Study Of The Interraction Between Akt And Islet-1 In Gcn5 Protein Complex During C3H10T1/2 Cells Specific Differentiation Into Cardiomyocytes

Objective:The mouse marrow mesenchymal stem cells C3H10T1/2was infected with the lentiviral vector containing Islet-1 gene and induce their specific differentiation into cardiomyocyte-like cells.Under these conditions,the changes in the binding of GCN5 and Islet-1 protein complex were investigated CoIP,to investigate the the interaction between Akt and Islet-1 in GCN5 protein complex during C3H10T1/2 cells specificdifferentiation into cardiomyocytes,and to further clarify the regulatory mechanism of differentiation of mesenchymal stem cells(C3H10T1/2).Methods:1.The well-formed mesenchymal stem cells C3H10T1/2 are spread in culture flasks at a density of 5×10~4 cells/vial,using the lentivirus carrying the GFP or Islet-1 gene infected cells until the cell alignment add to 20 to30%.After 96 hours,the cell infection rate was detected by flow cytometry;the protein levels and localization of Islet-1,cTnT,and Cx43 after infection were detected by Western blot and immunofluorescence;the morphological changes after infection were observed by inverted microscope and fluorescence microscope,the qRT-PCR techniques were used to detect the sequential mRNA expression of myocardial specific genes GATA4,Nkx2.5,and Mef2c.2.CCK-8 and Western blot results showed that the optimal activation concentration of IGF-1 was 220 ng/mL,and the optimal inhibitory concentration of MK-2206 was 15 nmol/L,and the drug had no effect on the protein levels of Islet-1 and GCN5.CO-IP results showed that the binding of Islet-1 to GCN5 was lower or higher than that of the untreated group when activated or inhibited by Akt(*P<0.05).Results:1.When C3H10T1/2 cells infected by lentiviral vector delivered with GFP and over-expressed Islet-1 were cultured 36 h,the cells expressing GFP stably were ninty percent by fluorescence microscopy.The Lv transfection efficiency of experimental and negative group were 96.7%and91.7%respectively.The morphology of infected Islet-1 cells was observed by inverted microscope and fluorescence microscopy.The expression of Islet-1,cTnT and Cx43 after infection was higher than that of negative controlgroupandblankgroupbyimmunofluorescenceand Western-bloting(*P<0.05).2.CCK-8 and Western-bloting results showed that the optimal activation concentration of IGF-1 was 220 ng/mL,and the optimal inhibitory concentration of MK-2206 was 15 nmol/L.Moreover,Islet-1expression was the highest at the 3rd week of cell culture,which is the optimal time for Islet-1 protein acquisition.The binding capacity of Islet-1and GCN5 was lower and higher than that of the untreated group under the situation of activation and inhibition of Akt,respectively(P<0.05).Conclusion:1.Successfully constructed the Islet-1 overexpression model of mesenchymal stem cell C3H10T1/2,and the Islet-1 gene-infected stem cells expressed the characteristics of cardiomyocyte-like cells,confirming that Islet-1 can induce mesenchymal stem cells to differentiate into cardiomyocyte-like cells.2.When Islet-1 promotes cells into cardiac-specific differentiation process,the activity of Akt is antagonistic to the protein binding capacity of Islet-1 and GCN5 during induction of mesenchymal stem cell C3H10T1/2into cardiomyocyte cells.