object of use of the aforementioned drug, or the object of application of the method, is a mammal.

[0028] Finally, the present invention also provides a method for preparing engineered exosomes MSC-sEVs, the method comprising the following steps:

[0029] 1) Cultivate human induced pluripotent stem cells in complete cell medium CCM for 2-3 days. When the cells grow to about 80% confluence, expand them to a 5-layer cell factory.

[0030] 2) After washing the cells with phosphate-buffered saline (PBS), the CCM was replaced with chemically defined protein-free (CDPF) medium.

[0031] 3) After 6 hours, the fresh CDPF medium was replaced, and about 600 ml of the supernatant was collected and centrifuged at 2,000 g for 20 minutes to remove cell debris.

[0032] 4) Recombinant lentivirus carrying the luciferase gene was used to infect iPSC-MSCs. The cells were seeded in a 12-well plate at a density of 3×10 4 cells / ml. After 6 hours of infection, the fresh medium was replaced. After 72 hours, puromycin was used for screening to obtain iPSC-MSCs stably expressing luciferase, which were cryopreserved to form a working cell bank.

[0033] 5) Purify MSC-derived small extracellular vesicles (MSC-sEVs) by anion exchange chromatography ( Figure 1 A).

[0034] Compared with the prior art, the present invention has the following advantages:

[0035] 1. Low immunogenicity and high non-tumorigenicity: Compared with cells, extracellular vesicles have the advantages of low immunogenicity, high non-tumorigenicity, high clinical safety, and low ethical risk.

[0036] 2. High safety: Since engineered MSC-sEVs do not use live cells but are in the form of extracellular vesicles, their clinical safety is higher, avoiding potential risks such as tumor transformation.

[0037] 3. Efficient DNA repair: By enhancing the DNA repair pathway, engineered extracellular vesicles exhibit more powerful functions than ordinary MSC-sEVs in the repair of ovarian cell damage, and are expected to significantly improve ovarian function and reproductive ability. In addition, due to the extremely unstable nature of NAD+ in vitro and its easy degradation, the closed structure of MSC-sEVs significantly extends the half-life of NAD+ in the extracellular microenvironment, enabling NAD+ to be effectively delivered to cells with low NAD+ levels, further promoting the recovery of their functions. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The following will combine the drawings and specific embodiments to detail the method for regulating the salt stress resistance of plants and / or cultivating salt stress-resistant plant varieties of the present invention and its beneficial effects.

[0039] Figure 1Characterization of iPSC-msc-sev is shown as follows: A. Schematic diagram of the isolation process of msc-sev, including cell culture, medium change, and anion exchange chromatography. B. Nanoparticle tracking analysis shows the size distribution of iPSC-msc-sev. C. Western blots show positive for sEV markers (ALIX, CD63, CD81) and negative for Calnexin. D. Transmission electron microscopy of iPSC-msc-sev. Scale bar: 200 nm. E. Nano- The msc-sev labeled with the luciferase detection system reached the ovary after 1.5 hours.

[0040] Figure 2 msc-sev improved ovarian degeneration and abnormal estrous cycles in the Cy-DOR model as shown: A. Experimental timeline and procedures for inducing Cy-DOR mice and treating with MSC-sEV, as well as sample collection. B. Representative estrous cycles of mice in the control group, Cy group, and Cy.MSC.sEVs group. C. Box plot showing the duration of each estrous stage. D. Representative images of ovarian HE staining. Scale bar, 200 μm. E. Quantification of primordial and atretic follicles. F. Quantification of the total number of follicles and corpora lutea. All experiments were repeated at least 3 times. Data are shown as mean ± SEM. Statistical analysis was performed using one-way ANOVA (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

[0041] Figure 3 msc-sev improved the number and fertility of oocytes as shown: A. Representative micrographs of oocytes in each group. Black arrows: abnormal oocytes. B. Quantitative analysis of the total number of ovulated oocytes and the total number of abnormal oocytes / total number of abnormal oocytes per mouse. C. Representative images of embryo implantation sites in each group. Mice were sacrificed 9.5 - 10.5 days after vaginal plug detection. Red arrows indicate embryos implanted in the uterus. D. Quantitative analysis of the number of implanted embryos per mouse. E. Representative images of the number of offspring in the first round of mating in each group. F. Quantitative analysis of the fertility parameters of the first litter. Total number and body weight. G. Representative pictures of the litters in the second round of mating in each group. H. Size of the offspring in the second round of mating, measured 4 weeks after the first litter was born. All experiments were repeated at least 3 times.

