CAJ | 학술논문

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CoCl2 100 μmol/ml的培养液培养48 h;PI 88组为含CoCl2 100 μmol/L和Hpa竞争性抑制剂PI-88 5 μg/ml的培养液干预48 h；空白对照组为等量PBS干预HREC 48 h.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)；PI-88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.
목적 관찰인시망막혈관내피세포(HREC)저양모형중을선간소매(Hpa)、혈관내피생장인자( VEGF)화RNA취합매-Ⅱ(Pol-Ⅱ)적표체변화,탐토저양성시망막신생혈관형성중Hpa화VEGF적상관성급가능궤제.방법 사용저양모의제록화고(CoCl2)조성HREC저양모형,분위4조,분별위정상대조조、저양유도조、린산감로무당류산염(PI-88)조화공백대조조.저양유도조위함CoCl2 100 μmol/ml적배양액배양48 h;PI 88조위함CoCl2 100 μmol/L화Hpa경쟁성억제제PI-88 5 μg/ml적배양액간예48 h；공백대조조위등량PBS간예HREC 48 h.면역형광염색법관찰정상대조조화저양유도조HREC중Hpa、VEGF이급PolⅡ적표체.단백질면역인적(Western blot)검측각조간Hpa화VEGF단백표체변화.결과 면역형광염색결과현시,저양유도조HREC세포질내Hpa형광화VEGF형광균교정상대조조증강；PI-88조세포질내VEGF형광교저양유도조감약.저양유도조세포핵내Hpa교정상대조조명현증강,차분포여Pol-Ⅱ상문합.Western blot검측결과현시,여정상대조조비교,저양유도조Hpa단백화VEGF단백표체균명현승고,차이유통계학의의(Hpa:F=-4.005,P＜0.05；VEGF:F=-4.063,P＜0.05)；PI-88조VEGF단백표체교저양유도조VEGF단백표체강저,차이유통계학의의(F=5.963,P＜0.05).결론 저양유도적HREC중,Hpa적표체증고,도치VEGF적증가,촉진료시망막신생혈관형성.
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 ＜ 0.05 ; VEGF: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.