中文摘要：

目的探讨人胰岛素样生长因子Ⅱ（IGF-Ⅱ）P4启动子调控单纯疱疹病毒胸苷激酶（HSV-tk）／丙氧鸟苷（GCV）自杀基因系统体外对人肝癌细胞的选择性杀伤效应。方法用分子生物学方法构建IGF-ⅡP4启动子和巨细胞病毒启动子（CMV）调控下HSVtk与增强型绿色荧光蛋白（EGFP）融合基因穿梭质粒，并用脂质体转染法将其转入肝癌细胞系HepG2及宫颈癌细胞系HeLa细胞中，在荧光显微镜下观察荧光表达情况。加入GCV使总浓度分别为0、1．0、10．0和100．0μg／ml，用四甲基偶氮唑盐实验检测HSV-tk／GCV系统对各种细胞的杀伤作用，用RTPCR法检测转染前后胸苷激酶（tk）和EGFPmRNA的表达情况。用方差分析进行统计学处理。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；pDC316tkEGFP-CMV和pDC316-tkEGFPP4转染的HepG2细胞均出现明显的细胞生长抑制现象，两者转染细胞的抑制率分别为6．95％±0．67％、24．99％±1．53％、49．68％±1．68％和71．85％±3．28％，4．83％±0．35％、17．34％±1．15％、30．17％±1．30％和40．39％±0．82％，转染后者对HepG2细胞的抑制率低（F=24．055，P〈0．05），pDC316-tkEGFPCMV转染的HeLa细胞也出现明显的细胞生长抑制现象，其抑制率分别是6．36％±0．83％、23．95％±1．72％、45．13％±1．64％和69．38％±3．17％，转染质粒pDC316-tkEGFP-P4的HeLa细胞未发现明显的细胞抑制现象。结论成功构建IGF-ⅡP4启动子调控携带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基础。

英文摘要：

Objective To investigate the selective killing effect of herpes simplex virus-thymidine kinase/ganciclovir （HSV-tk/GCV） suicide gene system controlled by human IGF-Ⅱ P4 promoter on HCC cells in vitro. Methods Recombinant shuttle plasmid vectors driven by IGF-Ⅱ P4 promoter and driven by CMV promoter were constructed by techniques of gene recombination. The recombinant shuttle plasmids were then transfected into HepG2 and HeLa cells by techniques of lipidosome transfection. EGFP expression was detected by fluoroscopy. Tk and EGFP mRNA expression were detected by RT-PCR. The selective killing effect after GCV application was determined with MTT method. Statistical analysis was performed with ANOVA analysis. Results Identification of pDC316-tkEGFP-P4 by enzyme digestion and sequencing analysis showed that the construction of the recombinant shuttle plasmid was correct. It was found that green fluorescence protein could only be seen in HepG2 cells, not in HeLa cells. The results of RT-PCRshowed only two bands could be seen in the samples of pDC316-tkEGFP-P4 transfected HepG2 cells. The growth of HepG2 ceils transfected with pDC316-tkEGFP-CMV and pDC316-tkEGFP-P4 were inhibited remarkably, the growth inhibition rates were 6.95%±0.67%, 24.99% ± 1.53%, 49.68% ± 1.68%, 71.85% ±3.28% and 4.83%± 0.35% vs 17.34%±1.15%, 30.17% ± 1.30%, 40.39% ±0.82% （F = 24.055, P 〈 0.05）, respectively. The growth of HeLa cells transfected with pDC316-tkEGFP-CMV were also inhibed, the growth inhibition rates were 6.36%±0.83%, 23.95%± 1.72%, 45.13% ±1.64% and 69.38%± 3.17%, respectively. The growth of HeLa cells transfected with pDC316-tkEGFP-P4 was not inhibed. The growth inhibition rates were 0.91 ±0.04, 1.18 ± 1.32, 1.19 ± 0.10 and 1.32 ±0.05 （F = 26.469, P 〈 0.01）, respectively. Conclusion The shuttle plasmid vector carrying the tkEGFP fusion protein gene driven by IGF-Ⅱ P4 promoter has been constructed successfully and its specific expression in HepG2 cells provided the basis for targeted gene therapy