优化阳离子脂质体介导的组织因子小干扰RNA转染HEK-293的转染条件

OPTIMIZATION OF TRANSFECTION CONDITIONS FOR TRANFECTING TF SIRNA TO HEK-293 CELLS MEDIATED BY CATIONIC LIPOSOME

目的:优化lipofectamine^(TM)2000介导的组织因子(TF)小干扰RNA(siRNA)的转染条件。方法:化学合成法合成特异的TFsiRNA,利用绿色荧光蛋白(GFP)进行标记,分别将1.0μL和1.5μL的lipofectamineTM2000与含有20、30、40、50、60pmol的TFsiRNA混合液制备成相应的转染混合物,转染人胚胎肾细胞株(HKE-293)24h后,在荧光显微镜下计数阳性细胞率。同时,四甲基偶氮唑盐(MTT)法检测每组细胞活性。结果:转染效率和细胞活性与lipofectamineTM2000以及siRNA有明显的交互作用,在1.0μL的lipofectamineTM2000和50pmolsiRNA转染HEK-293细胞时,细胞转染效率最高,转染效率达(86.5±2.4)%,在1.5μL的lipofectamineTM2000转染40pmolsiRNA时,细胞活性为(86.0±7.8)%。结论:经优化转染条件后的li-pofectamineTM2000可高效的将化学合成的siRNA转染入HKE-293细胞株,且细胞活性达85%以上,为建立稳定的沉默转染体系提供实验基础。

Objective：To optimize the transfection condition of tissue factor small interfering RNA（siRNA） transfected by lipofectamine^TM2000 in H EK-293（human embryonic kidney cell）. Methods. 1.0μL and 1.5 μL of Lipofectamine^TM2000 diluted with DMEM were mixed with different concentrations of fluorescin-labeled siRNA to form a group of mixtures to transfect Z93 cells. After 24 h transfection, the positive ceils were calculated to determine the percentage of the transfected under fluorescent microscope under each condition. The cell viability was test by using MTT assay at the same time. Results：No differences were found in transfection efficiency and cell viability whether using 1.0 μL or 1.5 μL Lipofectamine^TM2000, if the amount of siRNA was fixed. When siRNA was 50 pmol, the percentage of positive cells was as high as 80% The transfection efficiency could not be further increased by elevating the concentration of siRNA, and 60 pmol siRNA would bring about higher cytotoxicity. Conclusion： The transfection efficiency and cell viability are dependent on the lipofectamine^TM2000 and siRNA.