人肝癌细胞中缺氧反应元件对微环境的反应  

Response of hypoxia-response element in human liver cancer cells to different microenvironments

作　　者：王丰[1] 陈霞芳[1] 韦芳[1] 吴继红[1] 谢匡成[1] 田毓华[1] 易苗英[1] 李川源[2] 黄倩[1] 

机构地区：[1]上海交通大学附属第一人民医院中心实验室,上海200080 [2]美国科罗拉多大学健康科学中心放射肿瘤系,科罗拉多80010

出　　处：《肿瘤》2008年第5期367-371,共5页Tumor

基　　金：国家自然科学基金杰出青年项目(编号:30325043);国家重点基础研究计划(编号:2004CB518804);上海市卫生局百人计划(编号:99BR006);上海市青年科技启明星计划(编号:04QMX1417);上海市卫生局青年基金项目(编号:131014Y3)

摘　　要：目的:通过观察缺氧反应元件(hypoxia-response element,HRE)调控下,绿色荧光蛋白质(green fluorescent protein,GFP)构建的肝癌细胞Bel-7402/5HRE-EGFP在缺氧条件下,缺氧诱导因子1α(hypoxia-inducible factor 1α,HIF-1α)、血管内皮生长因子(vascular endothelial growth factor,VEGF)等表达水平的变化,研究HRE对微环境的反应特性。方法:以5个串连的HRE和巨细胞病毒(cytomegalovirus,CMV)的微小启动子为转录调控元件、GFP为报告基因构建表达载体,应用FCM和细胞免疫染色法观察缺氧、一氧化氮、过氧化物和酸性pH等微环境的变化对人肝癌细胞Bel-7402中HRE活性的影响,以及缺氧条件下HIF-1α、VEGF表达水平的变化。观察缺氧探针哌莫硝唑的染色强度和分布与HIF-1α、VEGF和GFP表达强度和分布之间的关系,探求裸鼠体内肿瘤组织缺氧对HRE活性及其相关基因表达的影响。结果:肝癌细胞中HRE对缺氧非常敏感,肿瘤细胞和组织缺氧时可上调HIF-1α和VEGF的表达,两者的分布也基本一致。结论:缺氧在调控肝癌血管生成因子表达方面可能起着重要的作用。Objective： Bel-7402 cells were stably transfected with a vector constructed with multiple copies of the hypoxia response-element （HRE） sequence of the human vascular endothelial growth factor （VEGF） gene and with the enhanced green fluorescent protein （EGFP） to establish a human hepatoma cell line Bel-7402/5HRE-EGFP. This paper aimed to study the responses of HRE in human liver cancer cells to different microenvironments by observing the changes in hypoxia-inducible factor 1 （HIF-1α） and vascular endothelial growth factor （VEGF） expression in Bel-7402/5HRE-EGFP cell lines under hypoxic conditions. Methods： The expression vector was constructed with 5 copies of HRE sequence and a minimal cytomegalovirus （CMV） as promoter and green fluorescent protein （GFP） as a reporter gene. The effect of different microenvironments such as hypoxia, H2O2 or acidic pH on the activity of HRE in Bel- 7402 cells and the changes in the expression levels of HIF-1α and VEGF under hypoxic condition were determined by using flow cytometry and immunohistochemical staining method. The association of the staining intensity and the distribution of pimonidazole, a hypoxic probe, with the expression and the distribution of HIF-1α, VEGF, and GFP were analyzed. The influence of hypoxia in tumor tissues of nude mice on the activity of HRE and expression of related genes were observed. Results： The HRE in liver cancer cells was very sensitive to hypoxia, which induces up-regulation of HIF-1α and VEGF expressions in tumor cells or in tumor tissues. Both distribution regions of HIF-1α and VEGF were almost the same. Conclusion： Hypoxia plays a pivotal role in controlling the expression of angiogenesisrelated factors in human liver cancer cells.

关 键 词：肝肿瘤 实验性 缺氧 缺氧诱导因子1 缺氧反应元件 绿色荧光蛋白质类 细胞 Bel-7402 

分 类 号：R735.7[医药卫生—肿瘤]