大鼠血管内皮细胞siRNA转染条件的优化

摘要:

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.

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.

作者:

郭昭涵 李银河 庞振凌 彭明星 黄冉涛 王燕华

机构地区:

南阳师范学院生命科学与技术学院

出处:

《Betway官方客服学报：自然科学版》 CAS 北大核心  2015年第6期107-111,共5页

基金:

国家自然科学基金项目(11101092) 国家自然科学基金(81260266) 河南省教育厅自然科学基础研究项目(12B130003) 南阳师范学院高层次人才科研启动项目(zx2013025)

关键词:

血管内皮细胞 脂质体 SIRNA 转染

Liposome siRNA transfection endothelial cell

分类号:

Q291 [生物学—细胞生物学] 

大鼠血管内皮细胞siRNA转染条件的优化.pdf