宫颈细胞系化学转染与电穿孔转染效率的比较与优化

Comparison and optimization of chemical and electroporation transfection e fficiencyin cervical cell lines

目的定量比较4种化学转染试剂在五株常用的宫颈(癌)细胞系中的DNA转染效率,以助于针对不同细胞系选择合适的转染试剂;同时探索电穿孔转染法在难转细胞系Ect1/E6E7、CaSki中的应用,选择最佳转染条件。方法以pcDNA3.1-EGFP为报告载体,采用FuGENE HD、jetPRIME、Lipofectamine 2000和Lipofectamine 3000四种转染试剂分别转染常用宫颈(癌)细胞系HeLa、SiHa、CaSki、C33A和Ect1/E6E7,转染24h后用倒置荧光显微镜观察记录,并采用Cellometer K2对转染效率进行定量分析。针对化学转染法难转细胞系Ect1/E6E7、CaSki,采用电穿孔转染法进行转染,优化转染条件,并对转染效率进行定量比较。结果jetPRIME和Lipofectamine 3000转染试剂在HeLa细胞中的转染效果很好,而对SiHa、C33A细胞转染效率相对较低,在CaSki、E6E7细胞中的转染效果很差。FuGENE HD转染试剂除了对HeLa、SiHa细胞转染效果较好之外,在其他3株细胞中的转染效果均不理想。Lipofectamine 2000在5株细胞中的转染效果也不理想。在穿孔电压170V、脉冲波长7ms、脉冲间隙10ms和驱动电压20V的条件下电穿孔转染法在Ect1/E6E7、CaSki细胞中的转染效率最佳且均高于化学转染法。结论针对不同的宫颈(癌)细胞系选择合适的转染方式和转染试剂可达到更高的转染效率。相对电转染,化学转染细胞存活率高,电转染更适用于难转细胞的稳定转染。

Objective To quantitatively compare the DNA transfection efficiency of four different chemical transfection reagents in 5 cervical cell lines including normal or cancer cell lines,so as to identify the appropriate transfection reagent for each cell line.In addition,to investigate the application of electroporation in the transfection of Ect1/E6E7 and CaSki cell lines,which were difficult to be transfected by chemical reagents,and then to help select the optimal transfection conditions.Methods 5 cervical cell lines including normal or cancer lines,namely HeLa,SiHa,CaSki,C33A and Ect1/E6E7,were transfected with pcDNA3.1-EGFP plasmid reporter,by using 4 liposome-based chemical reagents named FuGENE HD,jetPRIME,Lipofectamine 2000 and Lipofectamine 3000,respectively.24 hours after transfection,cells were photographed under an inverted fluorescence microscope,and then were harvested for quantitatively measure of transfection efficiency by using Cellometer K2 machine.For the 2 cell lines that were difficult to be transfected by chemical reagents,Ect1/E6E7 and CaSki,electroporation was conducted to optimize the transfection efficiency under various conditions and the transfection efficiency was quantitatively compared.Results jetPRIME and Lipofectamine 3000 reagents achieved high transfection efficiency in HeLa cells,medium efficiency in SiHa and C33A cells,but very low efficiency in CaSki and Ect1/E6E7 cells.FuGENE HD was suitable for HeLa and SiHa cells but not good for other 3 cell lines. Meanwhile, Lipofectamine 2000 was the worstchoice for all these cells. When performing electroporation, Ect1/E6E7 and CaSki both achieved the best efficiency at the condition of170V poration pulse voltage, 20V driving pulse voltage, 7ms pulse length, and 10ms pulse interval, which is much better than all above4 chemical reagents. Conclusion Higher transfection efficiency can be achieved by selecting appropriate transfection methods andtransfection reagents for different cervical (cancer) cell lines. Electroporation was an alternative method for those chemical transfectionreagents-resistant cell lines to get a better efficiency;however, chemically transfected cells have a high survival rate compared to electrotransfectedcells, therefore electroporation was usually applied for stable transfection for those chemical transfection reagents-resistantcell lines.