磷酸钙纳米介导自杀基因载体的构建及其应用研究

构建由CEA启动子、CMV增强子驱动的融合自杀基因PCDNA3.1(-)CVyCDglyTK表达载体和分别由CEA启动子和巨细胞病毒(CMV)增强子驱动的融合自杀基因PCDNA3.1(-)CEAyCDglyTK、PCDNA3.1(-)CMVyCDglyTK表达栽体.以磷酸钙纳米为载体分别转染CEA阳性的人结肠癌细胞株LOVO细胞和CEA阴性的HeLa细胞,Lovo细胞在感染以上3种质粒表达栽体后均有yCDglyTK mRNA表达,且对5-FC的敏感性明显增强:HeLa细胞在纳米PCDNA3.1(-)CMVyCDglyTK复合物感染后有yCDglyTK mRNA表达,对5-FC的敏感性增强,而在另外两种复合物感染后则没有yCDglyTK mRNA表达,5-Fc对其亦无杀伤作用,结果表明靶向性基因治疗栽体能使融合自杀基因在CEA阳性细胞中专一性表达,达到靶向治疗肿瘤的目的.

Construction of Calcium Phosphate Nanoparticles Mediating Fusion Suicide Gene yCDglyTK and its Application in vitro

The three expressing plasmids of pcDNA3.1 (-)CVyCDglyTK,pcDNA3.1 (-)CEAyCDglyTK,pcDNA3.1 (-)CMV-yCDglyTK were constructed with carcino-embryonic antigen (CEA) promoter and cytomegalovirus (CMV), CEA promoter, cytomegalovirus (CMV) enhancer, respectively. Calcium phosphate nanoparticles (CPNP) was used as vector to transfer the three plasmids into CEA-positive cells (Lovo) and CEA-negative cells (HeLa),respectively. The results showed that expression of yCDglyTK mRNA in all cells transferred by the three plasmids above,which significantly sensitized the cytotoxicity of prodrug 5-FC;expression of yCDglyTK mRNA in HeLa cells after transfected by CPNP- pCDNA3.1 (-)CMVyCDglyTK,which enhanced sensitivity of 5-FC,but the other plasmids had no expression of yCDglyTK mRNA and 5-FC had no killing effect, pcDNA3.1 (-)CVyCDglyTK might be a promising candidate vector for colon carcinoma gene therapy.