受缺氧反应元件调控的荧光素酶报告基因在缺氧肿瘤细胞内的表达活性研究  

The study on hypoxia response elements-regulated luciferase expression in hypoxic tumor cells

作　　者：沈晓娣[1] 张萍[1] 应磊[1] 葛盛芳[1] 钱关祥[1] 

机构地区：[1]上海交通大学基础医学院生物化学与分子生物学教研室,200025

出　　处：《肿瘤研究与临床》2007年第1期5-7,10,共4页Cancer Research and Clinic

基　　金：上海市科委重点基础研究基金资助项目（04JC14041）：国家基础研究资助项目（“973”项目,2004CB518804）;国家自然科学基金资助项目（30400536）

摘　　要：目的研究缺氧反应元件调控的荧光素酶报告基因在肿瘤细胞内的缺氧反应性。方法将pGL3—6HRE—TATA—luciferase—SV40pa报告载体以Lipofectamine2000介导瞬时转染Helas3和HepG2细胞，分别于缺氧（1％O2）和常氧（21％O2）培养后检测相对荧光素酶活性。结果pGL3—6HRE—TATA—luciferase—SV40pa报告载体转染细胞后，缺氧培养条件下相对荧光素酶的活性较常氧培养条件下显著上调，差异具有统计学意义（P〈0．01），并且该缺氧诱导性具有一定的时间依赖性，即Helas3细胞中，缺氧培养12—24h，相对荧光素酶的活性上升到了最高值；而HepG2细胞中，缺氧培养36h，相对荧光素酶的活性上升到了最高值。结论6HRE人工启动子在肿瘤细胞内具有显著的缺氧诱导性；6HRE人工启动子缺氧诱导性具有一定的时间效应，并且在不同肿瘤细胞中其诱导效应和时间效应可存在着差异。研究结果为实体肿瘤基因治疗奠定了理论和实验基础。Objective To study the hypoxic inducible activity of luciferase gene driven by hypoxia response elements in tumor cells. Method Luciferase activity was assayed under hypoxic （1% O2） or normoxic （21% 02） condition after pGL3-6HRE-TATA-luciferase-SV40pa was transiently transfected into HeLa S3 and HepG2 cells using Lipofeetamine 2000. Results Lueiferase activity was significantly increased under hypoxie condition compared to normoxic condition after pGL3-6HRE-TATA-luciferase-SV40pa was transfect- ed into cells（P 〈0.01）. We also found that the hypoxic inducible effect driven by 6HREs was in a time-dependent manner and was maximal after 12-24 hours incubated in hypoxic condition in HeLa S3 cell line, while 36 hours in HepG2 cell line by further studies. Conclusions Hypoxic inducible effect was significantly enhanced in tumor cells by hypoxia through the approach of 6HREs artificial promoter. The hypoxic inducible effect driven by 6HREs was in a time-dependent manner, which may be different in different tumor cell lines. Our study provided the theoretical and experimental basis for cancer gene therapies on malignant cancers.

关 键 词：细胞系 肿瘤 反应元件 细胞低氧 pGL3-6HRE-TATA-luciferase—SV40pa 

分 类 号：R730[医药卫生—肿瘤]