The Effects Of BMSCs On Granulosa Cells From The Aging Mice Via Mfn2/Mitochondria Pathway

Part I BMSCs and glanulosa cells isolation,culture and identificationObjectives: To obtain highly purified BMSCs and ovarian granulosa cells.Methods: Following approval from the Animal Research Center of Huazhong University of Science and Technology,the primary passage of BMSCs were isolated from the tibias and femurs of C57BL/6J(WT)mice under the strict sterile operation,then suspended in IMDM medium containing 10% fetal bovine serum,100u/ml penicillin,100u/ml streptomycin,followed by passaged and cultured under a regular condition.Well-conditioned cells in passage 4 were characterized by FCM for the existence of typical surface marker CD29,as well as the absence of the hematopoietic stem cells markers CD45 and CD34;The bilateral ovaries were aseptically derived from C57BL/6J(WT)mice at 40 weeks old,the follicles of which were manually separated under a stereoscopic microscope.The cell suspension was obtained via the common operation after degranulation and filtering,then planted on cell slides and cultured to acquire the well-conditioned cells for immunofluorescence analysis(Glanulosa cell marker FSHR,red fluorescence).Results: Flow cytometry assay confirmed that these forth passage cells expressed CD29(99.6%)positive and CD34(0.4%),CD45(1.5%)negative;Immunofluorescence analysis showed these extracted cells multi-shaped,basically,most of which dispayed red fluorescence mentioned above according to the three microscopic fields selected randomly.Conclusion: Purified BMSCs from passage 4 to 9 can be used for the subsequent experiments and the method above has been verified to be available for the extraction of ovarian granulosa cells.Part II The biological effects of BMSCs on granulosa cells extracted from the aging ovariesObjectives: To observe the effects of BMSCs on granulosa cells regarding cellular function,proliferation,apoptosis and so on.Methods: BMSCs from passage 4 or above were chosen in co-culture with the granulosa cells extracted from aging ovaries of mice by transwell for 72 h.Cells were divided into two groups: sole culture group(GCs)and co-culture group(BMSCs+GCs).After 72 h,we collected the culture medium in both groups,centrifuged to obtain the supernatant,and than detected the levels of estradiol(E2)and progesterone(P)by ELISA,respectively,for comparative analysis of the granulosa cells' function.The CCK-8 experiments were used to assay the cell vitality of granulosa cells as the indirect reflection of cell proliferation potential in two groups.Flow cytometry(FCM)analysis was applied to assess the cell apoptosis with annexin V/PI staining,and in addition,the apoptosis related genes were analyzed by western blot.Results: Compared with the GCs in sole culture group,the cells in co-culture group showed enhanced proliferation ability.And both FCM and Western blot revealed an unequivocal result,aside from the levels of hormone secretion to rise markedly in culture supernatant,was that BMSCs could attenuate the cell apoptosis and the Bcl-2levels as well as the Apoptosis Index(AI)was significantly increased(P<0.05),while the expressions of Bax,Caspase 3 and Caspase 9 were decreased in the co-culture group(P<0.05).Conclusion: BMSCs can reverse senescent cell function and overcome the cell apoptosis results from aging.Part III The mechanism of BMSCs effect on granulosa cells from the aging miceObjectives: To clarify that BMSCs can activate the expression of Mfn2 in granulosa cells extracted from the aging ovaries in mice,thereby regulate the mitochondrial structure and function.Methods: BMSCs from passage 4 or above were chosen in co-culture with the granulosa cells extracted from aging ovaries of mice by transwell for 72 h.Cells were divided into two groups: sole culture group(GCs)and co-culture group(BMSCs+GCs).After co-culture for 72 h,both of q RT-PCR and Western Blot were used to detect the expressions of Mfn2 m RNA and protein,respectively.Mitochondrial membrane potential was observed and measured by fluorescence microscope and flow cytometry with the Mitochondrial Membrane Potential Detection Kit(JC-1)and the ATP levels were detected with ATP Assay Kits(S0026).Results: In the co-culture group,Mfn2 m RNA expressions were on the rise and protein levels were increased markedly(P<0.05);the mitochondrial membrane potential(??m)was showed an obvious fluorescence enhancement and increased(P<0.05),when compared to that in sole culture group,as well as ATP levels.Conclusion: BMSCs may improve the aging cells function,proliferation and attenuate the cells apoptosis induced by senescence via up-regulating the Mfn2 expression and changing the mitochondrial function.