以HIF2α为靶点的化合物筛选的细胞模型构建  

Constructing a cell model for compounds screening targeted on HIF2α

作　　者：陈中原 皇昊 申淑娜 张博奇 樊范 杜珂欣 刘云章[1] 李筠[1] 卢玲[1] CHEN Zhong-yuan;HUANG Hao;SHEN Shu-na;ZHANG Bo-qi;FAN Fan;DU Ke-xin;LIU Yun-zhang;LI Yun;LU Ling(Key Laboratory of Marine Drugs,Ministry of Education,School of Medicine and Pharmacy,Ocean University of China,Qingdao 266003,China)

机构地区：[1]中国海洋大学海洋药物教育部重点实验室医药学院,山东青岛266003

出　　处：《中国海洋药物》2022年第6期69-75,共7页Chinese Journal of Marine Drugs

基　　金：国家自然科学基金项目(32171141)资助。

摘　　要：目的建立稳定表达血管内皮生长因子A(VEGFA)启动子区5×缺氧反应元件(5×HRE)和萤光素酶(Luc)报告基因的人肾透明细胞癌(786-O)单克隆细胞系,构建以缺氧诱导因子2α(HIF2α)为靶点的化合物筛选的细胞模型,并对其应用加以初步验证。方法采用基因重组技术,构建含有VEGFA启动子区5×HRE以及Luc报告基因的慢病毒载体,并感染786-O细胞,经过嘌呤霉素筛选获得能稳定表达Luc活性的786-O-5×HRE-Luc细胞系;采用等浓度梯度稀释获得单克隆细胞系,运用HIF2α抑制剂PT2385和PT2399处理,检测Luc活性变化,筛选对PT2385和PT2399反应更灵敏的单克隆细胞系。结果通过基因测序鉴定,VEGFA启动子区5×HRE和Luc报告基因慢病毒表达载体构建成功,经过嘌呤霉素(Puro)筛选获得稳定表达Luc的786-O-5×HRE-Luc细胞;PT2385和PT2399可剂量依赖性降低细胞Luc的活性;得到对PT2385和PT2399反应灵敏的单克隆细胞系。结论建立了786-O-5×HRE-Luc稳定表达5×HRE和Luc报告基因细胞系,为高通量筛选靶向HIF2α的药物提供一类新的细胞模型。Objective To establish a human clear cell carcinoma monoclonal cell line stably expressing human vascular endothelial growth factor A(VEGFA)promoter 5×hypoxia response element(5×HRE)and luciferase(Luc)reporter gene and to preliminarily verify its application targeting hypoxia inducible factor 2α(HIF2α).Methods Using genetic recombination technology,the lentiviral vector carrying 5×HRE and Luc reporter sequence were designed and constructed to infect 786-O cells and obtain 786-O-5×HRE-Luc cell lines by the selection with puromycin.Monoclonal cell lines were obtained by equal concentration gradient dilution,and treated with HIF2αinhibitors PT2385 and PT2399 to detect the change of Luc activity,and screened monoclonal cell lines that were more sensitive to PT2385 and PT2399.Results Lentivirus-infected 786-O cells were screened by puromycin,and the 786-O-5×HRE-Luc cells stably expressing firefly Luc was obtained.PT2385 and PT2399 decreased the Luc luminescence value of 786-O-5×HRE-Luc cells in a dose-dependent manner,which provided a new cell model for high-throughput screening of compounds targeting HIF2α.Conclusion The cell line of 786-O-5×HRE-Luc containing VEGFA promoter 5×HRE and Luc reporter gene has been successfully constructed,which provides a new cell model for high throughput screening of HIF2α-targeting drugs.

关 键 词：786-O细胞 缺氧诱导因子 缺氧反应原件 萤光素酶报告基因 

分 类 号：R965[医药卫生—药理学]