Biological material for repairing ovarian function as well as preparation method and application of biological material

Abstract

The invention provides a biological material for repairing an ovarian function as well as a preparation method and application thereof, and relates to the technical field of biological materials, a large number of experiments prove that specific biological protein components for reversing the ovarian function in extracellular vesicles are growth differentiation factor 9 (GDF-9) for repairing an ovary, and the biological material has the advantages that the ovarian repairing function mechanism of the specific biological protein components for reversing the ovarian function in the extracellular vesicles is found; according to the present invention, the GDF-9 extracellular vesicles (GDF-9Over-existed Extracellular vesicles, GOEs) are adopted as the active substance, such that the component is effectively extracted and purified so as to achieve the target of targeted therapy.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to a biomaterial for repairing ovarian function, a preparation method and an application thereof. Background Art

[0002] Ovarian function plays a decisive role in women's reproductive health. When ovarian failure occurs, women's fertility is significantly reduced. It is characterized by follicular atresia, which leads to anovulation, infertility, and low levels of sex hormones, which seriously affect women's physical and mental health. At present, the classic clinical treatment method is hormone replacement therapy (HRT), which relieves perimenopausal symptoms by exogenous supplementation of estrogen and progesterone. However, HRT cannot improve the cessation of follicular development or premature atresia, nor can it induce ovulation, and does not improve women's fertility, so it cannot fundamentally repair the failed ovaries. Therefore, how to repair ovarian function and restart the development, maturation, ovulation, secretion of sex hormones and improvement of fertility of ovarian follicles in the ovaries is a difficulty and hot spot in the treatment of ovarian failure. Regenerative medicine brings new hope for the treatment of ovarian failure. Mesenchymal stem cells have been confirmed to treat ovarian failure, but the immune response and tumor response brought by stem cells also limit their use in clinical practice.

[0003] In view of this, the present invention is proposed. Summary of the invention

[0004] One of the purposes of the present invention is to provide a biological material for repairing ovarian function, so as to at least solve one of the technical problems existing in the prior art.

[0005] The second purpose of the present invention is to provide a method for preparing the above-mentioned biological material for repairing ovarian function.

[0006] The third object of the present invention is to provide the use of the above-mentioned biological material for repairing ovarian function in preparing products for repairing ovaries.

[0007] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0008] The present invention provides a biological material for repairing ovarian function, comprising extracellular vesicles overexpressing GDF-9 as active substances.

[0009] Furthermore, the biomaterial is a film-type agent;

[0010] Preferably, the biological material is in the form of tablets, powders, emulsions or liquids.

[0011] The present invention also provides a method for preparing the above-mentioned biological material for repairing ovarian function, comprising the following steps:

[0012] (a) Preparation of extracellular vesicles overexpressing GDF-9;

[0013] (b) preparing the GDF-9 overexpressing extracellular vesicles obtained in step (a) as the biological material for repairing ovarian function.

[0014] Furthermore, the step (a) includes inducing mesenchymal stem cells to overexpress GDF-9 using GDF-9 mRNA, collecting cell supernatant, and obtaining extracellular vesicles overexpressing GDF-9;

[0015] The sequence of the GDF-9 mRNA is Homo sapiens growth differentiation factor 9 (GDF9), RefSeqGene on chromosome 5, Sequence ID: NG_047051.1: 5416-10700;

[0016] Preferably, the method for inducing GDF-9 overexpression in mesenchymal stem cells is selected from cell transfection or electroporation.

[0017] Furthermore, the step (b) comprises injecting the GDF-9 overexpressing extracellular vesicles prepared in step (a) as the inner aqueous phase of 0.1% polyvinyl alcohol into dichloromethane containing PLGA as the oil phase, injecting into a 0.7% PVA solution after ultrasonic treatment, and obtaining the biomaterial for repairing ovarian function after emulsification.

[0018] Furthermore, before step (b), the method further comprises the step of preparing the GDF-9 overexpressing extracellular vesicles into lyophilized powder.

[0019] In addition, the present invention also provides the use of the above-mentioned biological material for repairing ovarian function or the biological material for repairing ovarian function prepared by the above-mentioned preparation method in the preparation of products for repairing ovaries.

