The Protective Effects Of Human Cord Mesenchymal Stem Cell-derived Microvesicles On Cisplatin-damaged Granulosa Cells

Part I Isolation and purification of human cord mesenchymal stem cell-derived microvesiclesObjective: To establish an effective method for isolating and purifying mesenchymal stem cell-derived microvesicles.Methods: The human cord MSCs before passage 8 were cultured in serum-free medium,and medium supernatant were collected,then the MVs in supernatant were extracted and purified through three different ultracentrifugation,including Modified ultracentrifugation,Centrifugation and Sucrose/D2 O purifying.The morphological observation and protein quantification of MVs were carried out by transmission electronic microscope and Bradford method.Results: Compared with Centrifugation and Sucrose/D2 O purifying,More quantity of highly purified MVs could be obtained through the modified ultracentrifugation,and 1×106 cells might release 162μg MVs.The MVs was look like circular or elliptic single membrane vesicle structure with diameter ranged from 30 to 200 nm and low electron density shadow in the middle under transmission electron microscope.Conclusion: High concentration of MVs could be obtained from the medium of MSCs with the method of modified ultracentrifugation.Part Ⅱ The protective effects of human cord mesenchymal stem cell-derived microvesicles on cisplatin-damaged granulosa cellsObjective: To explore the protective effects of human cord mesenchymal stem cell-derived microvesicles on cisplatin-damaged granulosa cells(GCs).Methods: 1.The primary GCs were separated from 3 weeks female SD rats by mechanical method,and cultured in serum-free medium.The GCs were identified by FSHR immunohistechemical staining.The internalization of GCs on MVs were observed under fluorescent microscopy through co-culture of normal and cisplatin-damaged GCs with PKH26 labeled MVs.6h later,the location of PKH26 labeled MVs were observed both in normal GCs and cisplatin-damaged GCs under fluorescent microscopy.The different concentrations of MVs were co-cultured with cisplatin-damaged GCs to determine the optimal MVs concentration for protection of damaged GCs.2.The experiment consisted of three groups: normal GCs group,cisplatin-induced GCs group,MVs co-cultured with cisplatin-damaged GCs group.Respectively,the survival rate of GCs and the concentrations of E2 and P in medium supernatants were detected by MTT and chemiluminescence.The cell apoptosis rate of GCs were detected by FCM.Bax、Bcl-2、Caspase3、St AR m RNA expression were detected by q PCR.Results: 1.The primary GCs grow well in serum-free medium with the purity over 95%, proved by FSHR immunohistochemical staining.MVs of red fluorescence could be observed in cisplatin-damaged GCs after 6h co-culture.2.After co-culture with MSCs-MVs,The apoptosis cells of GCs were decreased and estradiol levels were increased significantly higher in the CTx group.Meanwhile,compared with CTx group,the m RNA levels of Caspase3 significantly decreased and ratio of Bcl-2 / Bax and St AR m RNA significantly increased.Moreover,the protective effect of MVs showed dose-dependent effect in cisplatin-damaged GCs.Conclusion: Human cord mesenchymal stem cell-derived MVs could be absorbed by cisplatin-induced GCs.MVs can alleviate the damage of GCs induced by chemotherapy and promote the proliferation and secretion function recovery.