RPS20 miRNA质粒载体的CT26细胞转染条件优化研究  

Optimization of Liposome-mediated Transfection of CT26 Cells Based on Functional Identification of Pi-deficiency Syndrome Relative Gene RPS20

作　　者：高小玲[1] 李茹柳[1] 郭文峰[1] 徐颂芬[1] 胡灿[1] 陈蔚文[1,2] 

机构地区：[1]广州中医药大学脾胃研究所,广州510405 [2]上海市高校中医内科学E-研究院,上海中医药大学,上海201203

出　　处：《中药新药与临床药理》2010年第3期318-322,共5页Traditional Chinese Drug Research and Clinical Pharmacology

基　　金：国家自然科学基金项目(90209004;90809001);上海市教育委员会E-研究院建设计划项目(E03008)

摘　　要：目的探讨阳离子脂质体法转染CT26细胞适宜的转染条件,为小鼠体内以RNA干扰法进行脾虚证相关基因RPS20的功能鉴定提供参考。方法以6孔培养板培养CT26细胞,利用阳离子脂质体转染pcDNATM6.2-GW/EmGFP-miR-neg Control plasmid进入CT26细胞,荧光显微镜、流式细胞仪两种方式观察不同剂量的质粒和转染试剂对转染效率的影响。结果荧光显微镜下细胞计数法检测转染效率,4.0μg和6.0μg质粒的转染效率无明显差异;流式细胞仪检测转染效率,以Lipofectamine2000 15.0μL+质粒4μg转染效率最高,平均为25.3%。结论 6孔培养板,4.0μg质粒+15.0 μL Lipofectamine2000为CT26细胞优化的转染条件。Objective To investigate the optimum transfection conditions of cationic liposome transfected CT26 cells（murine colon cancer cells）,thus to provide reference for functional identification of spleen deficiency-related gene RPS20 using RNA interfering methods in mice.Methods We transfected pcDNATM6.2-GW/EmGFP-miR-neg Control plasmid into CT26 cells which planted in six-well plate with cationic liposome,and observed the effect of plasmid and Lipofectamine2000 in various proportions on transfection efficiency by detecting fluorescence expression using fluorescence microscopy and flow cytometry.Results The results of detecting transfection efficiency by fluorescence microscope showed that plasmid 4.0μg and 6.0μg had no significant difference in transfection efficiency.The results of flow cytometry showed that the highest transfect efficiency（average being 25.3%）was obtained in the group of Lipofectamine2000 15.0μL＋plasmid 4.0μg.Conclusion The optimum transfection condition is tranfection into CT26 cells with plasmid 4.0μg＋Lipofectamine2000 15.0μL in six-well plate.

关 键 词：RNA干扰 CT26细胞 转染条件 

分 类 号：R342.3[医药卫生—基础医学]