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。
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 cytomctry 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.
出处
《广州中医药大学学报》
CAS
2009年第6期598-602,616,共6页
Journal of Guangzhou University of Traditional Chinese Medicine
基金
国家自然科学基金项目(编号:90809001)
上海市教育委员会E-研究院建设计划项目(编号:E03008)
