人脂肪酸合成酶FAS启动子的克隆及其在几种肿瘤细胞中的转录活性分析  

Clone of human fatty acid synthase promoter and analysis of its transcriptional activity in tumor cells

作　　者：陈衍[1] 刘文超[1] 贾军 秦鸿雁[3] 韩骅[3] 

机构地区：[1]第四军医大学附属西京医院肿瘤内科,陕西西安710032 [2]陕西省肿瘤医院,陕西西安710061 [3]第四军医大学遗传和发育教研室,陕西西安710032

出　　处：《现代肿瘤医学》2006年第12期1485-1488,共4页Journal of Modern Oncology

基　　金：国家自然科学基金项目(项目编号:30471944)

摘　　要：目的:克隆人脂肪酸合成酶基因FAS启动子的序列,构建重组荧光素酶表达载体pGL3-FAS,并分析其在几种肿瘤细胞中的转录活性,为其作为肿瘤靶向基因治疗的工具提供依据。方法:以人基因组DNA为模板,PCR扩增FAS启动子区680bp活性序列,经测序鉴定正确后,克隆至荧光素酶表达载体pGL3-enhancer中,构建成重组荧光素酶表达载体pGL3-FAS。将pGL3-FAS通过脂质体转染胃癌细胞系SGC-7901、人宫颈癌细胞系Hela、人乳腺癌细胞系SKBR3、人肝癌细胞系HepG2以及NIH3T3成纤维细胞系中,应用荧光素酶检测系统测定荧光素酶活性,通过内参校正得到相对转录活性。结果:成功扩增出大小约为680bp的FAS启动子序列,经测序鉴定与Genebank报道的一致。经酶切鉴定,成功构建重组荧光素酶表达载体pGL3-FAS。瞬时转染pGL3-FAS,发现其在SGC-7901、Hela、SKBR3、HepG2细胞中均具有较强的荧光素酶活性,且荧光素酶活性高于强启动子SV40驱动的pGL3-control载体;而在正常成纤维细胞中,转录活性较低。结论:FAS启动子在肿瘤细胞中具有强转录活性,而在正常细胞中转录活性很低,具有良好的肿瘤靶向性,为肿瘤靶向基因治疗提供理论依据。Objectlve：To clone the human fatty acid synthase gene promoter, construct the recombinant luciferase reporter plasmid pGL3 - FAS and detect its transcriptional activities in several tumor cell lines and in NIH3T3 fibroblast cells in order to provide theoretical evidence for its use as tool for tumor targeted gene therapy. Methods： The fragment of FAS promoter was acquired by PCR amplification from human genome DNA and cloned into the luciferase reporter plasmid pGL3 - enhance to construct pGL3 - FAS. pGL3 - FAS was transient transfected into human gastric carcinoma cell lines SGC -7901, human cervical carcinoma cell lines Hela, human breast cancer cell lines SKBR3, human hepatic cancer cell lines HepG2 and NIH3T3 fibroblast cells lines. The transcriptional activities of FAS promoter in these cells were detected by Luciferase Reporter Assay System. Results： The 680 bp hFAS promoter was successfully amplified by PCR and the sequence was accordance to the one published on Genebank. We successfully constructed the pGL3 - FAS recombinant luciferase reporter vector. The hFAS promoter showed high transcriptional activities in all tumor cell lines but little in NIH3T3 fibroblast cells. The transcriptional activities of FAS promoter in tumor cells were significantly stronger than SV40 strong promoter. Conclusion： FAS promoter possesses the tumorspecific high transcriptional activity in tumor cell lines. It may serve as a useful tool for tumor targeted gene therapy.

关 键 词：脂肪酸合成酶 启动子 转录活性 靶向治疗 

分 类 号：R730.54[医药卫生—肿瘤]