Akt Involvement In The Self-maintenance Of C3H10T1/2 During Islet-1 Induced Cardiomyocyte-like Differentiation

Part 1 Akt involvement in the self-maintenance of in C3H10T1/2during Islet-1 induced cardiomyocyte-like differentiationObjective: To investigate whether Akt is involved in the regulation of self-maintenance of C3H10T1/2 during Islet-1 induced C3H10T1/2cardiomyocyte-like differentiation.Methods: Islet-1 was overexpressed in C3 to construct a cardiomyocyte differentiation model.RT-qPCR was used to detect the gene expression of Akt,Sox2 and Nanog;western blotting was used to detect the protein expression levels;immunofluorescence was used to examine the localization.The Akt activator SC79 and the inhibitor MK2206 were applied to C3 cells respectively,the expression of p-Akt was detected by western blotting to find their optimal concentration.C3 was cultured by the optimal concentration drugs,and RT-qPCRwas used to detect the geneexpression of Sox2 and Nanog;western blotting was used to compare the protein levels;immunofluorescence was used to detect the localization.Results: C3 developed cardiomyocyte-like differentiation after overexpressing Islet-1;during the differentiation process,Akt activity and nuclear input decreased,but the gene expression,protein expression and cell localization of Sox2 and Nanog didn't change.The optimal concentration of SC79 and MK2206 were 4ug/ml vs 1umol/L.After activation and inhibition of Akt,the gene expression,protein expression and nuclear localization of Sox2 and Nanog did not change.Conclusion: Akt does not directly regulate the self-maintenance of C3H10T1/2 during Islet-1 induced C3 cardiomyocyte-like differentiation.PART 2 Akt involvement in the acanthopanax induced osteogenic differentiation of mesenchymal stem cellsObjective: To investigate whether Akt is involved in the regulation of self-maintenance of rat bone marrow stem cells(BMSCs)osteogenesis induced by acanthopanax.Methods: BMSCs were extracted from SD rats,and identified by flow cytometry of CD45,CD29,CD90 at the third generation.Acanthopanax were added to the classical osteogenic medium at the concentration of1×10-8mol/L.After 12 days of culturing by acanthopanax and classic osteogenic medium,RT-q PCR was used to detect osteogenic-related gene expression:RUNX,OSX,BSP,OCN;western blotting was used to detect the protein level of p-Akt,Sox2 and Nanog;on the 21 th day of culturing,mineralized calcium nodules were stained with alizarin red.Results: The surface antigens of the third generation BMSCs consistent with the stem cell identification criteria.The results of RT-q PCR showed that the expression of OSX,BSP,OCN in acanthopanax group was significantly higher than that in traditional osteogenic group and negative group,P<0.05;However,the RUNX expression in acanthopanax group was higher than that in negative group,but lower than that in traditional osteogenic group,P<0.05.The alizarin red staining indicated the number of mineralized calcium nodules of acanthopanax group was higher than that of traditional osteogenic group,suggesting that the osteogenesis effect of acanthopanax group is better than traditional osteogenic group.Western Blotting suggested that the p-Akt expression was much higher after osteogenesis;while Sox2 and Nanog expression did not change.Conclusion: acanthopanax may cooperate with dexamethasone to induce the osteogenesis of BMSCs,Akt were involved in the processes.