Discussion The Role Of Akt During Islet-1 Inducing C3H10T1/2 Cells To Differentiate Specifically Into Cardiomyocytes

ObjectiveMesenchymal stem cells of C3H10T1/2 cells were infected by lentiviral vector delivered with over-expressed Islet-1,which could induce C3H10T1/2 cells to differentiate into cardiomyocyte-like cells specifically.On this condition,through affect the activity of Akt and detect the change of myocardial early development related genes,in order to study the effect and mechanism of Akt during Islet-1 inducing C3H10T1/2 cells to differentiate into cardiomyocytes specifically.Methods1.Mesenchymal stem cells of C3H10T1/2 cells were plated in culture bottle at the density of 5×104/T13.training to cell alignment add to sixty or seventy percent,add lentiviral vector delivered with GFP and over-expressed Islet-1 into each bottle.After a special time,lentivirus transfection efficiency of cells was detected by flow cytometry detection technology;The protein expressions of Islet-1,cTnT and p-Akt/T-Akt were measured by Western-bloting and immunofluorescence.2.C3H10T1/2 cells infected by lentiviral with over-expressed Islet-1were dealed with Akt special inhibitors of MK-2206,The effect of MK-2206 on cell proliferation was tested by CCK-8 and the activity of Akt was detected by WB;The mRNA expression of GATA4,Nkx2.5 and Mef2 c,which were derived from C3H10T1/2 cells infected Lv as well as both infected Lv and dealed with MK-2206,was tested by real-time PCR.Results1.When C3H10T1/2 cells infected by lentiviral vector delivered with GFP and over-expressed Islet-1 were cultured ninty-six hours,the cells expressing GFP stably were ninty percent by fluorescence microscopy.The Lv transfection efficiency of experimental and negative group were 94.7%and 89.7%respectively.Immuno-fluorescence and Western-bloting could detect the expression of Islet-1 and c TnT protein.2.The results of CCK-8 and Western blot showed that,with the increasing of MK-2206 drug concentration,the inhibition rate of cell proliferation also increased,and the best inhibition concentration on the activity of Akt was 8 nm/L;With prolongation of time during Islet-1inducing C3H10T1/2 to differentiate into cardiomyocytes specifically,the protein expression of p-Akt/T-Akt gradually reduced,and the lowest was at the third week,then began to rise(*P<0.05);The gene expression of myocardial specific transcription factor GATA4,Nkx2.5 and Mef2 c gradually increased at the first three week,then began to decrease(*P<0.05);But,treated with MK-2206,the gene expression of these briefly increased significantly at the first week and then gradually reduced.Conclusions1.During Islet-1 inducing the Mesenchymal stem cells of C3H10T1/2cells to differentiate specifically into cardiomyocytes,Islet-1 could act on Akt to regulate C3H10T1/2 cells to differentiate into cardiomyocytes.2.During the Islet-1 inducing the C3H10T1/2 cells into cardiac-specific differentiation process,Akt inhibits to differentiate in the start-up phase of early differentiation,then promotes to the action of differentiation.