人snail基因rnai慢病毒载体的构建与鉴定 陈 丽,刘求真,邱际华，焦 锋 .docVIP

人Snail基因RNAi慢病毒载体的构建与鉴定 陈 丽 ,刘求真 ,邱际华，焦 锋，姚开泰 　(南方医科大学肿瘤研究所，广东 广州 510515) 摘 要:目的 构建人Snail基因慢病毒干扰载体及观察干扰前后对鼻咽癌5-8F细胞增殖和侵袭的影响。方法 设计Snail基因特异性RNAi靶序列,克隆至经双酶切后的plVTHM线性化载体，测序鉴定正确后，用病毒上清感染鼻咽癌细胞5-8F, 流式细胞仪分选获得稳定干扰Snail基因的细胞亚系。荧光定量PCR检测mRNA表达情况；MTT法和体外侵袭实验分别测定对细胞增殖和侵袭的影响。结果 plVTHM-siSnail慢病毒干扰载体构建正确。荧光定量PCR结果表明分选后的细胞Snail mRNA 水平表达显著降低。干扰后的细胞增殖减慢，穿透基膜的细胞数量减少, 差异具有统计学意义( P 0. 05)。结论 plVTHM-siSnail表达质粒可显著下调Snail基因在5-8F中的表达，在一定程度上抑制鼻咽癌细胞的增殖和侵袭。 关键词: Snail; RNA干扰; 鼻咽癌；增殖；侵袭 中图法分类号: [R34] Construction and identification of the lentiviral RNA interference vector of human Snail gene CHEN Li, LIU Qiu-zhen, QIU Ji-hua, JIAO Feng, YAO Kai-tai (Institute of Cancer Research ，Southern Medical University，Guangzhou 510515, China) 　Abstract: Objective To construct a recombinant lentiviral expression vector for RNA interference（RNAi）of human Snail gene, and to study its effects on proliferation and invasion of nasopharyngeal carcinoma cell line 5-8F . Methods The effective sequence of short hairpin RNAs (shRNA) targeting Snail gene was designed, and cloned into the linear pLVTHM vector , it was confirmed by DNA sequencing. 5-8F cells were infected with the viral supernatants and the cells with stable Snail gene knock-down were separated by Fluorescence Activated Cell Sorter (FASC). The 基金项目: 广东省科技计划项目(2007B031515006) Supported by the Planned Science and Technology project of Guangdong province ,China 作者简介：陈丽（1980-），女，湖北省随州市人，汉族，在读硕士研究生，主要从事肿瘤转移机制方面的研究。电话：（020E-mail:gmyychl@163.com 通信作者：姚开泰.电话：（020E-mail:ktyao@ expression of Snail mRNA was detected by Real time RT-PCR. MTT and cell invasion assay were used to detect the proliferation and invasion of 5-8F cells after plVTHM-siSnail transfection. Results The lentivirus vector plVTHM-siSnail was constructed successfully. The separated 5-8F-plVTHM-siSnail exhibited significant knock-down of Snail mRNA expression.The proliferation were more slowly and the cell population that cut through Matrigel were less a