目的:构建人胰岛素样生长因子Ⅱ基因(IGF-Ⅱ)P3启动子驱动单纯疱疹病毒胸苷激酶(tk)与增强型绿色荧光蛋白(EGFP)融合基因穿梭质粒,观察其在肝癌细胞系HepG2及宫颈癌细胞系HeLa细胞中的表达及其作用.方法:应用基因重组技术,构建IGF-Ⅱ P3启动子驱动EGFP与tk融合表达穿梭质粒载体,脂质体转染技术将其转入HepG2及HeLa中,48 h后荧光显微镜下观察荧光表达,RT-PCR法检测tk和EGFP mRNA表达,四甲基偶氮唑蓝(MTT)实验检测转染融合蛋白载体后给予不同浓度的GCV对HepG2及HeLa细胞毒作用.结果:酶切鉴定和测序分析证实重组质粒构建成功;转染此穿梭质粒的细胞中仅有HepG2细胞检测到绿色荧光;RT-PCR检测到tk和EGFP的mRNA仅在HepG2细胞中表达;GCV对转染了携此质粒载体的HepG2细胞有选择性细胞毒作用.结论:成功构建IGF-Ⅱ P3启动子驱动携带tk与EGFP融合基因穿梭质粒载体,转染后对人肝癌细胞系HepG2有选择性杀伤作用,为进一步靶向性肝癌腺病毒基因治疗奠定了基础.%AIM: To construct the shuttle plasmid vector for thymidine kinase (tk) and EGFP fusion protein gene driven by IGF - Ⅱ P3 promoter, and investigate the specific killing effect of the HSV - tk/GCV system on hepatocellular carcinoma(HCC) cells in vitro. METHODS: Recombinant shuttle plasmid vector was constructed by techniques of genetic recombination and screening, and identified by restriction digestion and sequencing analysis. Then the recombinant shuttle plasmid was transfected into HepG2 and HeLa cells by techniques of lipofectamine transfection and its expression was detected by fluorescence microscope and RT -PCR. Cell killing after ganciclovir(GCV) application was determined by MTT. RESULTS: Identification of pDC316 -tkEGFP- P3 by enzyme digestion and sequencing analysis showed that the length, inserted location and direction of the target genes which were inserted into the recombinant were correct. It was found that enhanced green fluorescence protein could only be seen in HepG2 cells, but not in HeLa cells. The results of RT -PCR showed that only two bands could be seen in the samples of pDC316 -tkEGFP- P3 transfected HepG2 cells. The MTT test showed the selective cytotoxicity of GCV to the transfected HepG2 cells. CONCLUSION: The shuttle plasmid vector carrying the tkEGFP fusion protein gene driven by IGF - Ⅱ P3 promoter has been constructed successfully and its specific expression in HepG2 cells provided a sound basis for targeted gene therapy for HCC.
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