缺氧诱导的肝癌靶向性基因治疗载体的构建和检测  

Construction and verification of a hypoxia-induced gene therapy vector targeting hepatocellular carcinoma

作　　者：禄韶英[1] 王燕[2] 孙学军[1] 白纪刚[1] 张伟[1] 

机构地区：[1]西安交通大学医学院第一附属医院普通外科,西安710061 [2]第四军医大学唐都医院中心实验室,西安710038

出　　处：《中国肿瘤生物治疗杂志》2006年第1期59-62,共4页Chinese Journal of Cancer Biotherapy

基　　金：国家自然科学基金(No.30400430)

摘　　要：目的:构建缺氧诱导的AFP基因启动子(HRE-AFPp)调控的基因表达载体,并检测该调控元件的特异性和对缺氧诱导的反应性。方法:用PCR方法从人类基因组中扩增VEGF基因缺氧反应元件(HRE)和AFP基因启动子(AFPp),将上述片段与含有报告基因(强化绿色荧光蛋白基因)载体pEGFP-1的多克隆位点连接,构建成为缺氧诱导的AFP基因启动子调控的基因表达载体(pEGFP-[HRE]AFPp)。用脂质体法将表达载体转染表达或不表达AFP的细胞系,G418筛选阳性克隆,荧光显微镜观测重组AFPp的活性,并用流式细胞术检测缺氧诱导是否对其有调控作用。结果:成功地将HRE、AFPp克隆到报告基因载体pWGFP-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,流式细胞术检测证实,16 h缺氧诱导能增强EGFP的表达(P<0．01)。结论:缺氧诱导的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 an element （HRE-AFPp） containing the hypoxia-induced enhancer （HRE） of the human VEGF gene and promoter of AFP gene （ AFPp）, and to verify the specificity and its activity to hypoxia stimulation. Methods： The DNA segments of AFPp and HRE were synthesized through PCR from human genome DNA. Gene fragments were then cloned into the multiple cloning sites of non-promoter EGFP vector pEGFP-1. The recombinant plasmid was transferred into positive or negative AFP cell lines （HepG2, SMMC-7721 and Cos-7 ） by means of lipofectaminc 2000TM. The expression of EGFP was tested by fluorescence microscope, The effect of hypoxia treatment on the expression of EGFP was tested by Flow Cytometry. Results： The length, position and orientation of HRE-AFPp were confirmed with restriction digestion and sequence analyses. The expression of EGFP was only detected in AFP-producing tumor cells, The expression rate of EGFP in pEGFP-HRE-AFPp and pEGFP-AFPp transfected HepG2 cells （ AFP positive） were 6.1% and 7.3%, respectively. Cos-7, SMMC-7721 cells （ AFP negative） and pEGFP-1, pEGFP-HRE transfected HepG2 cells did not express EGFP. EGFP expression in pEGFP-HRE-AFPp transfected cell line with and without hypoxic treatment were （35, 1 ±5.9） % and （ 12.5 ±3, 1 ） %, respectively （ P 〈0.01 ） ; and in pEGFP-AFPp transfected cell line with or without hypoxic treatment were （ 14.6 ±3.2）% and （ 13.4 ±2, 6） % . The expression of EGFP in pEGFP-HRE-AFPp and pEGFP-AFPp transfected cell lines were significantly different regardless of hypoxia treatment （P 〈 0. 01 ）. Conclusion： This recombinant vector constructed in this article can be used as a vector targeting for AFP-producing hepatocellular carcinoma and the activity of genes in this vector can be regulated by hypoxia treatment.

关 键 词：甲胎蛋白基因 缺氧反应元件 肝细胞癌 

分 类 号：R735.7[医药卫生—肿瘤] R730.54[医药卫生—临床医学]