靶向survivin的shRNA对人胆管癌QBC939细胞体外增殖及凋亡的影响  

Effect of shRNA targeting survivin gene on biological behavior of human cholangiocarcinoma cells

作　　者：卢昕[1] 郑启昌[2] 

机构地区：[1]武汉市第一医院胃肠外科,湖北武汉430022 [2]华中科技大学同济医学院附属协和医院肝胆外科,湖北武汉430022

出　　处：《现代肿瘤医学》2012年第8期1561-1566,共6页Journal of Modern Oncology

基　　金：武汉市卫生局科研基金资助项目[编号:武卫(2008)70号WX08D04]

摘　　要：目的:探讨靶向survivin的shRNA对人胆管癌QBC939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pSilence2.1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。RT-PCR及Western blot检测证实shRNA在mRNA及蛋白质水平抑制survivin表达,抑制率分别为66.2%和61.8%。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形成率明显减低(P<0.01)。流式细胞术测定细胞周期显示转染细胞G0/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术定量研究显示转染细胞凋亡比明显增多。结论:靶向sur-vivin的shRNA能有效抑制目的基因的表达,通过增加细胞凋亡在体外抑制人胆管癌细胞的生长。Objective：To study the influence of shRNA targeting survivin on the biological behavior of human cholangiocarcinoma cells in vitro. Methods： Double strand oligonucleotide with complementary sequence and enco- ding short hairpin RNA （shRNA）was synthesized and inserted into plasmid pSilence 2.1 to generate eukaryotic ex- pression vector. After stable transfection into cholangiocarcinoma cells, the hiologica/behaviors of the sttrvivin shRNA transfected cholangiocarcinoma cells were observed. Results： Neo gene expressed stablely in QBC939 cells transfect- ed with positive and negative plasmid. Survivin mRNA and protein expression inhibitory ratio reached 66.2% and 61.8% respectively. The QBC939 cells transfected with positive recombinant plasmid demonstrated significantly in- hibited cell growth in vitro by the cell growth curve and plate colony formation . The cell cycle analysis by flow cytom- etry （FCM）showed that the G0/G, phase cells significantly increased, whereas S phase cells markedly decreased in QBC939/Silence（ ＋ ）cells ,as compared with those in QBC939 and QBC939/Silence（ - ）cells. The results of obser- vation 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 （ ＋ ） ceils . Conclusion ： shRNA targeting survivin gene can specifically suppress survivin expression and cholangiocarcinoma cell growth in vitro by apoptosis. This pro- vides an innovative approach to cholangiocarcinoma gene therapy.

关 键 词：SURVIVIN SHRNA 胆管癌细胞 细胞凋亡 

分 类 号：R73-362[医药卫生—肿瘤] R735.8[医药卫生—临床医学]