[0042] Figure 4Shown are MSC-sEVs alleviating ovarian defects and gonadotoxicity: A. Immunohistochemical images of ovaries stained with AMH and DDX4 antibodies. Scale bars: 50 μm and 20 μm. B. Representative Western blot and quantitative analysis of AMH expression. GAPDH was used as a loading control. C. Quantitative analysis of AMH and DDX4 expression levels. D. Immunofluorescence images of ovaries stained with cleaved Caspase-3 antibody. Scale bar: 50 μm. E. Quantification of the apoptosis index, expressed as the ratio of cleaved caspase-3 fluorescence to total ovarian fluorescence. F. Representative Western blot of caspase-3 and cleaved caspase-3 expression. G. Quantification of the ratio of cleaved caspase-3 to total caspase-3. H. Serum estrogen and FSH levels were detected by ELISA. Data are presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). FSH: follicle-stimulating hormone.

[0043] Figure 5 Shown are MSC-sEVs enhancing the learning and memory abilities of the Cy-DOR model: A. Representative images of the behavior of each group in the Barnes maze. B. Quantitative analysis of the escape latency. C. Quantitative analysis of the number of times of exploring the safe hole, the latency to reach the safe hole, the travel distance before reaching the safe hole, and the major errors. The so-called "major errors" refer to the situation where the mouse first reaches the hole outside the safe hole. D. Representative images of the behavior of mice in each group in the elevated maze. E. Quantitative analysis of the number of entries into the open arms and closed arms. F. Quantitative analysis of the travel distance in the open arms and closed arms, the time spent in the open arms and closed arms, and the total travel distance. G. Representative Western blot images of NeuN, PSD95, cleaved-PARP1, γ-H2AX, XRCC1, and GAPDH. H-I. Quantitative analysis of NeuN, PSD95, cleaved PARP1, γ-H2AX, and XRCC1.

[0044] Figure 6Shown is the improvement of the Cy-DOR model by restoring NAD+ levels and increasing DNA repair as dynamic processes of MSC-sEVs: A. Levels of NAD+, NADH, total NAD+ plus NADH, and the NAD+ / NADH ratio in the short-term Cy-DOR model. B-C. Representative Western blot and quantitative analysis of cleaved PARP1 and PAR expression, with GAPDH as a loading control (short-term Cy-DOR). D. Levels of NAD+, NADH, total NAD+ plus NADH, and the NAD+ / NADH ratio in the long-term Cy-DOR model. E-G. Representative Western blot and quantitative analysis of PAR, γ-H2AX, and XRCC1 expression, with GAPDH as a loading control (long-term Cy-DOR). H. Ovarian immunofluorescence images stained with BRCA2 antibody. Scale bar: 40 μm. I. Ovarian immunofluorescence images stained with γ-H2AX antibody. Scale bar: 200 μm. J. Quantitative analysis of BRCA2 and γ-H2AX fluorescence intensity. Data are presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). Detailed implementation manners

[0045] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Apparently, the described embodiments are only a part of the embodiments of the present invention, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0046] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs.

[0047] Example 1 Preparation of msc-sev

[0048] MSCs were generated from human induced pluripotent stem cells (iPSCs) (urine cell-derived iPSCs (UC-IPSCs) were donated by the Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences (Guangzhou, Guangdong, China)), and the specific method was referred to CN201610107276.9. Cells were thawed from the working cell bank, seeded in culture dishes, and cultured in complete cell medium (CCM) for 2-3 days. When the cells grew to about 80% confluence, they were expanded to a 5-layer cell factory. After washing the cells with phosphate buffer (PBS), the CCM was replaced with chemically defined protein-free (CDPF) medium. The fresh CDPF medium was changed after 6 hours, and about 600 ml of supernatant was collected and centrifuged at 2,000 g for 20 minutes to remove cell debris.

