Yu Fengxu, Fang Yibing, Liu Xuan, Lai Qiancheng, Jia Jianbo, Liao Bin
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2013 Dec;27(12):1512-6.
To investigate the feasibility of recombinant lentivirus (LVs) mediated hyperpolarization-activated cyclic nucleotide-gated cation channel 4 (HCN4) gene transfecting rat bone mesenchymal stem cells (BMSCs) so as to construct the biological pacemaker cells.
Sprague Dawley rats at the age of 3-5 weeks were selected to isolate and culture BMSCs using modified whole bone marrow adherent culture method. LVs was used as carrier, and enhanced green fluorescent protein (EGFP) as marker to build LVs-HCN4-EGFP virus liquid. The BMSCs at passage 3 were transfected with LVs-HCN4-EGFP virus liquid (experimental group) and LVs-EGFP null virus liquid (control group). Fluorescence microscope was used to observe the green fluorescent protein expression after 24, 48, and 72 hours of transfection; Western blot method was used to detect the HCN4 protein expression. The electrophysiology was used to detect the pacemaker current in the experimental group.
After transfection, BMSCs in the experimental group showed normal morphology and good growth; scattered green fluorescence could be seen at 48 hours under fluorescence microscope, with a transfection efficiency of about 10%; the fluorescence expression increased slightly, with the transfection efficiency of 20% to 25% at 72 hours. While no expression of green fluorescence was seen in the control group. Western blot results showed that the same band expression as a relative molecular mass of HCN4 protein were found at 72 hours after transfection in the experimental group, only weak expression of protein band was seen in the control group; the gray value of the experimental group (33.75 +/- 0.41) was significantly higher than that of the control group (23.39 +/- 0.33) (t=17.524, P=0.013). In the experimental group, the pacemaker current was recorded, and it could be blocked by CsCl, in accordance with the characteristics of pacemaker current.
The recombinant LVs mediated HCN4 gene is successfully transfected into rat BMSCs, and the expression of HCN4 protein and the pacemaker current can be detected.
探讨重组慢病毒（LVs）介导超极化激活环核苷酸门控阳离子通道4（HCN4）基因转染大鼠骨髓间充质干细胞（BMSCs）以构建生物起搏器细胞的可行性。
选取3 - 5周龄的Sprague Dawley大鼠，采用改良全骨髓贴壁培养法分离培养BMSCs。以LVs为载体，增强型绿色荧光蛋白（EGFP）为标记构建LVs - HCN4 - EGFP病毒液。将第3代BMSCs分别用LVs - HCN4 - EGFP病毒液（实验组）和LVs - EGFP空病毒液（对照组）进行转染。转染24、48和72小时后，用荧光显微镜观察绿色荧光蛋白表达情况；采用蛋白质免疫印迹法检测HCN4蛋白表达。运用电生理学方法检测实验组的起搏电流。
转染后，实验组BMSCs形态正常，生长良好；荧光显微镜下48小时可见散在绿色荧光，转染效率约为10%；72小时荧光表达略有增加，转染效率为20% - 25%。而对照组未见绿色荧光表达。蛋白质免疫印迹结果显示，实验组转染72小时后出现与HCN4蛋白相对分子质量相同的条带表达，对照组仅见蛋白条带微弱表达；实验组灰度值（33.75±0.41）明显高于对照组（23.39±0.33）（t = 17.524，P = 0.013）。实验组记录到起搏电流，且可被CsCl阻断，符合起搏电流特征。
重组LVs介导的HCN4基因成功转染入大鼠BMSCs，且可检测到HCN4蛋白表达及起搏电流。
