In Vitro Transfection And Expression Of HHCN2 Gene Swine Mesenchymal Stem Cells

Background:The sinoatrial (SA) node dysfunction or atrial-ventricular (AV) conduction block causes improper heart rate (bradycardia). If such dysfunction is severe enough, it could be treated by implanting an electronic pacemaker, but still there are some limitations and inadequacies. Recently, progress in developing engineered cardiac biopacemakers with use of stem cells culturing and gene engineering has been made in experimental animal models. Especially, the hyperpolarization-activated cyclic nucleotide gated channel (HCN), the pacemaker channel that modulates cardiac automaticity via the hyperpolarization activated caution current (Ih) is the focus of this area. Objective:Two methods were applied separately, purify and proliferate the bone marrow mesenchymal stem cell (MSCs) which derived from the southern small swine of Yunnan province. One method is direct cell adherent, and the other one is direct cell adherent after density gradient centrifugation. Human HCN2 gene construct CMV-hHCN2-3xHA-IRES-EGFP plasmid was transferred into swine MSCs, and the expression of hHCN2 mRNA and protein in transfected MSCs were detected. Methods:MSCs were obtained in vitro by direct adherent or density gradient centrifugation method. The shapes of cells were observed through the microscope and its growth curves were measured. The plasmid that carried pacemaker gene was isolated and it's sequence was confirmed by DNA sequencing. The target gene was transfected into MSCs by using Lipofectamine 2000. The transfection efficiency was observed by fluorescence microscope. The expression of hHCN2 in the transfected cells was detected by RT-PCR and western blot. Results:The isolated MSCs cells proliferated slowly by direct adherent method, and the speed of that is faster than that of using density gradient centrifugation protocol. After three to four generations culture all of these cells grew into spindle shapes. DNA sequencing confirmed that hHCN2 correctly inserted into the plasmid construct. After three generation primary cells culture, the green fluorescence could be seen in transfected MSCs. Cell transfection efficiency reached to 15-20%. The overexpression of hHCN2 mRNA detected by RT-PCR, while protein of hHCN2 was detected by western blot. Conclusion:The growth speed of MSCs cultured by using density gradient centrifugation is faster than that of by using direct adherent. The protein overexpression of target gene was detected. These findings demonstrated that hHCN2 can be successfully delivered by Lipofectamine and was expressed in MSCs, which provided the materials for further studying of the biologic pacemakers.