新型非病毒载体介导融合自杀基因靶向治疗胃癌的实验研究

Novel non-viral vector-mediated fusion suicide gene targeted therapy of gastric carcinoma experimental study

目的构建靶向性基因治疗载体pcDNA3.1(-)CVyCDglyTK,并以新型磷酸钙纳米为基因治疗载体,研究该基因治疗体系在胃癌细胞中体外与体内专一性表达和杀伤作用。方法采用PCR、RT-PCR、融合PCR、酶切及连接等技术构建由CEA启动子、CMV增强子驱动的融合自杀基因pcDNA3.1(-)CVyCDglyTK表达载体和融合自杀基因pcDNA3.1(-)CMVyCDglyTK表达载体。以磷酸钙纳米为载体分别转染CEA阳性的人胃癌细胞株SGC7901细胞和CEA阴性的Hela细胞.采用RT—PCR、免疫荧光法检测感染细胞中yCDglyTK基因的表达,并在体外观测其对胃癌细胞的作用。结果 SGC7901细胞在感染以上两种质粒表达载体后均有yCDglyTK mRNA表达,Hela细胞在转染纳米-pcDNA3.1(-)CMVyCDglyTK复合物后有yCDglyTK mRNA表达,对5-FC的敏感性增强,而在转染纳米-pcDNA3.1(-)CVyCDglyTK复合物后则没有yCDglyTK mRNA表达,5-FC对其亦无杀伤作用。在体外实验中,经纳米转染pcDNA3.1(-)CVyCDglyTK筛选的阳性克隆组细胞存活率为8.0%,纳米-pcDNA3.1(-)CVyCDglyTK转染组为25.4%,而对照组的细胞存活率为99.0%。结论本实验构建的由CEA启动子、CMV增强子驱动的融合自杀基因yCDglyTK靶向性基因治疗载体,能使融合自杀基因在CEA阳性的胃癌细胞中专一性表达,从而达到靶向治疗胃癌的目的 。

【Objective】To constract the expression plasmid vectors of pcDNA3.1（-）CVyCDglyTK,and study the gene therapy system target therapy carcinoma in vitro mediated by calcium phosphates nanoparticles.【Methods】 Construct the expression plasmid vectors of pcDNA3.1（-）pcDNA3.1（-）CVyCDglyTK,pcDNA3.1（-）CMVyCDglyTK with PCR,RT-PCR,confusion PCR,enzyme restriction,ligation,transfection,CEA-positive SGC7901 cells and CEA-negative Hela cells was mediated by calcium phosphates nanoparticles;detect the expression of yCDglyTK by RT-PCR,immunofluorescence.【Results】The efficiency of expression yCDglyTK detected by in SGC7901 and hela cells,but have no CVyCDglyTK expression in hela cells.We discovered that 5-FC have high sensitive in expression yCDglyTK cells,After the administration of prodrug 5-FC,Study the therapeutic effect was evaluated in vitro.The rate of cell survival was 8%,25.36% and 99% in positive-yCDglyTK,nanoparticles-pcDNA3.1（-）CVyCDglyTK and control groups respectively.【Conclusion】Our study confirmed that the CEA and CMV fusion promoter could effectively drive the target gene to express only in CEA-positive gastric cancer cells.CVyCDglyTK /5-FC gene therapysystem driven by CEA promoter resulted in significant cytotoxicity,consequently to achachieved the purpose of targeted therapy.