不同方法HBx基因表达质粒转染肝癌HepG2细胞的效果比较

目的：比较Lipo2000、磷酸钙法和Lonza电转法转染乙型肝炎病毒X（HBx）基因的表达质粒pHA-HBx至HepG2肝癌细胞的转染效率及其细胞毒性。方法：分别用Lipo2000、磷酸钙法和Lonza电转法将绿色荧光蛋白（GFP）标记的pHA-HBx转染至HepG2肝癌细胞,以荧光显微镜及流式细胞仪检测转染效率,RT-PCR鉴定HBx基因在HepG2细胞基因组的整合,Annexin V/PI法检测细胞的存活情况。结果：RT-PCR结果显示,外源性HBx基因通过3种转染方法均可导入HepG2细胞并表达;Lipo2000和Lonza电转法的转染效率均高于磷酸钙法（均P<0.05）,而Lipo2000和Lonza电转法的转染效率间差异无统计学意义（P>0.05）;细胞存活率从高到低依次为Lonza电转法、Lipo2000、磷酸钙法（均P<0.05）。结论：Lonza电转法转染pHA-HBx至HepG2肝癌细胞转染效率高,且细胞毒性小,是较好的转染方法。

Comparison of different methods for HBx gene transfection into HepG2 cells

Objective: To compare the transfection efficiency and cytotoxicity among Lipo2000 reagent, calcium phosphate method, and Lonza electroporation in transfection of hepatitis B virus X (HBx) gene expression plasmid (pHA-HBx) into the HepG2 cells.Methods: Green fluorescent protein (GFP)-tagged pHA-HBx was transfected into the HepG2 cells by Lipo2000 reagent, calcium phosphate method, and Lonza electroporation-based delivery, respectively. Then, the transfection efficiency was determined by flow cytometry and fluorescence microscopy, the HBx gene integration into the genome of the HepG2 cells was identified by RT-PCR method, and the cell survival status was examined and analyzed by Annexin V/PI staining, respectively.Results: RT-PCR results indicated that exogenous HBx gene was integrated into the genome and expressed in the HepG2 cells after either of the three transfection methods. The transfection efficiency of Lipo2000 reagent and Lonza electroporation-based delivery were higher than that of calcium phosphate method (both P<0.05), but it had no statistical difference between the first two methods (P>0.05). The high-to-low order of cell survival rate was Lonza electroporation, Lipo2000 reagent and calcium phosphate method, respectively (all P<0.05).Conclusion: Lonza electroporation for pHA-HBx transfection into HepG2 cells has high transfection efficiency and less cytotoxicity, so it is a relatively satisfactory transfection method.