低氧诱导人视网膜血管内皮细胞中乙酰肝素酶和血管内皮生长因子相关性研究  

Correlation between heparanase and vascular endothelial growth factor in human retinal microvascular endothelial cells induced by hypoxia

作　　者：宋新[1] 胡洁[1] 袁玲[1] 李士清[1] 马红婕[1] 林少芬[1] 唐仕波[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：《中华眼底病杂志》2012年第1期57-61,共5页Chinese Journal of Ocular Fundus Diseases

基　　金：基金项目：眼科学国家重点实验室创新基金（2010C04）;广东省科技计划重点引进技术项目（2008B050100038）

摘　　要：目的观察人视网膜血管内皮细胞（HREC）低氧模型中乙酰肝素酶（Hpa）、血管内皮生长因子（VEGF）和RNA聚合酶-Ⅱ（Pol-Ⅱ）的表达变化，探讨低氧性视网膜新生血管形成中Hpa和VEGF的相关性及可能机制。方法使用低氧模拟剂氯化钴（CoCl2）造成HREC低氧模型，分为4组，分别为正常对照组、低氧诱导组、磷酸甘露戊糖硫酸盐（PI-88）组和空白对照组。低氧诱导组为含CoCl2100μmol／ml的培养液培养48h；PI-88组为含CoCl2100μmol／L和Hpa竞争性抑制剂PI-885μg／ml的培养液干预48h；空白对照组为等量PBS干预HREC48h。免疫荧光染色法观察正常对照组和低氧诱导组HREC中Hpa、VEGF以及PolⅡ的表达。蛋白质免疫印迹（Western blot）检测各组间Hpa和VEGF蛋白表达变化。结果免疫荧光染色结果显示，低氧诱导组HREC细胞质内Hpa荧光和VEGF荧光均较正常对照组增强；PI～88组细胞质内VEGF荧光较低氧诱导组减弱。低氧诱导组细胞核内Hpa较正常对照组明显增强，且分布与Pol-Ⅱ相吻合。Western blot检测结果显示，与正常对照组比较，低氧诱导组Hpa蛋白和VEGF蛋白表达均明显升高，差异有统计学意义（Hpa：F=-4．005，P〈0．05；VEGF：F=-4．063，P〈0．05）；PI88组VEGF蛋白表达较低氧诱导组VEGF蛋白表达降低，差异有统计学意义（F=5．963，P〈0．05）。结论低氧诱导的HREC中，Hpa的表达增高，导致VEGF的增加，促进了视网膜新生血管形成。Objective To investigate the effects of heparanase and vascular endothelial growth factor （VEGF） and their correlation in CoCl2-induced human retinal microvascular endothelial cells （HRECs） in an hypoxia model. Methods Human eyes were selected to establish CoCl2-induced HRECs hypoxia model in this study. Four experimental groups were studied： normal control group, hypoxia group （CoCl2 100 μmol/L, 48 hours）, PI 88 group （specific competitive inhibitor of heparanase： phosphomannopentaose sulfate, PI 88, 5 μg/ml, combined with CoCl2 100 μmol/L, 48 hours） and PBS control group. Heparanase, VEGF and Pol Ⅱ expression in HRECs of normal and hypoxia group were analyzed with immunofluorescence. Western blot was used to evaluate the expression of heparanase and VEGF in HRECs of normal, hypoxia, PI-88 and PBS control groups. Results Immunofluorescence studies showed that the expression of heparanase and VEGF in cytoplasm was intense in hypoxia HRECs, but faint in normal group. Heparanase was also observed in the nucleus of hypoxia HRECs. Western blot results showed that the expression of Hpa and VEGF protein was increased significantly in hypoxia group compared with normal group （Hpa.. F： =4. 005, P〈0.05VEGF..F=-4. 063, P〈0.05）, and VEGF was decreased in HRECs treated with PI-88 （F=5. 963, P〈0.05）. Conclusions Heparanase is up-regulated that resulted in increase of VEGF expression, therefore enhanced angiogenesis in CoCl2-induced hypoxia HRECs.

关 键 词：视网膜新生血管化/病理生理学 血管内皮生长因子类 缺氧 视网膜血管 内皮细胞 

分 类 号：R774.1[医药卫生—眼科]