慢病毒载体介导RNAi稳定抑制XIAP表达及对胰腺癌细胞增殖、凋亡影响的实验研究  

Experimental study on lentiviral vector-mediad RNA interference inhibiting XIAP expression and influencing proliferation and apoptosis of human pancreatic cancer cells

作　　者：易小平[1] 江春[1] 宰红艳[1] 邓公平[1] 欧阳洋[1] 周达全[1] 纪连栋[1] 李宜雄[1] 

机构地区：[1]中南大学湘雅医院普外科,湖南长沙410008

出　　处：《现代生物医学进展》2009年第22期4201-4205,4211,共6页Progress in Modern Biomedicine

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

摘　　要：目的:探讨运用慢病毒载体介导的RNA干扰技术对X-连锁凋亡抑制蛋白(XIAP)的抑制效率及对胰腺癌细胞增殖、凋亡的影响,建立XIAP表达稳定抑制的胰腺癌细胞株。方法:应用pGCSIL-PUR慢病毒载体构建针对XIAP的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,经嘌呤霉素(puromycin)筛选并扩大培养得到稳定克隆;实时荧光定量PCR和western-blot免疫印迹检测癌细胞内XIAP的表达;四甲基偶氮唑盐(MTT)比色法检测细胞增殖;caspase 3/7活性测定和DAPI染色检测细胞凋亡。结果:成功构建3个XIAP-ShRNA慢病毒载体(X1、X2、X3)及XIAP表达稳定抑制的胰腺癌细胞株,对XIAP的抑制效率均达70%以上;MTT检测显示X1、X3稳定抑制XIAP后胰腺癌细胞增殖明显减慢,但caspase 3/7活性及细胞凋亡并没有明显增加。结论:慢病毒载体介导的靶向XIAP的RNAi可有效抑制XIAP表达,降低胰腺癌细胞的增殖能力;成功建立的XIAP表达稳定抑制的胰腺癌细胞株为进一步研究打下基础。Objective：To investigate the possibility of XIAP inhibition by Lentiviral vector-mediated RNA interference and the influence on cell proliferation and apoptosis in pancreatic cancer cell line ,and to set up a pancreatic cancer cell line in which XIAP expression is stably suppressed. Methods： The Lentiviral vector of SiRNA targeted against XIAP （LV-XIAP-1, LV-XIAP-2,LV-XIAP-3） was constructed and transfected into the packagingcells 293T, and then collected supernatant with virus to transfect SW1990 cells. After seledtion by puromycin and culture expansion, the stable cell clones were attained; quantitative real-time fluorescent PCR and western-blot was used to detect the expression of XIAP. The effect of suppressing XIAP by RNAi on cell proliferation was quantified by methylthiazoletetrazolium （MTT） assay. Enzymatic activity of caspase-3/7 detecting and DAPI staining were employed to examine the apoptosis. Results： Three lentiviral vector-xiap-shRNA were constructed successfully, the xiap expression was decreased more than 70% in RNA and protein level when compared to contral. a pancreatic cancer cell line in which XIAP expression is stably suppressed was successfully set up. MTT showed that the cell number of XIAP expression suppression cell clones decreased significantly （P〈0.05）, but the activity of caspase-3/7 and apoptosis index did not increase significantly. Conclusions： Lentiviral vector-mediad RNA interference targeting against XIAP can effectively inhibit XIAP expression and cell proliferation. The pancreatic cell line in which xiap gene was stablely suppressed was successfully established, which paves a way for continuous studying of XIAP function and gene therapy.

关 键 词：胰腺癌 RNA干扰 X-连锁凋亡抑制蛋白 慢病毒载体 细胞增殖 

分 类 号：R576[医药卫生—消化系统]