GPC3真核表达载体的构建及其对肝癌细胞功能的影响  

CONSTRUCTION OF GPC3 GREEN FLUORESCENT PROTEIN EUKARYOTIC EXPRESSION VECTOR AND ITS INFLUENCE ON FUNCTION IN BEL-7404 CELLS

作　　者：凌永赤[1] 方天敏 刘宁[2] 赵荫农[2] 

机构地区：[1]广西崇左市扶绥县人民医院外一科,扶绥532100 [2]广西医科大学附属肿瘤医院肝胆外科,南宁530021

出　　处：《广西医科大学学报》2011年第3期348-351,共4页Journal of Guangxi Medical University

基　　金：广西卫生厅重点研究课题(No.广西卫生厅重200703号)

摘　　要：目的:通过构建GPC3增强型绿色荧光蛋白真核表达载体,研究磷脂酰肌醇蛋白聚糖-3(Glypican-3,GPC3)促细胞增殖效应的影响,探讨GPC3基因对肝癌细胞侵袭和转移能力的影响。方法:应用基因重组技术及限制性内切酶酶切构建并鉴定pEGFP-IRES-N1-GPC3增强型绿色荧光蛋白真核表达载体,经脂质体LipofectamineTM2000介导转染BEL-7404后,通过G418筛选出抗性克隆,应用逆转录—聚合酶链反应(RT-PCR)检测GPC3 mRNA在真核细胞中的表达,并在激光共聚焦显微镜下观察目的蛋白在真核细胞内的表达情况,采用免疫荧光法和流式细胞仪检测GPC3对细胞增殖效应的影响。Transwell小室实验检测BEL-7404肝癌细胞的侵袭能力。结果:限制性内切酶酶切分析、重组质粒测序鉴定表明为正确重组子,荧光显微镜下可见转染的真核细胞胞膜区发出强绿色荧光,RT-PCR法表明GPC3在真核细胞中成功表达,转GPC3的BEL-7404肝癌细胞与对照组相比有促细胞增殖及侵袭和转移效应。结论:构建完成真核表达重组质粒pEGFP-IRES-N1-GPC3;GPC3基因在BEL-7404中成功表达;GPC3可促进肝癌细胞的增殖,其通过增加细胞的侵袭能力而促进肝癌的转移。Objective： To construct a GPC3 green fluorescent protein eukaryotic expression vector pEGFPIRES-N1- GPC3, and analyze its effects on the proliferation, adhesion and invasion of effect of in human hepatoma cell line. Methods：A eukaryotic expression vector for GPC3 genes was constructed by recombinant DNA technique and was transfected into BEL-7404 cells by LipofectamineTM 2000; the cells stably expressing GPC3 were screened out by G418. The mRNA expression of GPC3 was detected by RT-PCR method and fluorescence microscope. The effects of GPC3 gene on the growth promoting effects of proliferation were examined by flow cytometer. Transwell system was used to assess the invasion of the cells. Resuits： The recombinant plasmid was verified to be correctly constructed by restriction endonuclease analysis and DNA sequencing. The green fluorescence was detected in the transfeeted BEL-7404 cells under fluorescence microscope. RT-PCR and Western blotting both confirmed that GPC3 was successfully expressed in BEI. 7404 cells. Transfection with CPC3 promoted the proliferation of BEL-7404 cells. Transfection with GPC3 significantly enhanced migration and invasion capacity. Conclusion：The eukaryotic expression vector pEGFP-IRES-N1- GPC3 has been correctly constructed and GPC3 protein has been successfully expressed in BEL-7404 cells. Transfection with GPC3 gene can enhance their multiplication and invasiveness. The present study provides an experimental basis for studying the invasion mechanism of liver cancer.

关 键 词：GLYPICAN 3 真核表达载体 肝癌细胞 增殖 

分 类 号：R735.7[医药卫生—肿瘤]