[0020] Furthermore, the overexpressed GDF-9 extracellular vesicles in the biomaterial bind to the ACVR1B receptor in ovarian cells, phosphorylating SMAD2 and transporting it into the nucleus, thereby repairing the ovary.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] Through a large number of experimental verifications, the present invention has found that the specific biological protein component in the extracellular vesicles that reverses ovarian function is the mechanism of action of Growth Differentiation Factor 9 (GDF-9) to repair the ovaries, and then the extracellular vesicles overexpressing GDF-9 are used as active substances to effectively extract and purify the components to achieve the purpose of targeted treatment. At the same time, the present invention also found that when the extracellular vesicles overexpressing GDF-9 are used as active substances to prepare a membrane-type agent, it is easier to break the membrane and release effective molecules after reaching tissues and organs. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 Provide a schematic diagram of the theoretical basis for the experimental example of the present invention.

[0025] Figure 2 Schematic diagram of EVs derived from umbilical cord mesenchymal stem cells provided in the experimental examples of the present invention; wherein, a, schematic diagram of the separation and purification of EVs; b, transmission electron microscopy showing the integrity and structure of EVs; c, Western-blot detection of the expression of CD63 and CD9 in EVs.

[0026] Figure 3 This is a graph showing the results of NTA detection of EVs concentration and size provided in the experimental example of the present invention.

[0027] Figure 4 Schematic diagram of the in vitro DO model for the potential of EVs to enhance the viability of GCs provided in the experimental examples of the present invention.

[0028] Figure 5 The flow cytometry provided in the experimental examples of the present invention detects GCs viability, apoptosis and death.

[0029] Figure 6 The flow cytometry provided in the experimental example of the present invention quantitatively shows the efficiency of EVs in treating DO in vitro; n = 3, data are expressed as mean ± SD; * and ** indicate that there is statistical significance between the two groups, and ns indicates that there is no statistical significance between the two groups. The viable cell rate of the group treated with EVs was significantly higher than that of the group not treated with EVs, and the dead cell rate and apoptotic cell rate were significantly lower than those of the group not treated with EVs. There was no significant difference in mechanical damage among the blank control, DO group and EVs treatment group.

[0030] Figure 7 The experimental examples of the present invention provide result graphs of the effects of GDF-9 on the survival and proliferation of GCs during EVs treatment; wherein, a, schematic diagram of in vitro induction of GCs; b, increase in the number of GCs after EVs treatment; c, higher cell counts of GCs overexpressing GDF-9 than in the control group, and lower cell counts of GCs inhibited by GDF-9 than in the control group; df, TUNEL assay showed reduced apoptosis of GDF-9 GCs compared with si-GDF-9 GCs treated with EVs; eg, higher expression level of Ki-67 in GDF-9 GCs compared with si-GDF-9 GCs treated with EVs.

[0031] Figure 8 The experimental examples of the present invention provide a verification result diagram that ACVR1B is the main regulatory factor of GCs; wherein, a, schematic diagram of the rat OD model, through total RNA sequence sampling and analysis; b, KEGG pathway enrichment reveals the signaling pathway of cytokine and cytokine receptor interaction; c, Volcano mapping of RNA-seq analysis; de, Western-blot detection of GDF-9, ACVR1B, SMAD2 levels and SMAD2 phosphorylation status; fg, Western-blot detection of inhibition of GDF-9 gene resulting in EVs unable to stimulate GDF-9 signal transduction, then ACVR1B and SMAD2 / p-SMAD2 protein expression levels are reduced.

[0032] Fig. 9 Schematic diagram of GOEs and animal experiment design and results provided for the experimental examples of the present invention.

[0033] Fig.10 The figure shows the verification results that the GDF-9 overexpressed EVs (GOEs) provided in the experimental example of the present invention can improve the ovarian function of DO rats, and the figure shows the weight results of rats before modeling and after drug administration.

[0034] Fig.11 The figure shows the verification results that GDF-9 overexpressing EVs (GOEs) can improve the ovarian function of DO rats provided in the experimental examples of the present invention; wherein, ad, ovarian size and body weight of rats after treatment; e, ovarian histology after treatment; the top row shows representative parts, and the black frame indicates the enlarged area; the arrow indicates PMF (primordial follicle), the arrow indicates SCF (secondary follicle), and the asterisk indicates ATF (atretic follicle); fh, the number of follicles in the control group, DO and GOEs treatment groups; n=3, and the data are expressed as mean±SD.

[0035] Fig.12The experimental examples of the present invention provide result graphs of follicle stimulating hormone receptor (FSHR) as a marker for detecting GCs expressing ACVR1B after GOEs treatment; wherein, a, IF detection of FSHR expression in ovarian GCs; b, IF detection of ACVR1B expression in ovarian GCs; c, IF detection of FHSR and ACVR1B expression in ovarian GCs; df, the number of FSHR and ACVR1B in the control group, DO and go treatment groups and their expression levels at mean fluorescence intensity; n=3, data are expressed as mean±SD.

