中文摘要：

目的 构建人胰岛素样生长因子Ⅱ基因（IGF-Ⅱ）P3启动子驱动的单纯疱疹病毒胸苷激酶（HSV-tk）与增强型绿色荧光蛋白（EGFP）融合基因穿梭质粒，观察其在肝癌细胞株HepG2及宫颈癌细胞株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HSV-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，48h后荧光显微镜下观察荧光表达，逆转录聚合酶链反应（RT-PCR）检测HSV-tk和EGFP的mRNA表达，四甲基偶氮唑蓝实验检测转染融合蛋白载体后给予不同浓度的更昔洛韦对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HSV-tk和EGFP的mRNA仅在HepG2细胞中表达；更昔洛韦（GCV）对转染了携此质粒载体的HepG2细胞有选择性细胞毒作用，1、10、和100μg／ml 3个浓度GCV作用72h后（实验均重复3次），对转染细胞的抑制率分别为19．8％±1．3％、36、2％±2．0％和48．7％±1．9％，与细胞病毒转染HepG2组（24．1％±1．9％、45．1％±1．7％、69．4％±3．6％）和此质粒转染HeLa细胞组（25．1％±1．6％、49．3％±1．1％、72．2％±2．9％）相比，差异有统计学意义（均P〈0．01）。结论成功构建IGF-Ⅱ P3启动子驱动携带HSV-HSV-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 construct a shuttle plasmid vector of fused herpes simplex virus thymidine kinase （HSV-tk） gene and enhanced green fluorescent protein （EGFP） gene driven by human insulin-like growth factor Ⅱ （ IGF-Ⅱ ） P3 promoter, and investigate the special killing effect of the HSV-tk/ganciclovir （GCV） system on hepatocellular carcinoma （HCC） cells. Methods An adenovirus shuttle plasmid, pDC316-tkEGFP-CMV containing fused genes tkEGFP and an adenovirus shuttle plasmid pDC316-tkEGFP-P3 driven by IGF-Ⅱ P3 promoter were constructed by techniques of gene recombination and screening, and identified by restriction digestion and sequencing analysis. Human hepatocellular carcinoma cells HepG2 and human cervical carcinoma cells HeLa were cultured and transfected with these 2 recombinant shuttle plasmids. RT-PCR was used to detect the mRNA expression of EGFP and HSV/tk. GCV of the final concentrations of 0, 1, 10, and 100 μg/ml respectively was added into the culture fluid of the HepG2 cells transfected with pDC316-tkEGFP-CMV or pDC316-tkEGFP-P3, and MTT method was used to detect the cell inhibition rate. Results Digestion and sequencing analysis showed that the recombinant plasmid pDC316- tkEGFP-P3 accorded with the design. Fluorescent microscopy showed that EGFP was expressed only in the HepG2 cells, but not in the HeLa cells. RT-PCR showed that mRNA expression of EGFP and HSV/tk could be seen in both HepG2 and HeLa cells transfected with pDC316-tkEGFP-CMV or pDC316-tkEGFP-P3, however, only in the pDC316-tkEGFP-P3 transfected HepG2 ceils, but not in the HeLa ceils transfected with pDC316-tkEGFP-P3. MTT assay showed that GCV dose-dependently inhibited the 2 cancer cells, the inhibition rates of GCV of the final concentrations of 1, 10, and 100 μg/ml were 24. 1%±1.9%, 45.1%±1.7%, and 69.4%±3.6% inthe HepG2 cells , and 25.1%±1.6%, 49.3%±1.1%, and 72.2%±2.9% in the HeLa cells. However, the inhibition rates of the pDC316-tkEGFP-P3-trasnsfected HepG2 cells by GCV of the final concentrations of