低氧诱导人视网膜微血管内皮细胞核中乙酰肝素酶对血管内皮生长因子基因转录调控研究  

Heparanase regulates transcription activity of VEGF gene in human tetinal microvascular endothelial cells induced by hypoxia

作　　者：王敬伟[1] 胡洁[1] 宋新[1] 冷炫[1] 田景毅[1] 吕林[1] 

机构地区：[1]中山大学中山眼科中心眼科学国家重点实验室,广州510060

出　　处：《中国实用眼科杂志》2013年第9期1195-1199,共5页Chinese Journal of Practical Ophthalmology

基　　金：国家自然科学基金面上项目（81271009）;广东省科技计划社会发展项目（20118020800005）;

摘　　要：目的通过观察低氧诱导人视网膜血管内皮细胞（HRECs）胞核中乙酰肝素酶（HPA）和RNA聚合酶II（Pol Ⅱ）的表达分布、两者相互结合及其与血管内皮生长因子（VEGF）基因启动子的作用，探讨在低氧诱导视网膜新生血管形成中HPA对VEGF基因转录调控的作用。方法用低氧模型模拟剂Cocl2建立低氧模型（含Cocl2100μmol／L的培养液培养48h）。免疫荧光染色法观察HPA与PolⅡ的表达；免疫沉淀法半定量分析HPA及PolⅡ的相互结合作用；染色质免疫沉淀（ChIP）联合实时定量PCR分析两组细胞中HPA与VEGF基因启动子之间的相互作用。结果（1）免疫荧光染色结果显示低氧诱导组HRECs胞核内HPA荧光和PolII荧光均较正常对照组增强，且低氧诱导组胞核内HPA分布与PolⅡ相吻合。（2）免疫沉淀结果表明在低氧诱导HRECs中HPA及PolⅡ相互结合共同作用，而对照组中HPA及PolⅡ则无结合。（3）ChIP联合实时定量PCR实验结果显示HPA及PolⅡ与VEGF启动子结合的高发生区段主要位于VEGF启动子序列-1165与-984之间，低氧诱导HRECs细胞核内HPA及PolII结合VEGF基因启动子水平均较正常组升高（t=-13．591，P=0．001；t=-3．188，P=0．049）。结论在低氧诱导的HRECs中，核内HPA与VEGF基因启动子直接结合，并参与了VEGF基因转录活性的调控。Objective To observe the nuclear expression and interaction of heparanase （HPA） and RNA polymerase Ⅱ （Pol Ⅱ） in human retinal microvascular endothelial cells （HRECs） under hy- poxia condition and to investigate the association of heparanase with the transcription activity of VEGF gene promoter. Methods Cultured HRECs were maintained for 48h in media with 100μmol/ L Coc12 or normal solution. The expression of heparanase, Pol Ⅱ in each group was analyzed with double immunofluorescence. Immunoprecipitation was applied to detect the interaction of heparanase and Pol Ⅱ proteins. Cells in both groups were used for Chromatin Immunoprecipitation （CHIP） with anti-HPA and anti-Pol Ⅱ antibodies to identify high-confidence HPA-binding regions across the entire VEGF gene promoter, and combine real-time PCR to demonstrate the interaction between heparanase and the VEGF gene promoter region. Results Double immunofluorescence studies showed that the expression of heparanase in cytoplasm was intense in hypoxia-induced HRECs, but faint in normal group; heparanase and Pol Ⅱ in nucleu was also intense in hypoxia HRECs, and the distribution of heparanase was consistent with that of Pol Ⅱ. Immunoprecipitation data showed that heparanase-1 combined with Pol Ⅱ in HRECs cells treated with Cocl2, while no interaction of two proteins exist- ed in normal HRECs cells. Real-time PCR-based ChIP results showed the high-confidence HPA-bind- ing regions was -1155 to -1018 （containing hypoxia response element） in VEGF gene promoter, and the cells treated with 100μmol/L Coc12 showed an increase of heparanase and Pol Ⅱ bonding in VEGF gene promoter region, compared with the normal cells （t =-13.591, P-0.001; t =-3.188, P=0.049, respectively）. Conclusions Nuclear heparanase is directly combined to VEGF gene promoter, and involved in the regulation of VEGF gene transcription in hrpoxia HRECs.

关 键 词：人视网膜血管内皮细胞（Human retinal MICROVASCULAR ENDOTHELIAL cells HRECs） 乙酰肝素酶（Heparanase HPA） 血管内皮生长因子（Vascular ENDOTHELIAL growth factor  VEGF） 染色质免疫沉淀法（Chromatin IMMUNOPRECIPITATION  ChIP） 

分 类 号：R774[医药卫生—眼科]