[0049] Next, to load luciferase into MSC-derived exosomes, iPSC-MSCs were infected with recombinant lentivirus carrying the luciferase gene. The cells were seeded in a 12-well plate at a density of 3×10 4 cells / ml, and the fresh medium was replaced 6 hours after infection. After 72 hours, puromycin screening was performed to obtain iPSC-MSCs stably expressing luciferase, which were cryopreserved to form a working cell bank.

[0050] Subsequently, MSC-derived small exosomes (MSC-sEVs) were purified by anion exchange chromatography ( Figure 1 A). An anion exchange chromatography kit provided by Mylab was used, and each Econo-Pac column containing anion exchange resin was pre-equilibrated with the equilibration buffer. The MSC supernatant was loaded onto the column, and eluted with the elution buffer in 8 fractions. The concentration of sEVs in each fraction was estimated by Bradford protein quantification and CD63 enzyme-linked immunosorbent assay (ELISA). The fractions with the peak sEV concentration were collected, dialyzed with PBS at 4°C, and further concentrated using a Pierce TM protein concentrator (30 kDa, Thermo Fisher Scientific). The final concentration of MSC-sEVs was determined by Bradford protein quantification and nanoparticle tracking analysis (Nanosight NS300, Malvern).

[0051] By nanoparticle tracking analysis, we determined their size distribution, ranging from 50 - 150 nm, with a peak at approximately 100 nm ( Figure 1 B). Using transmission electron microscopy (TEM), we determined the morphology of sEVs as bilayered and nanoscale particles ( Figure 1 C). Strong fluorescence was detected in the ovaries within 1.5 hours, indicating that sEVs could reach the ovaries ( Figure 1 D). Western blot results showed high levels of positive markers CD9, CD63, CD81, and ALIX and weak expression of calnexin in MSC-sEVs, demonstrating that they are typical sEVs. Overall, the isolated MSC-sEVs mainly had a peak near 100 nm, and other peaks were not significant. The sEVs were all positively labeled, and the negative labels were all weakly positive, indicating that the isolated MSC-sEVs were homogeneous, with good quality variation control and typical sEV characteristics.

[0052] Example 2 MSC-sEV improves the estrous cycle and follicular development in the Cy-DOR mouse model

[0053] In this invention, C57BL / 6J mice at 7 - 8 weeks old were selected to establish a DOR model related to chemotherapy. We first evaluated the role of MSC - sEVs in ovarian function in the decreased ovarian reserve (DOR) model. All mice were divided into a control group (control group), a DOR model group (Cy), and a DOR model treatment group (Cy.MSC.sEVs). The mice received tail - vein injection of msc - sev 2×10 10 150 ul or normal saline 150 ul from day 1 to day 6. From day 7, vaginal smears of the mice were collected and the estrous cycle was observed for 14 days. On day 21, the mice were euthanized and ovarian indexes and fertility indexes were analyzed. The experimental protocol was as Figure 2 shown in a. The estrous cycle was evaluated according to the cell types observed in the vaginal smear. Similar to the menstrual cycle in women, the normal estrous cycle is one of the main observations of normal ovarian function in female mice. The vertical axis represents proestrus (P), estrus (E), metestrus (M), and diestrus (D), and the horizontal axis represents the number of days. The estrous cycle of the control group was regular, with each cycle lasting 4 - 5 days. The distribution of mice in each stage was in good agreement with the typical duration of each stage. ( Figure 2 B). Compared with the Cy group, MSC - sEVs treatment increased the average number of days in the E stage by 1.12 days and decreased the duration of the M / D stage by 1.88 days ( Figure 2 C). These results indicate that msc - sev can restore the estrous cycle disrupted by Cy and increase the proportion of estrus in mice.

[0054] Through hematoxylin and eosin (HE) staining, obvious signs of recovery in the follicle distribution at each stage were observed after MSC - sEVs treatment ( Figure 2 D). Among them, the number of primordial follicles increased significantly by about 82.6%, and the total number of follicles increased by about 47.8%. In addition, the number of atretic follicles decreased by about 38.1% ( Figure 2 E - F, P < 0.0001). It is worth noting that there was no significant difference in the number of corpora lutea among the groups. The improvement in the number of follicles at the primordial, pre - antral, and secondary stages and the reduction of atretic follicles emphasize the potential of MSC - sEVs in promoting the early stage of follicle formation.

