MDR1启动子/荧光素酶质粒的构建及其在朊蛋白高表达胃癌细胞中的活性检测  

Construction of luciferase reporter gene vector containing MDRl promoter gene and MDR1 promoter activity in PrPc overexpressed gastric cancer cells

作　　者：葛伏林[1] 王婧[2] 周毅[2] 梁洁[1] 张燕齐[1] 郭雪艳[1] 丁杰[1] 

机构地区：[1]第四军医大学西京医院全军消化病研究所肿瘤生物学国家重点实验室,陕西西安710032 [2]解放军451医院消化内科,陕西西安710054

出　　处：《现代肿瘤医学》2007年第5期601-603,共3页Journal of Modern Oncology

基　　金：国家自然科学基金资助项目(30572134)

摘　　要：目的：构建含MDR1基因启动子的荧光素酶报告基因质粒,并检测其在朊蛋白高表达胃癌细胞系中的活性表达。方法：PCR克隆人MDR1基因启动子片段,通过亚克隆将启动子分别插入到pMD18-T载体和荧光素酶报告基因pGL3-Enhancer载体中,建立含MDR1启动子的荧光素酶报告基因质粒pGL-MDR1,并经测序及酶切确定扩增序列;脂质体基因转染法将pGL-MDR1转染入朊蛋白高表达胃癌细胞系SGC7901-PrP,并测定其荧光素酶活性。结果：PCR克隆出MDR1启动子经DNA测序证实序列正确,pGL-MDR1转染入朊蛋白高表达胃癌细胞系的荧光素酶活性,较转染入pcDNA3.1空载体细胞系相比升高3-5倍。结论：成功构建含MDR1启动子的荧光素酶报告基因质粒;上调朊蛋白表达可激活MDR1的转录活性。Objective： To construct the luciferase reporter vector containing MDR1 promoter gene and detect the Luc activity in PrPc overexpressed gastric cancer cells. Methods ：The promoter of the human MDR1 gene was amplified from human genomic DNA by PCR;then it was subcloned into pMD18 -T and then pGL3 - enhancer vector. The plasmid was determined by restriction enzyme digestion and DNA sequencing; then it was transfected into PrPc overexpressed gastric cancer cell line SGC7901 - PrP by Lipofectamine 2000. At last, the luc activity was detected. Resuits：After correctly constructed the pGL3 - MDR1 promoter plasmid, the luc activity in PrPc overexpressed gastric cancer cells were found to be 3 - 5 fold greater than those of empty vector control. Condusion： The luciferase reporter gene vector containing MDR1 promoter was successfully constructed and upregulation of PrPc expression could lead to the activation of MDR1 promoter in gastric cancer cells.

关 键 词：多药耐药基因1 启动子 朊蛋白 胃肿瘤 荧光素酶报告基因 

分 类 号：R735.2[医药卫生—肿瘤]