【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。更多还原

【Abstract】 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.更多还原