Tie2基因RNAi慢病毒载体的构建及其干预恶性黑色素瘤细胞的体外研究  

Construction of recombinant lentivirai vector of Tie2-RNAi and its influence on malignant melanoma cells in vitro

作　　者：单秀英[1] 刘照亮[1] 王彪[1] 郭国祥[1] 王美水[1] 庄福连[1] 蔡传书[2] 张明凤[3] 张彦定[3] 

机构地区：[1]福建医科大学附属第一医院整形外科,福州350005 [2]福建医科大学附属第一医院放疗科,福州350005 [3]福建师范大学生命科学院

出　　处：《中华整形外科杂志》2011年第4期277-283,共7页Chinese Journal of Plastic Surgery

基　　金：卫生部科学研究基金一福建省卫生教育联合攻关计划（WKJ2008-2-51）

摘　　要：目的 构建携带酪氨酸蛋白激酶受体2-小干扰RNA（Tie2-SiRNA）慢病毒载体,观察其对恶性黑色素瘤细胞的干扰作用.方法 将pSilencer 1.0-U6启动子-酪氨酸蛋白激酶受体2-小干扰RNA（pSilencer 1.0-U6-Tie2-siRNA）重组质粒经Xba Ⅰ酶切电泳鉴定后,与经Xba Ⅰ酶切电泳鉴定的带有加强绿色荧光蛋白的转移质粒（pNL-EGFP）载体连接,产生加强绿色荧光蛋白的转移质粒-u6启动子-酪氨酸蛋白激酶受体2-Ⅰ（pNL-EGFP-U6-Tie2-Ⅰ）、pNL-EGFP-U6-Tie2-Ⅱ慢病毒转移质粒,电泳筛选阳性克隆,测序鉴定.用连接成功的慢病毒转移质粒,分别与水疱性口炎病毒G蛋白（pVSVG）包膜质粒和pHelper包装质粒组成慢病毒三质粒转染系统,再共转染293T细胞,产生pNL-EGFP-U6-Tie2-Ⅰ、pNL-EGFP-U6-Tie2-Ⅱ慢病毒.收集病毒上清,测定病毒滴度.将收集的病毒上清感染恶性黑色素瘤细胞,通过实时荧光定量RT-PCR测定抑制Tie2基因表达的效率.结果 酶切电泳与测序鉴定证实成功构建了Tie2-SiRNA慢病毒载体,293T细胞测定病毒原液滴度为8.8×103/ml.实时荧光定量RT-PCR结果显示：Tie2-SiRNA慢病毒载体感染恶性黑色素瘤细胞,抑制了恶性黑色素瘤细胞中Tie2基因的表达,其中较高者可达68.55%,并且2种慢病毒载体之间差异无统计学意义（t=0.362,P＞0.05）.结论 成功构建了Tie2-SiRNA慢病毒载体,体外研究显示其能抑制Tie2 mRNA的表达,为下一步进行抑制肿瘤生成的动物实验研究奠定了基础.Objective To construct lentivector carrying Tie2-Small interfering RNA（SiRNA）,so as to study its influence on malignant melanoma cells.Methods Recombinant plasmid pSilencer 1.0-U6-Tie2-siRNA and plasmid pNL-EGFP were digested with Xbal.ligated a target lentiviral transfer plasmid of pNL-EGFP-U6-Tie2-Ⅰ or pNL-EGFP-U6-Tie2-Ⅱ,and then the electrophoresis clones was sequenced.Plasmids of pNL-EGFP-U6-Tie2-1 and pNL-EGFP-U6-Tie2-Ⅱ were constructed and combined with pVSVG and pHelper,respcectively,to constitute lentiviral vector system of three plasmids.The Lentiviral vector svstem was transfected into 293T cell to produce pNL-EGFP-U6-Tie2-Ⅰ and pNL-EGFP-U6-Tie2-Ⅱ lentivirus.Then the supernatant was collected tO determine the titer.Malignant melanoma cells were infected by both lentiviruses and identified by Realtime RT-PCR to assess inhibitory efficiency. Results The recombinant lentiviral vectors of Tie2-RNAi were constructed successfully which were analyzed with restriction enzyme digestion and identified by sequencing.And the titer of lentiviral vector was 8.8×103/ml,which was determined by 293T cell.The results of Reahime RT-PCR demonstrated that the lentiviral vectors of Tie2-RNAi could infect malignant melanoma cells and inhibit the expression of Tie2 genes in malignant melanoma cells（P〈0.01）.There was no significant difference in the expression level （P〉0.05）between the two lentiviral vectors of Tie2-RNAi.Conclusions Lentivector carrying Tie2-SiRNA can be constructed successfully and inhibit the expression of Tie2 gene in vitro significantly.The study will supply the theory basis for the further research on the inhibition of tumor growth in vivo.

关 键 词：受体 Tie2 RNA干扰 慢病毒载体 黑色素瘤 

分 类 号：R73[医药卫生—肿瘤]