Promotion Of Differentiating Bone Marrow Mesenchymal Stromal Cells (BMSCs) Into Cardiomyocytes Via With Pacing Function HCN2 And HCN4 Co-Transfection

Part ? Isolation,culture and induction differentiation of porcine bone marrow mesenchymal stem cells(BMSCs)Objective:To establish a method for in vitro isolation and culture of bone marrow mesenchyml stem cells(BMSCs),and conduct induction and differentiation,so as to explore the optimal sorting process of BMSCs and clarify the biological characteristics of BMSCs.Methods:After the porcine bone marrow was obtained,monocytes were isolated from the porcine bone marrow by erythrocyte lysis and immunomagnetic bead separation,respectively,and cultured.The bone marrow mesenchymal stem cells were detected by flow cytometry.The collected cells were divided into BMSC induction group and control group,and the bone marrow mesenchymal stem cells were transferred to the third generation.BMSC-induced group was treated with different induction methods and myocardial cell induction solution was added for 14-21 days.After the induction,the cell morphology of each group was observed.Results:Porcine bone marrow was collected by bone marrow aspiration for the isolation of bone marrow mesenchymal stem cells.Flow cytometry showed negative expression of CD45,CD11b and positive expression of CD90 in BMSCs.The results showed that the cultured porcine bone marrow stem cells accorded with the characteristics of stem cells.Conclusion:MSCs can be induced to differentiate into neuron-like cells,cardio-like cells and adipo-like cells,which confirms that MSCs has the characteristic of triple dermal cell differentiation,which lays the foundation for further induction.Part ? Effects of HCN2 and HCN4 on differentiation of bone marrow mesenchymal stem cells into cardiomyocytesObjective:Pig built up with bone marrow mesenchymal stem cells(MSCs)into myocardial cells between directional differentiation system,research of the construction of the myocardial cell sample with pacemaker current method,and the pacemaker channels of transfection cell protein expression and the If current dynamic characteristic testing,explore engineered and HCN4 between bone marrow mesenchymal stromal cells(BMSCs)to the effect of myocardial cell differentiation.To obtain the induction mechanism and method of stable and persistent cardiomyocytes.Methods:Bone marrow was extracted and bone marrow mesenchymal stem cells were isolated from miniature adult pigs.Cell surface antigens CD45,CD11b and CD90 were detected by flow cytometry.The HCN2 and HCN4 genes were co-transfected with BMSCs in the HCN2+HCN4 group,and BMSCs differentiation was induced by myocardial induction solution in the BMSCs induction group.Myocardial markers?-actin and cTnT were detected by immunofluorescence staining,and the protein expressions of?-actin,cTnT and Desmin were detected by Western blot.Whole-cell patch clamp technique was used to identify and detect the current activation curves of HCN2 and HCN4 channels and the inhibitory effect of CSCL on heterologous current expression.Results:Flow cytometry was used to detect negative expression of CD45,CD11b and positive expression of CD90.Immunofluorescence staining and Western blot results showed that the expressions of HCN2,HCN4,?-actin and cTnT were significantly increased in the HCN2+HCN4 group,which could be compared with those in the BMSC-induced group.The HCN2+HCN4 group can record the ionic current of the membrane channel similar to that of IF.Conclusion:The overexpression of HCN2 and HCN4 can significantly enhance the ability of MSCs to differentiate into cardiomyocytes in vitro,and the induced cardiomyocytes have pace-like ion current.Part ? Study on HCN1?HCN2 and HCN4 transfected BMSCs induced into pacemaker-like current cellsObjective:To study the construction method of pace-like cells,and to detect the expression of pace-like channel proteins and IF current dynamics characteristics of the transfected cells,we expect to obtain stable and durable pace-like cells.Methods:Biopacing target genes HCN1,HCN2 and HCN4 were transfected into porcine MSCs,respectively,and the transfected MSCs were induced into cells with initiation current.Results:If current can be detected successfully by whole cell patch clamp in transfected cells,which lays the experimental foundation for the establishment of a new and effective cell biological pacing technique.Conclusion:BMSCs transfected with HCN1,HCN2 and HCN4 genes could improve the induction and differentiation efficiency of pace-like cells.