Oct4-shRNA慢病毒载体沉默结肠癌Oct4基因并诱导细胞凋亡的初步研究.doc

Oct4-shRNA慢病毒载体沉默结肠癌Oct4基因 并诱导细胞凋亡的初步研究 文坤明1，张贵海2，陈旺盛1，钱江1，傅仲学1(1.400016重庆，重庆医科大学附属第一医院胃肠外科；2.563003遵义，遵义医学院附属医院肿瘤科) [ 摘要]：目的　探讨Oct4-shRNA慢病毒载体对结肠癌SW480细胞Oct4的抑制效率及对细胞凋亡的影响。方法　构建Oct4与GFP融合的过表达质粒及4对针对Oct4-shRNA的质粒，用Oct4过表达粒质分别与4个干扰质粒共转染293T细胞，采用western bolt检测GFP的表达（外源筛靶），确定出干扰效果最好的Oct4-shRNA慢病毒载体，用293Ｔ细胞包装后转染结肠癌SW480细胞（内源验证），实时荧光定量PCR和western bolt检测癌细胞内Oct4的表达；流式细胞仪测定肿瘤细胞凋亡。结果　通过外源筛靶筛选出干扰效果好的Oct4-shRNA慢病毒载体，采用293T细胞包装后，在SW480细胞内源验证，该载体对SW480细胞Oct4的mRNA和蛋白表达均有明显的抑制效果（P0.01）；与对照组相比，细胞凋亡明显增加（P0.01）。结论　Oct4-shRNA慢病毒载体可有效抑制结肠癌SW480细胞Oct4表达，并显著增加细胞凋亡。 [关键词]：结肠癌；RNA干扰；Oct4；慢病毒载体；细胞凋亡 [中图分类号]：R735.3+5 文献标识码：Ａ Oct4-shRNA lentiviral vectors silences colon cancer Oct4 gene and induces cell apoptosis Wen Kunming1, Zhang Guihai2, Chen Wangsheng1, Qian Jiang1, Fu Zhongxue1(1Department of Gastrointestinal Surgery, First Affiliated Hospital, Chongqing Medical University, Chongqing 400016,China;2 Department of Oncology, Affiliated Hospital of Zunyi Medical College, Zunyi 563003, China) [Abstract]: Objective To investigate the inhibition efficiency of Oct4 by lentiviral vector- mediated RNA interference and the effect on cell apoptosis in colon cancer cell line. Method Overexpression lentiviral vector plasmid of Oct4-GFP fusion and four Oct4-shRNA lentiviral vector plasmids was constructed, and each of the four Oct4-shRNA lentiviral vectors and Oct4-GFP overexpression plasmid were co-transfect respectively into 293T cells, then GFP protein expression in the cells was detected by western bolt（exogenous sieve target）. Through the previous experiment, it was determined which one of the four Oct4-shRNA lentiviral vectors was the best one in interference effect, then the Oct4-shRNA lentiviral vector was transfected into the packaging cells 293T, and at last the supernatant with virus was collected to transfect SW480 cells(endogenous authentication). Quantitative real-time fluorescent PCR and Western-blot were used to detect the expression of Oct4, and flow cytometry was use