Hho-1 Combined With GATA-4 Transduction Promotes Myocardial Transdifferentiation And Anti-apoptosis Of Mesenchymal Stem Cells

BackgroundAcute myocardial infarction is a serious threat to people’s health and lives of the elderly.In recent years,studies have shown that bone marrow mesenchymal stem cells(BMSCs)transplantation have huge potential for cardiomyocyte repair and regeneration,cardiac function recovery.However,the survival rate of transplanted BMSCs in the ischemia hypoxia microenvironment of the host is too low and the rate of myocardial transdifferentiation is also poor,which limits its curative effect.The combination of cell therapy and gene modification provides a new strategy for the repair of damaged cardiac tissue and improve the outcome of transplantation.Heme oxygenase-1(hHO-1)has a good anti-oxidation and anti-apoptotic effect.GATA-4 is an important transcription factor in the differentiation and development of heart cells.GATA-4 can enhance the protective factors of paracrine cells and up-regulate the expression of cardiomyocyte-specific genes and promote the differentiation of BMSCs into cardiomyocytes.The effect of single gene modified BMSCs is low efficiency because of various factors affecting the cell transplantation curative effect.In this study,hHO-1 combined with GATA-4 modified rat BMSCs were took to hypoxia serum-free conditions simulated ischemia hypoxia microenvironment in vivo to explore it was superior to the hHO-1 or GATA-4 single gene modified rat BMSCs.ObjectiveTo explore the hHO-1 gene combined with GATA-4 gene modified rat bone marrow mesenchymal stem cells(BMSCs)tolerance to hypoxia ischemic injury and Myocardial TransdifferentiationMethods(1)BMSCs were extracted from the bone marrow of SPF Sprague Dawley rats,using the whole bone marrow adherence method to isolate and culture BMSCs in vitro;the cultured cell was identified by committed differentiation in adipogenic medium and osteogenic medium as well as detect the cell surface antigen by flow cytometry.(2)hHO-1 gene and GATA-4 gene recombinant adenovirus transfected BMSCs;Western blot to determinate the optimal time of gene expression.(3)The genetically modified rat BMSCs were took to hypoxia serum-free conditions simulated ischemia hypoxia microenvironment in vivo;using CCK-8 kit and trypan blue staining to detect the 12,24,48,72h survival rates in hypoxia ischemia respectively;flow cytometry were used to detect apoptosis in hypoxia ischemia for 24h.(4)cardiomyocyte-specific protein cardiac troponin I(cTnI)were detected by Western blot and cellular immunofluorescence.Results(1)The BMSCs isolated and cultured in vitro is high purity with great proliferation ability.The results of flow cytometry showed the third generation cells have specific surface markers of BMSCs:The positive rate of CD29 was 98.0%、CD45 was 0.5%and CD90 was 99.2%.mineralized nodule formation is visible by alizarin red staining after 3 weeks of osteogenic induction;lipid droplets is visible by oil red O staining after 3 weeks of adipogenic induction,.All thoes results show that rat BMSCs was successfully cultured and amplified in vitro.(2)Western blotting showed that hHO-1 protein expression increased with time and reached the maximum at day 4.The expression of target in GATA-4 protein group increased with time and reached the maximum at day 5;hHO-1 + GATA-4 group,and reached the maximum at the 4th to 5th day.(3)The results of trypan blue staining showed that the survival rates of the hHO-1+GATA-4 group after 12,24,48 and 72h culture with Ischemia and hypoxia were higher than the hHO-1 group(P<0.05)and the GATA-4 group(P<0.05).The results of CCK-8 showed that the survival rates of the hHO-1+GATA-4 group after 12,24,48 and 72h culture with Ischemia and hypoxia were higher than the hHO-1 group(P<0.05)and the GATA-4 group(P<0.05).After 24h culture with Ischemia hypoxia,detected by flow cytometry,the survival rates of the hHO-1+GATA-4 group were higher than the hHO-1 group(P<0.05)and the GATA-4 group(P<0.05).(4)Western blotting showed that there was no expression of cTnI in control group,but cTnI expression in GATA-4 group and hHO-1 + GATA-4 group,but there was no significant difference between the two groups.Cell immunofluorescence The positive rate of cTnI positive cells in GATA-4 group was 31.03%± 1.91%,the positive rate of cTnI positive cells in hHO-1 + GATA-4 group was 31.47%±1.93%,There was no significant difference in cTnI expression between the GATA-4 group and the hHO-1+GATA-4 group.ConclusionCompared with transfection of hHO-1 or GATA-4 single gene,hHO-1 combined with GATA-4 transduction can increased BMSCs survival in hypoxia ischemic significantly,but myocardial transdifferentiation does not increase significantly.