摘要
构建了新型联合基因载体pcDNA3.1(-)VEGF-siRNA/yCDglyTK,研究其在人胃癌细胞系SGC7901细胞中的表达和杀伤作用.构建靶向血管内皮生长因子(VEGF)的干扰质粒pGenesil-VEGF-siRNA,采用PCR法从中扩增siRNA表达框(含U6启动子),亚克隆至双自杀基因载体pcDNA3.1(-)CV-yCDglyTK,构建联合基因质粒pcDNA3.1(-)VEGF-siRNA/yCDglyTK;通过酶切、测序等鉴定重组质粒;以磷酸钙纳米颗粒为载体,将干扰质粒、双自杀基因质粒及联合基因质粒转染SGC7901细胞,RT-PCR、Western-blot验证目的基因表达;MTT法检测转染细胞对5-氟胞嘧啶(5-FC)的敏感性.结果表明:酶切及测序证实联合基因载体pcDNA3.1(-)VEGF-siRNA/yCDglyTK构建成功;SGC7901细胞转染联合基因质粒后,RT-PCR、Western-blot证实融合自杀基因表达,而VEGF基因表达下调;在前体药物5-FC作用下,转染联合基因组细胞存活率最低,与其他组比较有统计学差异.成功构建联合基因载体pcDNA3.1(-)VEGF-siRNA/yCDglyTK,初步验证靶向VEGF的RNA干扰与双自杀基因系统具有协同效应.
This research aimed to construct a new combination gene vector: pcDNA3.1 (-)VEGF-siRNA/ yCDglyTK, study its expression quality and lethal effet in human gastric cancer cell line SGC7901. First, RNA interference (RNAi) targeting vascular endothelial growth factor (VEGF) was applied to construct interfering plasmid pGenesil-VEGF-siRNA. Then, the siRNA expression cassette (including U6 promotor ) was amplified by PCR and subcloned into pcDNA3.1 (-)CV-yCDglyTK to build a new combination gene plasmid: pcDNA3.1 (...
出处
《生物化学与生物物理进展》
SCIE
CAS
CSCD
北大核心
2010年第5期503-509,共7页
Progress In Biochemistry and Biophysics
基金
国家自然科学基金(30800518)
教育部博士点新教师基金(200805331090)资助项目~~
关键词
RNA干扰
自杀基因
胃癌
联合基因治疗
RNA interference
suicide gene
gastric cancer
combination gene therapy
