体外培养猪骨髓间充质干细胞hHCN2基因的转染及表达

背景：通过超极化活化的阳离子电流调制心脏自律性成为该领域研究的焦点。目的：将起搏基因质粒CMV-hHCN2-3xha-IRES-EGFP转染到猪骨髓间充质干细胞,检测其在骨髓间充质干细胞中核酸和蛋白水平是否表达。方法：采用单纯直接贴壁法或密度梯度离心结合直接贴壁法分离、纯化、增殖获得骨髓间充质干细胞,脂质体2000转染质粒CMV-hHCN2-3xha-IRES-EGFP至骨髓间充质干细胞。结果与结论：单纯直接贴壁法收集到的细胞增殖速度慢,而密度梯度离心结合直接贴壁法细胞增殖速度较快,原代细胞培养第3代后转染48h荧光显微镜下可见骨髓间充质干细胞发出荧光,对照组无荧光。细胞转染率达15%-20%。RT-PCR检测及Westernblot印迹检测证明了hHCN2基因在骨髓间充质干细胞有表达。密度梯度离心结合直接贴壁法较单纯直接贴壁法培养骨髓间充质干细胞的培养效率更高。转染目的基因hHCN2的骨髓间充质干细胞蛋白有表达。

Transfection and expression of swine bone marrow mesenchymal stem cells hHCN2 gene cultured in vitro

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.