【摘要】 【目的】探索影响大鼠小肠上皮细胞(IEC-6)RNA干扰效率的转染条件,为进一步以RNA干扰(RNAi)技术构建不同程度表达靶蛋白的细胞模型打下基础。【方法】以24孔培养板培养IEC-6细胞,利用阳离子脂质体转染FAM-siRNA片段进入IEC-6细胞,采用荧光显微镜和流式细胞仪检测FAM-siRNA和转染试剂Lipofectamine 2000不同剂量比例对转染效率的影响。【结果】(1)采用荧光显微镜检测转染效率,以评分进行秩和检验,结果仅有转染试剂或仅有FAM-siRNA或两者均无的组别评分均为0,转染试剂加FAM-siRNA组别均有或强或弱的荧光表达。(2)采用流式细胞仪检测转染效率,以Lipofectamine 2000 1.0μL+FAM-siRNA 160 nmol/L组转染效率最高(平均76.3%),而空白对照组约为1%。【结论】FAM-siRNA160 nmol/L(20μmol/L、4.8μL,终体积为0.6 mL)+Lipofectamine 2000 1.0μL组合为IEC-6细胞RNA干扰时转染效率最高的转染条件。更多还原

【Abstract】 Objective To investigate the transfection condition for affecting RNA interference efficiency on rat intestinal epithelial cells(IEC-6) cells,and to lay a foundation for the further research of constructing cell model of target protein different expression by the method of RNA interference.Methods IEC-6 were cultured in 24-well plate.With the help of cation liposome,the transfection of FAM-siRNA fragment into ICE-6 was carried out,and the effect of FAM-siRNA and Lipofectamine 2000 in different proportions on the transfection efficiency was examined with fluorescence microscope and flow cytometry.Results(1)The results of scoring of transfection efficiency under fluorescence microscope showed that the score was zero when Lipofectamine 2000 or FAM-siRNA was used alone,or both of them missed,but there was weak or strong fluorescence expression when Lipofectamine 2000 and FAM-siRNA used together.(2) The results of detecting transfection efficiency by flow cytometry showed that the transfect efficiency was the highest(76.3% on average) in the group of Lipofectamine 2000 1.0 μL+FAM-siRNA 160 nmol/L,while was only 1% in the blank group.Conclusion Under the transfection condition of FAM-siRNA 160 nmol/L(20 μmol/L,4.8 μL)+ Lipofectamine 2000 1.0 μL,IEC-6 has the highest transfection efficiency during RNA interference.更多还原