[0036] Fig.13 The result diagram of the combination of immunohistochemistry (IHC) and immunofluorescence (IF) analysis provided in the experimental example of the present invention; wherein, a, ovarian ACVR1B immunohistochemistry diagram, wherein the black dotted box is used as an enlarged part to emphasize, and the black arrow points to the positive result; b, ovarian ACVR1B immunofluorescence diagram, wherein the white dotted box is used as an enlarged part to emphasize, and the white arrow points to the red fluorescence positive result; c, d, are quantitative analysis diagrams of 13a, b, indicating that the expression of ACVR1B in the ovaries of DO rats treated with GOEs is higher than that of DO rats not treated with GOEs. DETAILED DESCRIPTION

[0037] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0038] Generally, the nomenclature and its technology used in conjunction with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally carried out according to conventional methods as well-known in the art, and as described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's specifications, as generally achieved in the art, or as described herein. The nomenclature and its laboratory procedures and technology used in conjunction with analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein are those well-known and commonly used in the art.

[0039] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] According to one aspect of the present invention, a biomaterial for repairing ovarian function is provided, comprising a membrane-type agent having extracellular vesicles overexpressing GDF-9 as an active substance.

[0041] Through a large number of experimental verifications, the present invention has found that the specific biological protein component in the extracellular vesicles that reverses ovarian function is the mechanism of action of Growth Differentiation Factor 9 (GDF-9) to repair the ovary, and then the extracellular vesicles that overexpress GDF-9 are used as active substances to effectively extract and purify the component to achieve the purpose of targeted treatment ( Figure 1 ).

[0042] Since lipids are membrane-type exosomes, they are more in line with biological characteristics and can be easily compatible with cells in tissues and organs to achieve targeted treatment purposes. Therefore, when extracellular vesicles overexpressing GDF-9 are used as active substances to prepare membrane-type agents, it is easier to break the membrane and release effective molecules after reaching tissues and organs. Therefore, in some preferred embodiments, the biomaterial is a membrane-type agent.

[0043] Optionally, the biomaterial is in the form of tablets, powders, emulsions or liquids. In order to be more suitable for clinical applications, the biomaterial is preferably in the form of tablets. After the extracellular vesicles form a membrane, they need to be frozen and stored to maintain stability. When placed in the pelvic cavity, the tablet form can be directly attached to the surface of the ovary, and when it encounters water and the temperature rises to body temperature, the extracellular vesicles become active.

[0044] According to a second aspect of the present invention, there is provided a method for preparing the above-mentioned biomaterial for repairing ovarian function, comprising the following steps:

[0045] (a) Preparation of extracellular vesicles overexpressing GDF-9;

[0046] (b) preparing the GDF-9 overexpressing extracellular vesicles obtained in step (a) as the biological material for repairing ovarian function.

[0047] In some specific embodiments, step (a) comprises inducing GDF-9 overexpression in mesenchymal stem cells using GDF-9 mRNA, collecting cell supernatant, and obtaining extracellular vesicles overexpressing GDF-9.

[0048] The sequence of the GDF-9 mRNA is Homo sapiens growth differentiation factor 9 (GDF9), RefSeqGene on chromosome 5, Sequence ID: NG_047051.1: 5416-10700.

[0049] The method for inducing mesenchymal stem cells to overexpress GDF-9 is not limited, and any conventional molecular biological method in the art that can achieve the induction function can be used, such as cell transfection or electroporation. As a preferred solution, the electroporation method can be used instead of the stem cell transfection method, which can effectively improve the efficiency of carrying effective molecules.

[0050] In some specific embodiments, the step (b) comprises injecting the GDF-9 overexpressing extracellular vesicles prepared in step (a) as the inner aqueous phase of 0.1% polyvinyl alcohol into dichloromethane containing PLGA as the oil phase, injecting into a 0.7% PVA solution after ultrasonic treatment, and obtaining the biomaterial for repairing ovarian function after emulsification.

[0051] Specifically, the extracellular vesicles overexpressing GDF-9 were injected into dichloromethane (DCM) containing PLGA as the oil phase as the inner aqueous phase of 0.1% polyvinyl alcohol (PVA). Then, the entire content was ultrasonically treated at 4°C. The emulsion was then immediately injected into a solution of 0.7% PVA. The secondary emulsion was emulsified on a high-speed homogenizer for 12 minutes to obtain a final water / oil / water (w / o / w) mixture, which was the biological material for repairing ovarian function.

