自杀基因协同表达促肝癌细胞凋亡的研究

Co-expression of Suicide gene Promotes the Apoptosis of HepG2 Cells

目的:观察偶联去唾液酸糖蛋白(AF)的人端粒酶的逆转录酶启动子(hTERTp)驱动的自杀基因(tk/cd)协同表达对肝癌细胞株HepG2的靶向杀伤作用。方法:细胞培养,PCR方法构建AF-pGL3-hTERTp-cd与AF-pGL3-hTFERTp-tk质粒,通过瞬时转染方法联合转染肝癌细胞HepG2和正常肝细胞L-02,MTT法观察药物浓度对肝癌细胞存活率的影响,流式技术观察其对肝癌细胞生长和凋亡的影响。结果:MTT法显示GCV、5-FC对自杀基因协同转染的HepG2细胞有较强的细胞毒作用,联合作用可降低药物浓度。流式结果表明在hTERTTp驱动下自杀基因协同作用于肝癌细胞后细胞总的凋亡率为87%,高于单—AF-pGL3-hTERTp-cd组(77%),以及单一AF-pGL3-hTERTp-tk组(70%),对正常肝细胞L-02的生长影响较小。结论:偶联有AF的hTERTp驱动的自杀基因tk/cd协同表达可增强肝癌细胞的自杀效果,并促使其凋亡的作用。

Objective： To observe the target effect of co-expression of suicide gene CD and TK under the hTERT promoter that has been conjugated with AF on HCC cell line HepG2. Methods： AF-pGL3-hTERTp-CD plasmid was constructed with PCR method. We used the united plasmid including AF-pGL3-hTERTp-CD and AF-pGL3-hTERTp-TK to transfect the HepG2 cells and L-02 cells by transient transfection methods. The cell growth rate and apoptosis at different drug concentrations were observed with MTT and flow cytometry. Resuits： MTT showed that the united plasmid had strong cytoxic effect and can reduce the CD and GCV concen- tration level. The total apoptosis rate was 87% in HepG2 cells compared with that of cells transfected with AF-pGL3-hTERTp-CD （77%） and AF-pGL3-hTERTp-TK （70%）. The united genes was more effective than single ones in targeted therapy against HepG2 cells, but no obvious influence was observed on L-02 cells. Con- clusion： With the hTERT promoter and AF, the united genes can enhance the suicide activities of liver cancer cells and are effective in targeted gene therapy against HCC.