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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-ⅡP3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-ⅡP3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。
OBJECTIVE: To construct the shuttle plasmid of human insulin-like growth factor Ⅱ gene (P3-Ⅱ) promoter and to promote herpes simplex virus thymidine kinase (tk) and enhanced green fluorescent protein (EGFP) fusion gene to observe its expression in hepatoma cell line HepG2 and The expression of cervical cancer cell line HeLa and its role. Methods: Recombinant plasmid was transfected into HepG2 and HeLa cells by lipofectamine 2000. After 48 h, the expression of EGFP was observed by fluorescence microscope. The expression of RT-PCR was detected by RT- The expression of tk and EGFP mRNA was detected by PCR, and the cytotoxicity of different concentration of GCV on HepG2 and HeLa cells was detected by MTT assay. Results: The recombinant plasmids were successfully constructed by restriction enzyme analysis and sequencing analysis. Only HepG2 cells transfected with this shuttle plasmid detected green fluorescence. The mRNA of tk and EGFP was only detected in HepG2 cells by RT-PCR. HepG2 cells transfected with this vector had selective cytotoxicity. CONCLUSION: IGF-ⅡP3 promoter is successfully constructed to drive shuttle plasmid vector of fusion gene of tk and EGFP, which can selectively kill human hepatocellular carcinoma cell line HepG2 after transfection, which lays a foundation for further targeted gene therapy of hepatocellular carcinoma.