The Effects And Its Mechanism Of Mesenchymal Stem Cell-derived Microvesicles On Ovarian Granular Cells

Part I Isolation and purification of mesenchymal stem cell-derived microvesiclesObjective To establish an effective method for isolating and purifying mesenchymal stem cell-derived microvesiclesMethods The human cord blood MSCs before passage 8 were cultured in serum-ftee medium, and medium supernatant were collected after 24h,48h and 72h respectively, and then the MVs in supernatant were extracted and purified by ultracentrifugation and density gradient centrifugations. The morphological observation and protein quantification of MVs were carried out by transmission electronic microscope and Bradford method.Results The MVs was look like circular or elliptic under the transmission electron microscope with diameter ranged from 30 to 1000 nm and low electron density shadow in the middle. Following the incubation time prolonged, the amount of MVs increased significantly, approximately 8×105 cells might release 10 μg MVs.Conclusion Highly purified MVs could be obtained by the ultracentrifugation combined with density gradient centrifugations, prolonged serum-free culture time appropriately could increase amount of MVs significantly.Part II The internalization of MVs and its effect on proliferation and secretion of ovarian granular cellsObjective To explore the internalization of MVs and its effect on proliferation and secretion of ovarian granular cells (GCs).Methods:GCs were separated from 3 weeks female SD rats by mechanical method, the purity of GCs were identified by FSHR immunohistechemical staining. The internalization of GCs on MVs was observed under fluorescent microscopy after 3h co-culture of GCs and PKH26 labeled MVs. MVs of different concentration (0、10、 15、20、25μg/ml) were co-cultured with GCs for 48h, then the concentrations of E2 in medium supernatants and the cell cycle phases of GCs were detected by chemiluminescence and FCM, respectively.Results:GCs of rats were separated successfully by mechanical method with the purity over 95%,proved by FSHR immunohistochemical staining. MVs could be absorbed by GCs after 3h co-culture. Proliferation index(PI)、s-phase fraction (SPF) of GCs and the concentration of E2 in medium supernatants were all presenting a trend rising first then going down, and increased significantly when GCs were co-cultured with the MVs under the concentrations of 15μg/ml (P<0.05).Conclusion Highly purified GCs could be separated successfully by mechanical method. Human cord blood mesenchymal stem cell-derived MVs could be absorbed by GCs. and promote the proliferation and secretion function of GCs. GCs could obtain the most vigorous proliferative and secretary potential under 15μg/ml MVs.