[0055] Example 3 MSC - sEVs can improve the number of oocytes and fertility outcomes

[0056] To study the role of MSC - sEVs in oocytes in the DOR model, we continued to analyze the number and quality of oocytes induced by superovulation in mice. We found that compared with the Cy group (P < 0.0001), the number of oocytes in the MSC - sEVs group increased by about 86.9%, and the number of abnormal oocytes decreased significantly by about 40.8% ( Figure 3A-B, P < 0.01). Since preserving ovarian reserve does not necessarily mean producing better-quality oocytes, we conducted a breeding experiment on mice. Male and female mice were allowed to mate freely, and then the embryos implanted in the uterus were analyzed. MSC-sEVs treatment increased the number of implants from an average of 5 in Cy-induced to an average of 7 ( Figure 3 C-D, P < 0.01). Subsequently, the number and weight of offspring were recorded in two consecutive breeding cycles to evaluate the fertility of the mice. In two breeding cycles, the number of live-born pups in MSC-sev-treated Cy mice was significantly higher than that in the Cy group, approximately 75% and 64.2% ( Figure 3 E-H, P < 0.01). Notably, there was no significant difference in body weight among the mice born after MSC-sEVs treatment. There was no significant difference in body weight between the mice born after msc-sev treatment and the normal mouse group. Our results indicate that MSC-sEVs can restore oocyte quality and reproductive potential in Cy-DOR mice.

[0057] Example 4: MSC-sEVs can enhance ovarian function and serum hormone levels

[0058] We evaluated the levels of anti-Müllerian hormone (AMH) and DEAD-box helicase 4 (DDX4), both of which are markers related to ovarian reserve function. Immunofluorescence images showed that MSC-sev treatment increased the level of AMH compared with the Cy group (P < 0.01). At the same time, the expression of DDX4 also increased in the msc-sevs group ( Figure 4 A-C). Similarly, we observed a significant decrease in the expression ratio of cleaved caspase-3 to total caspase-3 ( Figure 4 D, F, G, P < 0.0001). Compared with the control group, the apoptosis index increased to 12.4% in the cyclophosphamide group and decreased to 4.2% in the msc-sev treatment group ( Figure 4 E, P < 0.0001). Further evaluation of the effect of MSC-sEVs on serum hormones showed that compared with the Cy treatment group, the serum E2 level was significantly increased and the FSH level was significantly decreased in the MSC-sEVs group (Fig. 4H, P < 0.05)

[0059] Example 5: MSC-sEVs can improve the behavior and cognitive function of the Cy-DOR model

[0060] To investigate whether mesenchymal stem cell-sEVs can repair ovarian and brain functions, we conducted a series of neurobiological experiments to evaluate the effect of mesenchymal stem cell-sEVs on learning and memory.

[0061] In the Barnes maze test, the latency to escape into the safe hole was recorded daily ( Figure 5 A). The data showed that there were no significant differences between the Cy group and the MSC-sEVs group on the first, second, and fourth days. However, the latency in the MSC-sEVs group was significantly shortened on the third and fifth days, reaching 36 seconds on the fifth day ( Figure 5 B). On the test day, the number of times the MSC-sEVs group explored the safe hole increased to 3 times, which was significantly higher than that of the Cy group (P < 0.05). In addition, the time to first reach the safe hole was significantly shortened to 9 seconds, and the distance to first reach the safe hole was shortened to 931.9 mm (P < 0.05). The number of errors was also significantly reduced (P < 0.01), but there was no difference in the total moving distance among the groups ( Figure 5 C). In the elevated maze test measuring anxiety and fear responses, MSC-sEV treatment significantly increased the number of entries into the open arms (P < 0.05), but there was no significant improvement in other indicators ( Figure 5 D-F). These results indicate that learning and memory abilities were significantly enhanced after MSC-sEV treatment. Mesenchymal stem cell-sEVs treatment significantly increased the expression of NeuN and PSD95 in the mouse brain ( Figure 5 G-H, P < 0.05), decreased the expression of cleaved-PARP1 (P < 0.0001) and γ-H2AX (P < 0.01), and enhanced the expression of XRCC1 ( Figure 5 I, P < 0.01).

