The Research Of Lentivirus-mediated Gene HCN₄ Transfect Bone Mesenchymal Stem Cells

Abstract:Objective:To investigate the feasibility of production of the biological pacemaker cells from rat bone marrow mesenchymal stem cells (BMSCs), by transfection with hyperpolarization-activated cyclic nucleotide gated cation channels 4 (HCN4) gene. Methods:1. Separation, purification and cultivation of BMSCs:BMSCs were isolated and puried by the modified method of whole bone marrow adherent culture and cultured in DMEM medium with 20% fetal bovine serum (FBS). The cells were serial subcultured, 3rd～5th generation cells were used in the experiments.2. Detection of HCN4-EGFP fusion gene expression in human embryonic kidney epithelial cell (HEK293T):recombined plasmid (LVs-HCN4-EGFP) was transfected into HEK293T with LipofectamineTM 2000. The expression of fusion gene was detected by enhanced green fluorescent protein (EGFP) as a tracer gene.3. Recombined lentivirus particles of LVs-HCN4-EGFP were packaged in HEK293T cells, with plasmid and lentiviral packaging mixture (pHelper1.0 plasmid and pHelper2.0 plasmid).4. The HEK293T cells were infected with lentiviral particles containing HCN4 to verify the reliability of the lentiviral vector:HCN4 gene was adopted as target gene of biological pacemaker, and EGFP gene as a tracer gene. The HEK293T cells were transfected with HCN4 lentiviral vector (LVs-HCN4-EGFP) and the control vector (LVs-EGFP) respectively, and EGFP expression was observed in transfected HEK293T cells. 5. Establishment of HCN4 stable transfection HEK293T cells line by G418 selection, and expression of HCN4 protein in transfected HEK293T cells was detected by Western blot. HCN4 channel current (If) in transfected HEK293T cells was recorded with whole cell patch clamp experiments.6. The HCN4 lentiviral vector (LVs-HCN4-EGFP) and the control vector (LVs-EGFP) respectively transfected BMSCs. The expression of HCN4 protein in transfected BMSCs cells was detected by Western blot. The whole cell patch clamp experiment with transfected BMSCs is being performed. Results:1. BMSCs with high purity were obtained by the modified method of whole bone marrow adherent culture.2. The recombined plasmid (LVs-HCN4-EGFP) was successfully transfected into HEK293T with LipofectamineTM 2000. After 24 hours culturing, a large number of EGFP (green) was detected by the fluorescence microscope.3. HCN4 expression lentivirus (LVs-HCN4-EGFP) was successfully constructed.4.48 hours after transfection of LVs-HCN4-EGFP, strong EGFP expression in HEK293T cells was observed with the fluorescence microscope. The efficiency of transfection was as high as 80～90%. Then, we established HCN4 stable transfection HEK293T cell lines with G418 selection.5. Western blot analysis suggested HCN4 expression in HCN4 stable transfection HEK293 cells was increased significantly, compared with control group (with null vector transfection). The whole cell patch clamp recorded an inward current in response to hyperpolarization which was time-dependent and voltage-dependent. The maximum activation voltage is about-100 mV. The was block by specific blocking agent 7288.6. After 24hrs after infection of LVs-HCN4-EGFP lentiviral vector, BMSCs were also found EGFP positive, although efficiency of infection was only 10-20%, much lower than that in HEK293 cells. Western blot analysis suggested that HCN4 expression was increased significantly in HCN4 lentivirus infected BMSCs, compared with control group (null vector infected BMSCs). The whole cell patch clamp with HCN4 lentivirus infected BMSCs is to be performed in following study. Conclusion:1. Expression of HCN4 channel protein in HEK293T cells was increased significantly by transfection of HCN4 lentivirus, and If current is recorded on the HEK293T cells. It suggested that some electrophysiological character of pacemaker cells (If current) appeared in HEK293T cells, resulted from overexpression of HCN4 gene by HCN4 lentivirus transfection.2. HCN4 protein expression was also increased in BMSCs by lentivirus-mediated transfection of HCN4. It is postulated that If current will be created in BMSCs by HCN4 gene transfection, although it is needed to be verified with cell patch clamp study.