Abstract:

BACKGROUND: Recently, the pacemaker channel that modulates cardiac automaticity via the hyperpolarization activated caution current is the focus of this area.
OBJECTIVE: To transfer the gene constructs CMV-hHCN2-3xHA-IRES-EGFP plasmid into the swine bone marrow mesenchymal stem cells (BMSCs) and to observe the expression of nucleic acid and protein in BMSCs.
METHODS: BMSCs were separated, purified and proliferated in vitro by direct adherent or density gradient centrifugation combined with direct adherent method. The target gene CMV-hHCN2-3xHA-IRES-EGFP was transfected into BMSCs using lipofectamine 2000.
RESULTS AND CONCLUSION: The growth speed of BMSCs cultured by using density gradient centrifugation was faster than that by direct adherent method. There was fluorescence of BMSCs under fluorescence microscope after the third generation of BMSCs transfected for 48 hours, there was no fluorescence in the control group. The transfection rate of the cells was 15%-20%. The expression of human hyperpolarization-activated cyclic nucleotide gated channel 2 (hHCN2) was observed on BMSCs by RT-PCR and Western blot. The growth speed of BMSCs cultured using density gradient centrifugation combined with direct adherent was faster than that by direct adherent method. The protein overexpression of hHCN2 gene was detected.