外源性脯氨酸羟化酶对人RPE细胞中缺氧诱导因子通路的负向调节作用  

Negative regulation of exogeneous polyi-4-hydroxylase domain proteins on hypoxia-inducible factor pathwayin human RPE cells

作　　者：马昱[1] 唐少华[1] 姜燕荣[2] 石璇[2] 

机构地区：[1]北京积水潭医院眼科,100044 [2]北京大学人民医院眼科,100034

出　　处：《中华实验眼科杂志》2016年第3期234-238,共5页Chinese Journal Of Experimental Ophthalmology

摘　　要：背景目前抗VEGF药物的应用已广泛用于眼部新生血管性疾病发病的治疗，但有部分患者的疗效并不理想，因此研究VEGF的上游基因缺氧诱导因子-1（HIF—1）及其限速酶脯氨酸羟化酶（PHDs）在新生血管形成中的作用具有重要的临床意义。目的探讨外源性PHDs在HIF-1激活通路中的负性调节作用。方法用Hela细胞提取RNA，采用逆转录PCR法从cDNA克隆PHD1、PHD2和PHD3，通过限制性内切酶构建pFLAG—PHD1、pFLAG—PHD2和pFLAG—PHD3质粒并通过基因测序进行鉴定。分别将人RPE细胞株（ARPE-19）在体积分数21％O2（常氧组）、1％O2（低氧组）和缺氧模拟剂（CoCl2，缺氧组）条件下进行培养，将pFLAG—PHD1、pFLAG—PHD2和pFLAG—PHD，质粒分别转染至培养的ARPE-19细胞中，pFLAG—CMV2转染作为空白对照。采用Westernblot法测定和比较转染细胞在不同氧环境培养下PHD1、PHD2和PHD3蛋白的表达强度；采用双荧光素酶报告系统评估各组细胞中HIF-1的转录活性。结果Westernblot法检测显示常氧组、低氧组和缺氧组ARPE-19细胞中均有PHD1、PHD2和PHD3蛋白的表达，各组细胞中PHD2的表达条带均强于PHD1和PHD3蛋白，差异均有统计学意义（P〈0．05）。pFLAG—CMX转染后常氧组细胞中内源性HIF-1活性反应低，低氧组和缺氧组细胞中HIF-1的转录活性明显升高，与空白对照组相比，pFLAG—PHD1、pFLAG—PHD2、pFLAG—PHD3转染后常氧组细胞中内源性HIF-1活性反应无明显变化（F=0．48，P〉0．05），而低氧及缺氧组细胞中HIF-1活性明显下降，与空白对照组比较差异均有统计学意义（F=24．30，112．67，均P〈0．05）。相同培养条件下，pFLAG—PHD：转染后细胞中HIF-1活性明显低于pFLAG—PHD，和pFLAG—PHD，转染细胞，差异均有统计学意义（均P〈0．05）。结论PHDs对人RPE细胞中HIF-1的激活通路有明显的负向调节作用，其对低氧细胞和缺氧细胞中HIBackground Anti-VEGF drugs are generally applied in the treatment of ocular neovascular diseases. However,the therapy effect is unsatisfactory in some patients. Studing the effect of hypoxia-indueible factor-1 （HIF-1） , a upstream regulatory gene of VEGF, and its limiting enzyme prolyl-4-hydroxylase domain proteins （PHDs） is of important clinical significance. Objective This study was to investigate the negtive regulation of exogeneous PHDs on HIF-1 pathway in human RPE cells. Methods pFLAG-PHD1 ,pFLAG-PHD2 and pFLAG-PHD3 plasmids were constructed by extracting RNA from Hela cell line and coloning PHD1, PHD2 and PHD3 using reverse transcription PCR with restriction enzyme. The plasmids were identified by gene sequencing. ARPE-19 cells were cultured at 21% 02 （ normoxia group） , 1% 02 （ hypoxia group） , or in hypoxia-mimieking agents （ CoC12 , anoxia group ） , respectively, and then were transfeeted with plasmids encoding FLAG-tagged PHDl , PHD2, PHD3 and pFLAG- CMV2 transfeeted cells served as blank control. The expressional intensities of PHD1, PHD2 and PHD3 in the cellswere detected and compared among different groups by using Western blot assay. The transcriptional activity of HIF-1 in the cells was evaluated with dual luciferase reporter assay. Results Western blot assay showed that PHD1 , PHD2 and PHD3 all were expressed in ARPE-19 cells in the normoxia group, hypoxia group and anoxia group. The expression was strong in PHD2 protein and was weak in PHD3 protein,a statistically significant difference was found between PHD2 protein expression and PHD1 or PHD3 expressions （all at P〈0. 05）. Endogenous HIF-1 activity was elevated in pFLAG-CMX transfected ceils in the hypoxia group and anoxia group than that in the normoxia group. Compared with pFLAG-CMX transfected ceils, no obvious normoxia group,however, HIF-1 activity was declined in change was seen in the endogenous HIF-1 activity in the the hypoxia group and anoxia group after pFLAG-PHD1, pFLAG-PHD2 or pFLAG-PHD3 transfection. Under

关 键 词：细胞缺氧 血管内皮生长因子 基因表达调控 缺氧诱导因子-1 脯氨酸羟化酶/代谢 人 转录因子 视网膜色素上皮细胞 

分 类 号：R77[医药卫生—眼科]