[0052] Optionally, before step (b), the method further comprises preparing the GDF-9 overexpressing extracellular vesicles into lyophilized powder. Lyophilized powder has the advantages of being easy to carry and transport, and has enhanced stability and is convenient for clinical use.

[0053] According to the third aspect of the present invention, the present invention also provides the use of the above-mentioned biological material for repairing ovarian function or the biological material for repairing ovarian function prepared by the above-mentioned preparation method in the preparation of products for repairing ovaries.

[0054] The inventors of the present invention discovered for the first time that the extracellular vesicles overexpressing GDF-9 in the biomaterial bind to the ACVR1B receptor in the ovarian cells, phosphorylating SMAD2 and transporting it into the nucleus, thereby realizing the mechanism of repairing the ovary.

[0055] The extracellular vesicle itself is a double-layer lipid structure with a pro-cytotoxic effect, so it can bind to cells. The ACVR1B receptor on the surface of granulosa cells is a target receptor, which can add the modified molecule Actin A on the surface of the vesicle. Actin A is a member of the BMP / TGF-β family and can specifically bind to ACVR1B. This allows the extracellular vesicle to be targeted to the ovarian granulosa cells. The vesicle membrane is taken up and out of the granulosa cells, GDF-9 activates the SMAD2 pathway to phosphorylate, and the cells proliferate and express, achieving the purpose of repairing ovarian function.

[0056] The present invention is further described below by way of examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or directly purchased from the market.

[0057] Example 1

[0058] This embodiment provides a biomaterial for repairing ovarian function, which is prepared by the following method:

[0059] (a) Extract the supernatant of mesenchymal stem cells, filter the cells and impurities with a 0.22μm filter membrane, and then perform low-temperature differential centrifugation: at 4°C, centrifuge the supernatant at 330g for 10min, remove the bottom debris, and retain the supernatant; centrifuge at 2000g for 10min, remove the bottom debris, and retain the supernatant; centrifuge at 0.22μm to remove impurities and retain the supernatant; centrifuge at 100000g for 85min, and retain the precipitate, which is the extracellular vesicle. Resuspend the exosomes in PBS. Add GDF-9 mRNA and electroporation buffer Bio-Rad Gene Pulser Electroporation Buffer, blow evenly, put into the electroporation cup, put into the Lonza 4D-Nucleofector electroporator at 4°C, and let it stand until the membrane is closed, which is the GDF-9 overexpression exosome particles.

[0060] (b) Extracellular vesicles overexpressing GDF-9 were injected into dichloromethane (DCM) containing PLGA as the oil phase as the inner aqueous phase of 0.1% polyvinyl alcohol (PVA). Then, the entire content was ultrasonically treated at 4°C. The emulsion was then immediately injected into a solution of 0.7% PVA. The secondary emulsion was emulsified on a high-speed homogenizer for 12 minutes to obtain a final water / oil / water (w / o / w) mixture, which was the biomaterial for repairing ovarian function.

[0061] Example 2

[0062] This embodiment provides a biomaterial for repairing ovarian function, which is prepared by the following method:

[0063] (a) Lentivirus transfection: 1. Construct GDF-9 mRNA lentivirus vector plasmid 2. After mesenchymal stem cells adhere to the culture dish, add appropriate amount of virus suspension, incubate at 37°C, and replace the culture medium every 24 hours. GDF-9 overexpressing mesenchymal stem cells are obtained after 72-96 hours.

[0064] (b) Extracellular vesicles overexpressing GDF-9 were injected into dichloromethane (DCM) containing PLGA as the oil phase as the inner aqueous phase of 0.1% polyvinyl alcohol (PVA). Then, the entire content was ultrasonically treated at 4°C. The emulsion was then immediately injected into a solution of 0.7% PVA. The secondary emulsion was emulsified on a high-speed homogenizer for 12 minutes to obtain a final water / oil / water (w / o / w) mixture, which was the biomaterial for repairing ovarian function.

[0065] Experimental example

[0066] Test method:

[0067] 1. Collection, purification and characterization of EVs

[0068] Human umbilical cord mesenchymal stem cells (Huc-MSCs) were cultured in medium containing 10% fetal bovine serum (FBS) in a standard cell culture incubator set at 37°C and 5% CO2. When the cells reached 80-90% density, they were subcultured and the cells were washed with phosphate-buffered saline (PBS) instead of medium every 30 minutes to remove residual FBS. After 3 days of culture, the conditioned medium was harvested. Subsequently, the collected medium was filtered through a 0.22 μm filter to eliminate cell debris and contaminants. The filtered supernatant was centrifuged at 4,000×g for 30-60 minutes at 4°C using a centrifugal filter device with a molecular weight cutoff of 100 kDa, or until the desired volume was reached. The concentrated EVs were washed with phosphate-buffered saline (PBS) and centrifuged at 100,000×g for 70 minutes at 4°C to pellet the EVs. The supernatant was carefully discarded and the EVs pellet was resuspended in an appropriate volume of PBS or other suitable buffer. Transmission electron microscopy (TEM) and nanoparticle tracking analysis (TA) were used to analyze the size distribution and morphology of EVS.

