The Role Of P110α/Akt/Foxo3a In Marginal Cells Under Oxidative Stress

Part I Primary Culture of Rat Stria Vascularis Marginal Cell in vitro and Establish the Cochlear Marginal Cell oxidative stress ModelObjective:Primary culture of stria vascularis marginal cells (MCs) from neonatal SD rats and established the stria vascularis marginal cell oxidative stress model.Methods:The strias vascularis of3-day old SD rats were dissected and digested, from which the MCs were suspended. The cell suspension were plated with Epithlial Cell Medium-animal(EpiCM-animal) and incubated at an atmosphere of37℃and5%CO2. Identify the MCs from the morphology and immunohistochemistry. Treated the MCs with glucose oxidase and selected the optimal concentration and duration of glucose oxidase by CCK-8. Using flow cytometry detected the level of ROS and apoptosis of MCs being treated under the optimal concentration and time duration.Results:under a microscope, MCs can be seen as polygons and grow together to make cell islands after plated for24hours. The cell islands appear like cobblestones. The fibroblasts can be removed by purification. Besides, detecting CK-18fluerscent signal under confocal microscope, MCs can be identified for high express of CK-18. Detecting cell activities by CCK-8, cell activities were suppressed when treated with glucose oxidase. We chose the50U/L (p<0.05) as the previous concentration level for treatment. Under this concentration, we found the cell activities were suppressed at4hr (p<0.05) compared with their control groups respectively. The level of ROS and apoptosis of MCs treated with50U/L glucose oxidase for4hr were increased compared with the control group.Conclusion:Primary culture of strias vascularis marginal cells can be successfully done. Meanwhile treated with50U/L glucose oxidase for4hours, an MC oxidative stress model was successfully established. Part Ⅱ Expression of and Foxo3a in Marginal Cells and Screen1v-p110α-siRNAs to Knockdown p110aObjective:The expression and the distribution of p110α and Foxo3a in marginal cells. Screen the best one of1v-p110a-siRNA from three siRNA.Methods:Using an immunofluorescence method, we detected the distribution of p110α and Foxo3a in the MCs by laser-scanning confocal microscope (LSCM). Three type of siRNA of rat p110α were designed and synthesized according to the sequence of p110α in the Genbank, named lv-p110α-siRNA-1to1v-p110α-siRNA-3, respectively,1v-p110α-siRNA-1,1v-p110α-siRNA-2,1v-p110α-siRNA-3and1v-CON(negative control scramble siRNA) were transfected into MCs respectively. Screen the effective siRNA by detecting the expression of mRNA and protein of p110α.Results:In MCs the p110α were expressed in both the cytoplasm and nucleus while Foxo3a mainly in nucleus. The expression of p110a was signigicantly decreased in the1v-p110α-RNAi-2group(p<0.05).Conclusion:The p110α and Foxo3a were expressed in MCs. the1v-p110α-RNAi-2was selected since its best effectiveness of knockdown the target gene in MCs. PartⅢ Role of p110α/Akt/Foxo3a in Marginal Cells Under Oxidative StressObjective:To investigate the role of p110α/akt/Foxo3a in marginal cells under oxidative stress.Methods:We set up four groups, as follows:group1:untransfected and untreated with glucose oxidase; group2:untransfected but with50U/L glucose oxidase for4hr; group3: transfected with1v-CON before treatment with50U/L glucose oxidase for4hr; and group4: transfected with1v-p110α-siRNA before treatment with50U/L glucose oxidase for4hr. Then, we tested the expression of p110α, p-Akt, SOD2and Foxo3a by western blot and real-time PCR. Meanwhile we test ROS and apoptosis by flow cytometry in those four groups.Results:We found that the mRNA and protein expression levels of p110α and p-akt increased in our oxidative stress model, while Foxo3a and SOD2decreased. After gene knockdown, the expression level of p110α and p-akt decreased, Foxo3a and SOD2increased in group4compared with group2and group3. Through detecting ROS and apoptosis, the levels of ROS and apoptosis decreased in group4compared with group2and3.Conclusion:Knockdown p110α might protect MCs under oxidative stress. Through the relative transcription factors, it can relive the production of ROS and apoptosis of MCs.