靶向survivin的shRNA对人胆管癌细胞体外增殖及凋亡的影响

摘要

目的:探讨靶向survivin的shRNA对人胆管癌细胞体外增殖及凋亡的影响.方法:合成具有互补序列的能够编码短发卡RNA(shRNA)的双链寡核苷酸并构建pSilencer2.1真核表达质粒,经稳定转染QBC939细胞后对转基因后胆管癌细胞的生物学行为进行观察.结果:neo基因稳定表达于转染阳性质粒及阴性对照质粒的QBC939细胞中.通过RT-PCR及western blot检测证实shRNA在mRNA及蛋白质水平抑制survivin表达率分别达68.9 ％和61.3％.细胞计数及平板克隆形成实验显示转染细胞生长速度及细胞克隆明显减缓(P＜0.01).流式细胞术测定细胞周期显示转染细胞G1期细胞明显增多,S期细胞明显减少.DNA ladder、透射电镜观察及流式细胞定量研究显示转染细胞凋亡明显增多.结论:靶向survivin的 shRNA能有效抑制目的基因的表达,通过增加细胞凋亡在体处抑制人胆管癌细胞的生长,为胆管癌的基因治疗提供一定的实验基础.%Objective: To study the influence of shRNA targeting survivin on the biological behavior of human cholangiocarcinoma cells in vitro. Methods: Double strand oligonucleotide with rncomplementary sequence and encoding short hairpin RNA (shRNA) was synthesized and inserted into plasmid pSilencer 2.1 to generate eukaryotic expression vector. After stable transfection into cholangioearcinoma cells, the biological behaviors of the survivin shRNA transfeoted cholangiocar-cinoma cells were observed. Results: Neo gene expressed stablely in QBC939 cells transfected with positive and negative plasmid. Survivin mRNA and protein expression inhibitory ratio reached 68.9% and 61.3% respectively. The QBC939 cells transfected with positive recombinant plasmid demonstrated significantly inhibited cell growth in vitro by the cell growth curve and plate colony formation .The cell cycle analysis by flow cytometry(FCM) showed that the G1 phase cells signif-icantly increased, whereas S phase cells markedly decreased in QBC939/Silence(+) cells, as compared with those in QBC939 and QBC939/Silence(-) cells. The results of observation by DNA ladder, transmission electro microscopy and the quantitative analysis of apoptotic cells by FCM showed that the apoptotic cells markedly increased in QBC939/Silence(+) cells.Conclusion: rnshRNA targeting survivin gene can specifically suppress survivin expression and cholangiocarcinoma cell growth in vitro by apoptosis. This provides an innovative approach to cholangiocarcinoma gene therapy.