[0069] 2. Establishment of DO model in vitro

[0070] After the rat ovaries were extracted from the animals, they were immediately immersed in sterile PBS containing 1% gentamicin. Subsequently, the ovaries were cut into smaller tissue fragments and enzymatically hydrolyzed in 2% collagenase IV solution for 1 hour to promote cell dissociation. The separation process was terminated by adding an equal amount of culture medium containing 20% ​​fetal bovine serum (FBS). After centrifugation, the supernatant was carefully aspirated and the resulting cell spheres were resuspended in culture medium supplemented with 10% FBS. The suspended cells were then seeded into T-175 culture flasks and maintained in a standard cell culture environment at 37°C and 5% CO2. The culture medium was changed every 24 hours to ensure optimal cell growth and viability. After 5 days of incubation, the granulosa cell (GC cell) characteristics of the cultured cells were analyzed by flow cytometry. Cisplatin was used as an inducer and administered at a concentration determined by preliminary cell counting experiments.

[0071] 3. Total RNA Sequencing

[0072] Ovarian samples were obtained from DO rats and control rats and immediately immersed in cold, sterile phosphate-buffered saline (PBS) to preserve the integrity of RNA. Total RNA extraction was performed using TRIzol reagent (ThermoFisher Scientific, catalog number: 15596018) according to the manufacturer's instructions. Subsequently, the quality and quantity of the extracted RNA were assessed using a NanoDrop ND-1000 spectrophotometer (NanoDrop, Wilmington, DE, USA) and an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA).

[0073] 4. Induction of rat DO model

[0074] Animal care and experimental procedures strictly adhered to the guidelines set by the Animal Welfare Ethics Committee of Beijing Hospital. Female Sprague-Dawley rats aged 5 to 7 weeks were used to establish the DO rat model. This was achieved by administering cisplatin at a dose of 3 mg kg / day for three consecutive days. On the other hand, control rats received an injection of physiological saline solution.

[0075] 5. Weight and ovarian values

[0076] After euthanasia, the ovaries of rats in each experimental cohort were weighed to ensure consistency in subsequent analyses. After euthanasia, the ovaries were carefully dissected bilaterally and the bicornuate uterus and adipose tissue were removed to facilitate clear separation. The isolated ovaries were then carefully placed on pads for standardized measurements and photographic documentation. To accurately determine ovarian mass, each ovary was individually weighed using a precision balance.

[0077] 6. Follicle Analysis Histological sections of ovarian tissue were carefully examined under a microscope equipped with scanning and analysis software SlideViewer (2.6.0)

[0078] Using the established criteria from previous studies by the inventors' research team (Table 1), follicles at different developmental stages were systematically classified, including primordial, secondary, and atretic follicles. Subsequently, a comprehensive follicle quantification process was performed, with three independent researchers meticulously counting the number of follicles in every ten consecutive tissue sections. The resulting datasets were rigorously statistically analyzed using GraphPad Prism software (9.5.0), enabling robust interpretation and inference generation.

[0079] Table 1

[0080]

[0081] 7. Western blots

[0082] GC cells were lysed using cell lysis buffer at 4°C for 30 minutes and then centrifuged at 15,000rpm for 10 minutes. The resulting supernatant was collected and quantified using a BCA detection kit. Equal amounts of protein were loaded onto polyacrylamide gels for SDS PAGE separation. The separated proteins were then transferred to a polyvinylidene fluoride (PVDF) membrane. Subsequently, the PVDF membrane was incubated with the primary antibody at 4°C overnight and then incubated with the secondary antibody at room temperature for 1 hour. The blot was visualized using an enhanced chemiluminescence kit.

