应用RNAi技术沉默survivin基因对肝癌细胞系BEL-7404生长的影响  

Growth effect of liver cancer cell line BEL-7404 by silencing survivin gene with RNAi

作　　者：廖艳[1] 袁晟光[2] 廖维甲[2] 覃理灵[2] 梅铭惠[2] 陈谦[2] 

机构地区：[1]桂林市疾病预防控制中心,广西桂林541001 [2]桂林医学院附属医院肝胆外科研究室

出　　处：《中国实验诊断学》2011年第12期1991-1994,共4页Chinese Journal of Laboratory Diagnosis

基　　金：国家自然科学基金项目(30360116);广西科学研究与技术开发计划项目(桂科攻0428005-7);广西壮族自治区教育厅面上项目(200911MS163);广西壮族自治区卫生厅计划课题(Z2009030)

摘　　要：目的为研究生存素survivin基因的功能及肝细胞癌(HCC)的基因治疗策略。构建针对survivin基因的siR-NA的表达载体,转染肝癌细胞系BEL-7404后评价其对survivin基因表达的抑制作用。方法依据survivin基因设计siRNA片断,构建pGenesil-1/survivin表达载体,脂质体LipofectamineTM 2000转染BEL-7404细胞。实时定量聚合酶链反应从基因转录水平检测survivin基因的表达率,应用免疫组织化学的方法检测肝癌细胞系BEL-7404中survivin蛋白表达抑制情况。结果本实验已成功构建了针对survivin基因的pGenesil-1/survivin质粒。实时定量PCR检测显示,BEL-7404细胞经特异性siRNA作用后survivin基因的表达受抑制,其Ct值由34.2降低到22.8,免疫组织化学结果显示,pGenesil-1/survivin介导的siRNA能抑制BEL-7404细胞中survivin蛋白的表达。结论构建的siRNA能特异地抑制sur-vivin靶基因在肝癌细胞株BEL-7404中的表达,为肿瘤的生物学治疗提供了新的方法和材料。Objective To study function of survivin gene and gene therapy stratagem of hepatocellular carcinoma（HCC）.To construct siRNA expression vector against survivin gene,evaluation survivin gene expression inhibitory action after into liver cancer cell line cell BEL-7404.Methods The siRNA part design based on surviving gene,pGenesil-1/ survivin expression vector were transfected into BEL-7404 cells by using LipofectamineTM 2000.The expression rate of survivin was measured by real-time relative quantitative PCR,The protein level of survivin in the culture supernatant was measured with a enzyme-linked immunosorbent assay（ELISA）.Results The recombinant plasmid pGenesil-1/ survivin was constructed successfully.The result real-time of PCR revealed that the threshold cycle value was reduced form 34.2 to 22.8 after BEL-7404 cell were inhibitory by siRNA,ELISA showed that pGenesil-1/ survivin vector mediated survivin siRNA could inhibit the expression of survivin proteinic in BEL-7404 cells.Conclusion siRNA targeting survivin mRNA can specially suppress the expression of targeting gene in BEL-7404 cells,which provides a new method and material to the biological therapy of cancer.

关 键 词：RNA干扰 生存素 BEL-7404细胞 基因表达 

分 类 号：R735.7[医药卫生—肿瘤]