可调控的肝癌特异性基因治疗载体的构建

Construction of a regulatable gene therapy vector specific for hepatocellular carcinoma

目的 构建新型AFP顺式作用元件调控的基因表达载体 ,检测该调控元件的特异性和可调控性。方法 PCR扩增截短的AFP基因启动子、增强子 ,将上述片段与含有报告基因强化绿色荧光蛋白基因的载体pEGFP 1的多克隆位点连接 ,构建成为AFP基因顺式作用元件调控的肝癌特异性EGFP表达载体 (pEGFP 1 EP)。用脂质体法将表达载体转染表达或不表达AFP的肿瘤细胞系 ,荧光显微镜观测重组AFP顺式作用元件的启动活性 ,并用流式细胞术检测全反式视黄酸对它的抑制作用。结果 成功地将AFP基因启动子、增强子克隆到报告基因载体pEGFP 1的多克隆位点 ,酶切鉴定和DNA序列分析无误 ,荧光显微镜观测证实EGFP能在AFP阳性肝癌细胞特异性表达 ,1× 10 -7M全反式视黄酸对其活性有明显的抑制。结论 AFP顺式作用元件修饰的基因治疗载体 ,在基因转录水平特异性调控目的基因的表达 ,为下一步将其作为肝癌基因治疗载体奠定了基础。

Objective To construct a gene modified hepatocellular carcinoma (HCC) specific EGFP expression vector regulated by abbreviated cis acting element of AFP. Methods The DNA segments of minimum essential AFP enhancer and promoter were synthesized through PCR from Genome DNA of HepG2 cells. The gene fragments were then cloned into the multiple cloning site of promoterless EGFP vector pEGFP 1. Recombinant plasmid was transfected into AFP positive or negative cell lines by the means of lipofectamine. The expression of EGFP was tested by fluorescent microscope and flow cytometry. Results By the methods of restriction digestion and sequence analysis, we confirmed that the length, position and orientation of inserted genes of cis acting element of AFP were all correct. The activity of AFP gene promoter was significantly suppressed by the adding of 2 5×10 -7 M all trans retinoic acid (P<0 05). Conclusions This recombinant expression vector can be used as a gene therapy vector for HCC. The expression of tumor killing gene will be confined just within the site of tumor and the activity of which can be suppressed by all trans retinoic acid.