[0083] 8. Flow cytometry

[0084] To comprehensively characterize the viability and cellular responses of granulosa cells (GCs) following cisplatin induction and treatment with extracellular vesicles (EVs), flow cytometric analysis was performed using a Beckman Coulter flow cytometer. This analytical approach allows for the assessment of the survival status, apoptosis occurrence, mechanical damage, and cell death kinetics of GCs. Specifically, Annexin V-FITC and propidium iodide (PI) apoptosis kits were used to identify apoptotic and necrotic cell populations. This methodological framework enables the quantitative assessment of cellular responses to cisplatin induction and subsequent EVs treatment, providing important insights into the mechanisms of therapeutic interventions aimed at improving ovarian dysfunction.

[0085] 9. Immunohistochemistry and Immunofluorescence

[0086] Tissue sections were dewaxed and dehydrated through a series of solvent treatments. Antigen retrieval was then performed to improve the efficiency of immunostaining. After antigen retrieval, sections were washed with PBS, incubated in 3% H2O2 PBS buffer for 10 min at room temperature to block endogenous peroxidase activity, and then washed thoroughly. Subsequently, tissue sections were incubated overnight at 4°C with primary antibodies against ACVR1B, FSHR, or SMAD2. After incubation with the primary antibodies, sections were washed and then incubated with the corresponding secondary antibodies for 1 h at room temperature. Visualization of bound antibodies was achieved using 3.3'-diaminobenzidine (DAB) or 4',6-diamino-2-phenylindole (DAPI). Subsequently, counterstaining was performed, and the stained sections were quantitatively analyzed using ImageJ software.

[0087] 10. Statistical analysis

[0088] Statistical analysis was performed using GraphPad Prism software (Version 9.5). Data are presented as mean ± SD of at least three independent experiments that consistently produced comparable results. For comparisons between two groups, a two-tailed unpaired Student's t-test was used. For comparisons involving more than three groups, a one-way ANOVA followed by a Bonferroni post hoc test was performed. Statistical significance was defined as P < 0.05.

[0089] Test results:

[0090] 1. EVs derived from umbilical cord mesenchymal stem cells can be separated and purified by ultrafiltration technology

[0091] EVs are small, membrane-bound structures released by cells into the extracellular environment. EVs are loaded with a variety of biomolecules including proteins, lipids, nucleic acids, and metabolites, acting as mediators of intercellular communication and regulatory processes. In this study, the present invention focused on the isolation and improvement of EVs from HucMSCs ( Figure 2 Using transmission electron microscopy (TEM), we carefully examined the morphological features of the isolated EVs and confirmed their integrity and structure ( Figure 2 b). Subsequent evaluation by Nano Sight helped determine EVs concentration and size, key parameters for elucidating their biophysical properties ( Figure 3 In addition, EV-specific markers, such as CD63 and CD9, were identified by Western blot analysis, confirming the purity and authenticity of the isolated EV population ( Figure 2 Through this systematic approach, this study carefully characterized the nature and composition of EVs produced by hucmSCs, laying the foundation for further investigation of their functional roles and therapeutic potential.

[0092] EVs show the potential to enhance granulocyte cell viability after cisplatin-induced granulocyte injury in vitro

[0093] To explore the functional significance of EVs in cell dynamics associated with survival and apoptosis, this experiment used human granulosa cells (GCs) as an in vitro experimental model. In addition, this experiment used cisplatin at a concentration of 2.7 μg / ml to establish a model that simulates dysfunctional ovaries ( Figure 4 ), and this was confirmed by cell counting kit-8 (CCK8) detection. Then, the cells were divided into three groups in this experimental example: control group, DO group, and EVs-treated DO group. Flow cytometry was used to evaluate cell viability, apoptosis, and death in each group ( Figure 5 In the EVs-treated DO group, the number of live cells was significantly higher than that in the DO group and close to that in the control group ( Figure 6 In addition, in the EVs-treated DO group, the number of dead cells was significantly reduced compared with the untreated DO group and was similar to that of the control group ( Figure 6 In addition, no significant difference was observed in the number of mechanically damaged cells among the three groups ( Figure 6 However, the number of apoptotic cells in the EVs-treated DO group was not significantly different from that in the untreated group ( Figure 6 d) in.

[0094] 3. GDF-9 plays a key role in improving GCs viability and proliferation under EVs stimulation

[0095] Compared with the control group, EVs showed the ability to significantly increase the number of GCs ( Figure 7 a and b in the figure). EVs contain multiple molecules. To investigate the target molecules in EVs for the treatment of ovarian dysfunction (DO), this experiment involved transfecting granulosa cells with GDF-9 mRNA to induce GDF-9 overexpression, or transfecting granulosa cells with si-GDF-9 mRNA to inhibit GDF-9 expression. After EVs treatment, the cell count of GCs with GDF-9 overexpression was higher than that of the control group. However, EVs treatment failed to increase the number of GCs in the case of inhibiting GDF-9 gene expression ( Figure 7 In addition, the apoptosis of GDF-9GCs treated with EVs was reduced compared with that of si-GDF-9GCs treated with EVs, which was confirmed by TUNEL assay. In addition, the expression level of proliferation marker Ki-67 was higher in GDF-9GCs compared with si-GDF-9GCs ( Figure 7 dg in ).

