An Animal Experiment Study On The Treatment Of Premature Ovarian Insufficiency With Exosomes Derived From Umbilical Cord Mesenchymal Stem Cells

BackgroundPremature ovarian insufficiency,as one of the most common endocrine diseases in gynecology,can lead to the loss of female fertility and low estrogen status,and seriously affect the quality of life of women.Currently,the most commonly used hormone replacement therapy has obvious limitations.It can only improve a series of uncomfortable symptoms caused by low estrogen level in POI patients,but cannot fundamentally improve ovarian function.A number of studies have shown that mesenchymal stem cells can improve the ovarian function of POI mice caused by various factors.Currently,there are 10 registered clinical studies at home and abroad,but the exact mechanism has not been clarified.Exosomes,as paracrine products of MSCs,play an important role in tissue protection and repair in myocardial infarction,stress urinary incontinence,stroke and other diseases,but there are few reports on their role in POI.Our previous studies have shown that human umbilical cord mesenchymal stem cell-derived exosomes can reduce chemotherapy-induced apoptosis of ovarian granulocytes in vitro.In this study,animal experiments were used to further explore whether UCMSCS-EXOS can improve the ovarian function of POI mice,so as to provide a positive and effective new method for the treatment of POI.ObjectiveTo evaluate whether UCMSCs-Exos can improve the ovarian function of POI mice,and to lay an experimental foundation for the application of UCMSCs-Exos in the clinical treatment of POI.Methods1.Establishment of POI mice model:40 female ICR mice aged 6-7 weeks were randomly divided into 4 groups with 10 mice in each group.Control group and drug administration groups with different gradients were set:low dose group（5 mg/kg）,middle dose group（7.5 mg/kg）,high dose group（10 mg/kg）,observe the estrus cycle of mice change,by ELISA to detect serum FSH and E₂ levels,ovarian tissue after HE staining to observe the organizational structure,counting number of follicles at different levels,confirm the best dose of cisplatin modeling.2.UCMSCs-Exos extraction:UCMSCs were obtained by enzymatic hydrolysis,and were confirmed to conform to the biological characteristics of MSCs by flow cytometry and multidirectional differentiation induction.Exosomes from the supernatant of UCMSCs culture were extracted by hypervelocity centrifugation.The surface markers,morphology and particle size of UCMSC-Exos were identified by Western Blot,Transmission electron microscopy（TEM）and laser particle size analyzer.3.Animal experimental study on improving chemotherapy-induced POI by UCMSCs-Exos:18 female ICR mice aged 6-7 weeks were randomly divided into three groups:control group,model group and treatment group.The control group was intraperitoneally injected with normal saline,and the rest was given a single intraperitoneal injection of cisplatin 7.5 mg/kg to form a model.After modeling,mice in the control group and model group were injected with 200μL PBS through caudal vein on the 1st,5th and 10th day,respectively,and mice in the treatment group were injected with 200μL UCMSCS-Exos suspension with a concentration of 625μg/m L through caudal vein.On the 13th day,blood samples and ovarian tissues were collected,and the improvement of ovarian function of mice was evaluated by observing the general condition of mice,the size of ovarian tissues,immunohistochemical determination of Caspase 3 expression difference in ovarian tissues,counting of follicles at all levels,and determination of FSH and E₂ levels.Results1.Compared with the control group,the general state,estrous cycle,hormone level and follicle number of mice were changed to varying degrees after single intraperitoneal injection of different doses of cisplatin,but the low-dose group recovered faster,while the medium-dose and high-dose groups suffered significant ovarian function damage,among which 2 mice died in the high-dose group during the observation period.After comprehensive evaluation,single intraperitoneal injection of cisplatin in 7.5 mg/kg mice was consistent with the clinical pathological process of POI,which could establish a stable and effective POI mouse model.2.UCMSCs were fusiform and adherent,and CD73,CD90,and CD105 were highly expressed by flow cytometry,while CD34,CD11b,CD19,CD45 and HLA-DR were almost not expressed.Calcium salt granules were observed in osteogenic induction,and red lipid droplets were observed in adiposiogenic induction.Under TEM,the UCMSCs-Exos extracted by the hypervelocity centrifugalization method showed a typical double-layered disc-like vesicle structure,with an average diameter of 166.5 nm.The surface of UCMSCs-Exos expressed specific marker proteins CD 9 and Flotillin 1,but did not express endoplasmic reticulum marker protein Calnexin.3.Compared with model group,treatment group mice after caudal vein injection of UCMSCs-Exos,estrus cycle of mice was significantly shortened,FSH level declined obviously,E₂ level increased,the number of primary follicles and growing follicles increased,atretic follicles decreased,and the positive rate of granulosa cell Caspase 3 decreased significantly,indicating that ovarian function of POI mice was improved after UCMSCs-Exos injection through caudal vein.Conclusion1.A single intraperitoneal injection of cisplatin 7.5 mg/kg could successfully establish a mouse POI model.2.High purity and good quality UCMSCs-Exos were obtained by hypervelocity centrifugation,which can be used for further animal experiments.3.UCMSCs-Exos injection through caudal vein can effectively improve ovarian function in POI mice.