化学合成siRNA转染人羊膜的WISH细胞的效率检测

Chemosynthesis siRNA transfection efficiency in human amnionic WISH cells

目的:检索中国期刊全文数据库2005/2008相关文献显示,RNA干扰研究中,转染效率的高低直接决定siRNA的表达效果,目前关于应用小分子干扰RNA对人羊膜WISH细胞转染效率的检测研究少见。实验应用化学合成siRNA转染人羊膜WISH细胞,通过流式细胞仪检测转染效率,并初步检测转染对细胞凋亡的影响。方法:实验于2007-03/12在广州医学院试验医学研究中心完成。①实验材料:实验中应用的人羊膜WISH细胞株购于中国科学院上海生命科学研究所。②实验方法:应用0,5,10,20,30nmol/L化学合成的CY3-Negative siRNA转染人羊膜WISH细胞,转染后24h应用荧光显微镜和流式细胞仪定性、定量评估其转染效率;20nmol/L CY3-Negative-siRNA转染细胞24h后,应用Annexin V-EGFP试剂盒检测细胞早期凋亡情况。结果:荧光显微镜显示,5,10,20,30nmol/L CY3-Negative siRNA转染细胞内均可见红色荧光,对照组无荧光信号。20,30nmol/L CY3-Negative siRNA的转染效率明显高于5,10nmol/L组(P<0.05);20nmol/L和30nmol/L siRNA组转染效率差异不显著(P>0.05)。检测20nmol/L Negative siRNA转染细胞24h后的细胞早期凋亡情况,转染组与对照组细胞早期凋亡率分别为1.96%和2.36%,两组之间细胞早期凋亡率差异不显著。结论:①化学合成的siRNA可以成功转染人羊膜WISH细胞,转染后对细胞的早期凋亡率无影响。②20nmol/L的siRNA即可获得理想的转染效果。

AIM： Researching the pertinent literature in China Journal Full-text Database （CJFD） published between 2005 and 2008 indicated that the inhibitory effect of small interference RNA （siRNA） was determined by the transfection efficiency in RNA interference. At present, there are few reports about detection of the transfection efficiency for human WISH cells with chemosynthesis siRNA. In this study, we use the flow cytometer detected the transfection efficiency and the effect on apoptosis of WISH cells after chemosynthesis siRNA transfected human WISH cells. METHODS： The experiment was performed at the Experimental Medical Research Center of Guangzhou Medical College from March to December 2007. ①WISH cells were bought from Shanghai Life Science Academy of Chinese Academy Of Sciences. ② WISH cell line was transfected with CY3-Negative siRNA （0, 5, 10, 20, 30 nmol/L）. Twenty-four hours later, transfection efficiencies of different protocols were evaluated by fluorescent microscopy and flow cytometer. Apoptosis of WISH cells was detected with Annexin V-EGFP kit after 24-hours 20 nmol/L CY3-Negative-siRNA transfection. RESULTS： Red fluorescence was discovered under the fluorescence microscope with 5, 10, 20, 30 nmol/L CY3-Negative siRNA transfected WISH cells, and no signals were discovered in control group. The transfection efficiency of 20, 30 nmol/L CY3-Negative siRNA was remarkably higher than 5, 10 nmol/L CY3-Negative siRNA transfection groups （P 〈 0.05）. There was no significant difference in transfection efficiency between 20 nmol/L and 30 nmol/L siRNA groups （P 〉 0.05）. The apoptosis of WISH cells was detected after 24-hours transfection with 20 nmol/L Negative siRNA. The apoptotic rates were 1.96% and 2.36% in the transfection and control groups, respectively. There was no significant difference in apoptosis of WISH cells between transfection group and control group. CONCLUSION.①The chemosynthesis siRNA can transfect human amnion-derive WISH cells successfully, and there is no effect on the apoptosis of WISH cells after transfection. ②Better transfection efficiency can be obtained in 20 nmol/L siRNA transfected human WISH cells.