[0096] 4. Total RNA-seq analysis shows that ACVRIB is a major regulator of GCs

[0097] In order to find out the target genes related to ovarian function in the ovary, this experiment used the cisplatin-induced ovarian dysfunction model based on previous studies and performed total-RNA sequencing analysis on normal ovaries and OD ( Figure 8 a in the figure). The differentially expressed gene, activin A receptor type 1B, encoding the type I receptor of the transforming growth factor β (TGF-β) superfamily ligand, was identified as a key gene for the interaction between cytokines and cytokine receptors. KEGG pathway enrichment and pathway integration analysis showed that after ACVR1B phosphorylation, SMAD2 interacted with SMAD4 to form a complex ( Figure 8 Subsequently, the complex translocates to the nucleus where it regulates the transcription of target genes involved in various cellular processes, such as cell proliferation, differentiation, and apoptosis.

[0098] Furthermore, to elucidate the gene expression patterns under different conditions, Western blot analysis was performed in this experimental case to evaluate the levels of GDF9, ACVR1B, SMAD2, and the phosphorylation status of SMAD2. GDF-9 protein levels were significantly increased after EVs treatment of GCs. In contrast, when the GDF-9 gene was inhibited by the GDF-9 antagonist follistatin, EVs were unable to stimulate GDF-9 signaling. Similarly, when the GDF-9 gene was inhibited, the protein expression levels of ACVR1B and SMAD2 / p-SMAD2 were reduced. This observation supports the view that EVs enhance GC function through the GDF-9 / ACVR1B / SMAD2 signaling pathway ( Figure 8 d and e).

[0099] Subsequently, a reverse experiment of the GDF-9 / ACVR1B / SMAD2 pathway was formulated. GCs were stimulated or inhibited by GDF-9 or ACVR1B genes and then treated with EVs. Protein levels in these cells were then assessed by western blot analysis. GCs were genetically manipulated against GDF-9 and ACVR1B, and then functional genes were reintroduced via repair constructs. Notably, upon restoration of GDF-9 expression in GCs, reactivation of the ACVR1B-mediated signaling pathway was observed, as evidenced by phosphorylation of the downstream effector SMAD2. When ACVR1B was stimulated by ALK-4 in GCs, but GDF-9 was knocked out, a significant loss of SMAD2 protein expression in GCs was observed. Knockout of ACVR1B in granulosa cells (GCs) using the inhibitor SB431542 resulted in the inability of GCs to express the specific protein GDF-9 ( Figure 8f and g). This observation highlights the key role of ACVR1B in regulating GDF-9 expression in GCs, underscoring its importance in mediating granulosa cell function and reproductive processes. This compelling evidence underscores the hierarchical relationship in the GDF-9 / ACVR1B / SMAD2 pathway, confirming that GDF-9 is an upstream regulator that initiates the ACVR1B-mediated signaling cascade in GCs.

[0100] 5. EVs upregulate GDF-9 to restore ovarian function in rats

[0101] Stimulating the GDF-9 gene in HucMSCs followed by the isolation of GDF-9-containing extracellular vesicles (GDF-9 overexpressing EVs, GOEs) provides a promising avenue for therapeutic intervention in the cisplatin-induced DO rat model. In order to comprehensively investigate the effects of GOEs on ovarian dysfunction, a controlled animal experimental study was designed in this experimental case. Three different groups were established, including a control group, a DO group, and a GOEs-treated DO group ( Fig. 9 ). After administration of purified GOEs to DO rats, body weight, ovarian size, and body weight were significantly increased compared to untreated DO rats ( Fig.10 , Fig.11 In addition, histological examination showed that the number of primary follicles (PMF) and secondary follicles (SCF) increased significantly, while the number of atretic follicles (ATF) decreased significantly in the GOEs treatment group ( Fig.11 eh in ). Together, these compelling findings suggest that GOEs have a multifaceted mechanism of action, including promoting follicular development, inhibiting follicular atresia, and enhancing overall ovarian function. This robust therapeutic effect highlights the potential utility of GOEs as a new and promising therapeutic modality to alleviate ovarian dysfunction and restore ovarian health characteristic of DO. This experimental example provides valuable insights into the therapeutic potential of GOEs and highlights their promising role in addressing ovarian dysfunction, thereby providing new avenues for the development of targeted interventions aimed at improving reproductive health outcomes in affected individuals.

