Zhou Hong-ke, Yang Dong-hua, Tang Shao-hui, Huang Wei, Lu Xiao-hua
Department of Gastroenterlolgy, First Affiliated Hospital, Jinan University, Guangzhou 510630, China.
Zhonghua Yi Xue Za Zhi. 2006 Jan 10;86(2):106-10.
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.
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 microg/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.
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 cells, but not in the HeLa cells 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 microg/ml were 24.1% +/- 1.9%, 45.1% +/- 1.7%, and 69.4% +/- 3.6% in the 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-transfected HepG2 cells by GCV of the final concentrations of 1, 10, and 100 microg/ml wee 19.8% +/- 1.3%, 36.2% +/- 2.0% and 48.7% +/- 1.9% respectively, all significantly lower than those of the pDC316-tkEGFP-CMV-transfected HepG2 cells (all P < 0.01), and no significant cell inhibition was found in the HeLa cells transfected with pDC316-tkEGFP-CMV.
A shuttle plasmid vector containing the tkEGFP fusion protein gene driven by IGF-II P3 promoter has been constructed successfully and its specific expression in HepG2 cells provides a sound basis for targeted gene therapy for HCC.
构建人胰岛素样生长因子II（IGF-II）P3启动子驱动的单纯疱疹病毒胸苷激酶（HSV-tk）基因与增强型绿色荧光蛋白（EGFP）基因融合的穿梭质粒载体，并研究HSV-tk/更昔洛韦（GCV）系统对肝癌（HCC）细胞的特异性杀伤作用。
采用基因重组和筛选技术构建含融合基因tkEGFP的腺病毒穿梭质粒pDC316-tkEGFP-CMV及IGF-II P3启动子驱动的腺病毒穿梭质粒pDC316-tkEGFP-P3，经酶切及测序分析鉴定。培养人肝癌细胞HepG2和人宫颈癌细胞HeLa，并用这两种重组穿梭质粒进行转染。采用RT-PCR检测EGFP和HSV/tk的mRNA表达。将终浓度分别为0、1、10和100μg/ml的GCV加入转染了pDC316-tkEGFP-CMV或pDC316-tkEGFP-P3的HepG2细胞培养液中，采用MTT法检测细胞抑制率。
酶切及测序分析表明重组质粒pDC316-tkEGFP-P3符合设计要求。荧光显微镜观察显示EGFP仅在HepG2细胞中表达，而不在HeLa细胞中表达。RT-PCR显示转染pDC316-tkEGFP-CMV或pDC316-tkEGFP-P3的HepG2和HeLa细胞中均可见EGFP和HSV/tk的mRNA表达，但仅在转染pDC316-tkEGFP-P3的HepG2细胞中，而不在转染pDC316-tkEGFP-P3的HeLa细胞中。MTT法检测显示GCV对两种癌细胞均有剂量依赖性抑制作用，终浓度为1、10和100μg/ml的GCV对HepG2细胞的抑制率分别为24.1%±1.9%、45.1%±1.7%和69.4%±3.6%，对HeLa细胞的抑制率分别为25.1%±1.6%、49.3%±1.1%和72.2%±2.9%。然而，终浓度为1、10和100μg/ml的GCV对转染pDC316-tkEGFP-P3的HepG2细胞的抑制率分别为19.8%±1.3%、36.2%±2.0%和48.7%±1.9%，均显著低于转染pDC316-tkEGFP-CMV的HepG2细胞（均P<0.01），且转染pDC316-tkEGFP-CMV的HeLa细胞未发现明显的细胞抑制作用。
成功构建了IGF-II P3启动子驱动的含tkEGFP融合蛋白基因的穿梭质粒载体，其在HepG2细胞中的特异性表达为肝癌的靶向基因治疗奠定了良好基础。
