大鼠血管内皮细胞siRNA转染条件的优化

目的:旨在优化siRNA转染大鼠血管内皮细胞的转染条件.方法:将0.75μL和1.5μL的脂质体LipofectamineTM3000分别与20、40、60、80nmol带荧光标记的FAMsiRNA组合,转染6、12、18、24h后,用荧光显微镜计数阳性细胞率、MTT法检测各浓度条件下内皮细胞的存活率,筛选最优转染条件.结果:(1)转染12h后,用荧光显微镜检测20、40、60、80nmol各组,均可观察到绿色荧光(2)siRNA浓度为60nmol/L,脂质体为1.5μL的组合转染效率最高,继续增加siRNA的浓度,转染效率提高不明显.(3)转染时间超过24h,各组细胞荧光减弱,细胞死亡率显著增加.结论:结果表明,以1.5μL LipofectamineTM3000与60nmol/L的siRNA浓度组成转染混合物转染12h可以实现对大鼠血管内皮细胞高效转染并保持较高的细胞活性.

Optimization of Transfection Conditions for Tranfecting siRNA to Rats Vascular Endothelial Cells

Objective:This work aims to optimize the transfection parameters for transfecting siRNA(small interfering RNA)to rats vascular endothelial cells(EC).Methods:Using the 0.75μL and 1.5μL of LipofectamineTM3000 diluted with FAM-siRNA(20nmol、40nmol、60nmol、80nmol)were mixed with different concentrations of fluorescin-labeled siRNA to form a series of mixtures to transfect EC cells.At the time point of 6h,12 h,18h,24 hafter transfection,under fluorescent microscope to observe and determine the percentage of the cells transfected under each condition.The cell viability was determined by using MTT assay to achieve the optimal conditions.Results:(1)Green fluorescence was discovered under the fluorescence microscope after transfection 12 h.(2)The highest transfection efficiency can be obtained in 60nmol/L siRNA with the1.5μL of Lipofectamine transfected EC cells.No differences were found in transfection efficiency and cell viability whether using the higher concentration of siRNA.(3)Twenty-four hours later,the transfection efficiency could not be further increased by elevating the concentration of siRNA,and the cell death rate increased significantly.Conclusions:The results showed that the transfection efficiency and cell viability are dependent on the correct LipofectamineTM3000(1.5μL)-siRNA(60nmol/L)ratio after transfection 12 h.