摘要
目的探讨RNA干扰（RNAi）沉默survivin基因对肺腺癌A549细胞的影响。方法应用RNAi技术，以化学合成的survivin小干扰RNA（siRNA）体外转染A549细胞，采用MTT法及流式细胞仪检测转染前后A549细胞增殖、凋亡和细胞周期的变化，通过RT-PCR及Western blot分析survivin mRNA和蛋白的表达情况。结果survivin siRNA对A549的生长抑制作用呈浓度依赖性（P〈20．05），并能引起G1期细胞比率的增加和S期细胞相应的减少，诱导一定数量的细胞凋亡，转染后36、60、84、108、132h的IC50值分别为152．4、151．2、136．0、144．0、150．4nmol／L。100～200nmol／L siRNA转染组细胞的survivin表达在蛋白及RNA水平都显著低于对照组（P〈0．01），但空白对照组与空质粒组间无明显差异（P〉0．05）。结论应用siRNA沉默survivin基因可以下调肺腺癌A549细胞survivin的表达，进而抑制细胞生长、增殖并诱导细胞凋亡。RNAi的抑制效果具有特异、高效和持久的特点。
Objective To assess the growth inhibition effect of survivin specific siRNA （small interference RNA） on human lung adenocarcinoma A549 cell line in vitro. Methods A549 ceils were transfected with chernosynthetic survivin specific siRNA by using RNAi （RNA interference） technique. The effects of siRNA on cell proliferation, cell cycle and apoptosis were analyzed by MTT method and flow cytometry. The expression changes in survivin mRNA and protein were detected by RT-PCR and Western blot respectively. Results The chemosynthetic survivin specific siRNA could not only inhibit significantly the growth and proliferation of A549 cell, but also produce an increased G1 fraction and a decreased S fraction, and induce apoptosis in certain extent. The inhibitory effect showed concentration dependent. The IC50（50% inhibitory concentration） values of siRNA at 36h, 60h, 84h, 108h and 132h were 152. 4 nmol/L, 151.2 nmol/L, 136. 0 nmol/L, 144. 0 nmol/L and 150. 4 nmol/L, respectively. These IC50 values had no significant difference by statistic analysis （P〉0. 05）. The mRNA and protein expressions of survivin in cells transfected by 100-200nmol/L siRNA were significantly lower than those of untransfected cells （P〈0. 01）, but there was no significant difference between blank control group and empty plasmid control group （P 〉0. 05）. Conclusion Silencing survivin gene by the RNAi technique can decrease effectively the expressions of survivin gene and protein, suppress significantly the growth and proliferation of A549 cells, and induce apoptosis of A549 cells. The inhibitory effect of RNAi is characterized by its specificity, high efficiency and durability. This may lay an experimental foundation for further study of gene therapy in lung.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2007年第6期595-597,共3页
Medical Journal of Chinese People's Liberation Army