[0062] Example 6: MSC-sEVs alleviate Cy-DOR by maintaining NAD+ levels and DNA repair

[0063] Within two days of mesenchymal stem cell-sEVs treatment, we observed a significant decrease in the elevated levels of cleaved-PARP1 and PAR levels ( Figure 6 B-C, P < 0.01). However, this early treatment did not significantly alter NAD+ levels, increase NADH, or affect the NAD+ / NADH ratio ( Figure 6 A). NADH is the reduced form of NAD+, and the NAD+ / NADH ratio is an important component of the cellular redox state, reflecting cell metabolism. Notably, after 21 days of treatment with mesenchymal stem cell-SEVs, the NAD+ content (P < 0.0001), the NAD+ / NADH ratio (P < 0.01), and the total amount of NAD+ plus NADH were all significantly restored, while the NADH level was significantly decreased (P < 0.05) ( Figure 6 D). In addition, PAR, which was initially reduced by Cy treatment, increased after MSC-sEVs treatment, showing a reverse trend compared to two days ago ( Figure 6E, P < 0.001). In addition, the DNA damage marker γ-H2AX was significantly reduced after Cy treatment but increased after MSC-sEVs treatment ( Figure 6 F, I, J, P < 0.001). The levels of XRCC1 and BRCA2, which are involved in DNA damage repair and responsive to PARP1, also increased after MSC-sEVs treatment ( Figure 6 G, H, J, P < 0.05). These findings indicate that mesenchymal stem cell-sEVs can not only alleviate early DNA damage and apoptosis but also restore long-term redox balance and DNA repair, highlighting their role in enhancing cellular metabolic health and DNA repair capacity.

[0064] The foregoing description of the disclosed embodiments enables those skilled in the art to practice or use the present invention. Various modifications to the above-described embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Thus, the invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An application of MSC-sEVs, characterized in that: Application of MSC-sEVs in the preparation of drugs for the treatment of diminished ovarian reserve.

2. The use according to claim 1, characterized in that The treatment of reduced ovarian reserve is to improve or enhance at least one of the following symptoms: 1) Improve estrous cycle and / or follicle development; 2) Improve oocyte quantity and / or fertility outcomes; 3) Enhance ovarian function and / or serum hormone levels.

3. An application of MSC-sEVs, characterized in that: Application of MSC-sEVs in the preparation of drugs to improve behavioral and cognitive functions.

4. The use according to claim 3, characterized in that The improved behavior and cognitive function is the enhancement of learning and memory abilities.

5. A method for improving follicle development without disease treatment, characterized in that: Use MSC-sEVs or drugs prepared based on MSC-sEVs to improve follicle development.

6. A method for improving ovulation quantity without disease treatment, characterized in that: Using MSC-sEVs or drugs prepared based on MSC-sEVs can improve the number of ovulation in animals.

7. A method for improving reproductive quantity without disease treatment, characterized in that: Using MSC-sEVs or drugs prepared based on MSC-sEVs to improve animal reproductive numbers.

8. Use of the method according to any one of claims 5 to 7 in scientific research experiments or animal breeding production.

9. A method for preparing MSC-sEVs, the method comprising the following steps: 1) Cultivate human induced pluripotent stem cells in complete cell culture medium (CCM) for 2-3 days, and expand to a 5-layer cell factory when the cells grow to about 80% confluence; 2) After washing the cells with phosphate-buffered saline (PBS), CCM was replaced with chemically defined protein-free (CDPF) medium; 3) After 6 hours, fresh CDPF medium was replaced, and about 600 ml of supernatant was collected and centrifuged at 2000 g for 20 minutes to remove cell debris; 4) iPSC-MSCs were infected with recombinant lentivirus carrying luciferase gene; cells were cultured at 3×10 4 The cells / ml density was inoculated in a 12-well plate, and fresh medium was replaced 6 hours after infection. Puromycin was used for selection 72 hours later to obtain iPSC-MSCs that stably expressed luciferase, which were then frozen to form a working cell bank; 5) Purification of MSC-derived small exosomes (MSC-sEVs) by anion exchange chromatography.

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