[0102] 6. GOEs may enhance ovarian function by stimulating the expression of ACVRIB in GCs

[0103] In addition, the present study used a combination of immunohistochemistry (IHC) and immunofluorescence (IF) analysis to further explore the effects of GOEs on ACVR1B expression in DO GCs. The use of IHC and IF techniques provided a comprehensive assessment of ACVR1B expression in GCs, providing complementary perspectives on the observed effects ( Fig.13The results of both methods consistently showed that ACVR1B expression was significantly upregulated in GCs of the control group and GOEs-treated DO group compared with that of untreated DO ovaries ( Fig.13 c, d). This strong evidence strongly supports the hypothesis that GOEs treatment enhances ACVR1B expression in DO ovarian GCs, thus delineating a promising therapeutic trajectory to alleviate ovarian dysfunction. By adopting this multifaceted approach, this experimental example not only enhances the validity and reliability of the findings, but also provides a deeper understanding of the complex molecular mechanisms of GOEs-mediated regulation of ACVR1B expression in the context of ovarian physiology and pathology.

[0104] In addition, if Fig.12 As shown, to improve the accuracy and specificity of our analysis, the follicle-stimulating hormone receptor (FSHR) was integrated as a marker to distinguish GCs within the ovarian tissue microenvironment. FSHR is a key receptor expressed on the surface of GCs that coordinates key processes related to follicle development and ovulation. The use of FSHR as a recognition marker facilitates the precise identification and visualization of GCs within ovarian tissue sections, allowing for the detailed characterization of ACVR1B expression dynamics within this cellular environment. This strategic integration of FSHR not only optimized the accuracy and resolution of our study, but also provided valuable insights into the complex molecular crosstalk that underpins the effects of GOEs on ovarian function. Through this meticulous and comprehensive approach, our study provides compelling evidence delineating a critical role for GOEs in regulating ACVR1B expression in GCs, thereby paving the way for the development of targeted therapeutic interventions aimed at improving ovarian dysfunction and promoting reproductive health outcomes.

[0105] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A biological material for repairing ovarian function, characterized in that: It includes extracellular vesicles that overexpress GDF-9 as active substances.

2. The biomaterial according to claim 1, characterized in that The biological material is a film-type agent; Preferably, the biological material is in the form of tablets, powders, emulsions or liquids.

3. The method for preparing the biological material for repairing ovarian function according to claim 1 or 2, characterized in that: The steps include: (a) Preparation of extracellular vesicles overexpressing GDF-9; (b) preparing the GDF-9 overexpressing extracellular vesicles obtained in step (a) as the biological material for repairing ovarian function.

4. The preparation method according to claim 3, characterized in that: The step (a) comprises inducing mesenchymal stem cells to overexpress GDF-9 using GDF-9 mRNA, collecting cell supernatant, and obtaining extracellular vesicles overexpressing GDF-9.

5. The preparation method according to claim 4, characterized in that: The sequence of the GDF-9 mRNA is Homosapiens growth differentiation factor 9 (GDF9), RefSeqGene on chromosome 5, Sequence ID: NG_047051.1: 5416-10700.

6. The preparation method according to any one of claims 3 to 5, characterized in that: The method for inducing mesenchymal stem cells to overexpress GDF-9 is selected from cell transfection or electroporation.

7. The preparation method according to claim 3, characterized in that: The step (b) comprises injecting the GDF-9 overexpressing extracellular vesicles prepared in step (a) as the inner aqueous phase of 0.1% polyvinyl alcohol into dichloromethane containing PLGA as the oil phase, injecting into a 0.7% PVA solution after ultrasonic treatment, and obtaining the biological material for repairing ovarian function after emulsification.

8. The preparation method according to claim 7, characterized in that: Before step (b), the method further comprises preparing the GDF-9 overexpressing extracellular vesicles into lyophilized powder.

9. Use of the biological material for repairing ovarian function according to claim 1 or 2, or the biological material for repairing ovarian function prepared by the preparation method according to any one of claims 3 to 8 in preparing products for repairing ovaries.

10. The use according to claim 9, characterized in that: The overexpressed GDF-9 extracellular vesicles in the biomaterial bind to the ACVR1B receptor in ovarian cells, phosphorylating SMAD2 and transporting it into the nucleus, thereby repairing the ovary.

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