慢病毒载体介导RNAi靶向抑制胰腺癌细胞survivin表达并诱导细胞凋亡的实验研究  

Lentiviral vector-mediad RNA interference targeted against survivin inhibits survivin expression and induces cell apoptosis of human pancreatic cancer in vitro

作　　者：易小平[1] 江春[1] 宰红艳[1] 邓公平[1] 李宜雄[1] 

机构地区：[1]中南大学湘雅医院普通外科,湖南长沙410008

出　　处：《中国普通外科杂志》2010年第3期227-233,共7页China Journal of General Surgery

基　　金：国家自然科学基金资助项目(30872492);湖南省自然科学基金资助项目(08JJ3042)

摘　　要：目的探讨运用慢病毒载体介导的RNA干扰技术对survivin的抑制效率及对胰腺癌细胞SW1990凋亡的影响。方法应用pGCSIL-neo质粒构建3对针对survivin的慢病毒ShRNA载体,转染包装细胞293T,收集病毒上清转染胰腺癌细胞系SW1990,实时荧光定量PCR和western-blot免疫印迹检测癌细胞内survivin的表达;测定caspase3/7活性和DAPI染色检测细胞凋亡。结果成功构建3个survivin-ShRNA慢病毒载体,LV-shRNA-survivin-1对survivn的mRNA和蛋白表达抑制效率分别为73.50%和87.64%;与对照组相比,其caspase3/7活性升高14.5倍,细胞凋亡明显增加(11.95%)。结论慢病毒载体介导的Suvivin-ShRNA靶向转染可有效抑制survivin表达,并显著增加胰腺癌细胞的凋亡。Objective To investigate the possibility of survivin inhibition by lentiviral vector-mediated RNA interference and the influence on cell apoptosis in pancreatic cancer cell line.Methods The lentiviral vector of SiRNA targeted against survivin（LV-shRNA-survivin-1,LV-shRNA-survivin-2,LV-shRNA-survivin-3） was constructed and transfected into the packaging cells 293T,and then the supernatant with virus was collected to transfect SW1990 cells.Quantitative real-time fluorescent PCR and Western-blot were used to detect the expression of survivin.DAPI staining and detection of enzymatic activity of caspase 3/7 were employed to examine cell apoptosis.Results Three lentiviral vector-survivin-shRNA were constructed successfully.In the LV-shRNA-survivin-1 group,the survivin mRNA and protein expression inhibitory rate was 73.50% and 87.64% respectively;when compared to control group,the activity of caspase-3/7 increased significantly,which showed a 14.5-fold increase,and apoptosis increased 11.95%.Conclusions Lentiviral vector-mediad RNA interference targeted against survivin can effectively inhibit survivin expression and increase cell apoptosis significantly.

关 键 词：胰腺肿瘤 RNA干扰 survivin 慢病毒载体 细胞凋亡 

分 类 号：R735.9[医药卫生—肿瘤]