CAJ | 학술논문

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) 人視網膜血管內皮細胞(Human retinal microvascular endothelial cells,HRECs)%乙酰肝素酶(Heparanase,HPA)%血管內皮生長因子(Vascular endothelial growth factor,VEGF)%染色質免疫沉澱法(Chromatin Immunoprecipitation,ChIP)
인시망막혈관내피세포(Human retinal microvascular endothelial cells,HRECs)%을선간소매(Heparanase,HPA)%혈관내피생장인자(Vascular endothelial growth factor,VEGF)%염색질면역침정법(Chromatin Immunoprecipitation,ChIP)
Human retinal microvascular endothelial cells (HRECs)%Heparanase (HPA)%Vascular endothelial growth factor (VEGF)%Chromatin Immunoprecipitation (ChIP)

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Cocl2100 μmol/L的培养液培养48 h).免疫荧光染色法观察HPA与Pol Ⅱ的表达;免疫沉淀法半定量分析HPA及Pol Ⅱ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Pol Ⅱ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Pol Ⅱ相互结合共同作用,而对照组中HPA及Pol Ⅱ则无结合.(3) ChIP联合实时定量PCR实验结果显示HPA及Pol Ⅱ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Pol Ⅱ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.
목적 통과관찰저양유도인시망막혈관내피세포(HRECs)포핵중을선간소매(HPA)화RNA취합매Ⅱ (Pol Ⅱ)적표체분포、량자상호결합급기여혈관내피생장인자(VEGF)기인계동자적작용,탐토재저양유도시망막신생혈관형성중HPA대VEGF기인전록조공적작용.방법 용저양모형모의제Cocl2건립저양모형(함Cocl2100 μmol/L적배양액배양48 h).면역형광염색법관찰HPA여Pol Ⅱ적표체;면역침정법반정량분석HPA급Pol Ⅱ적상호결합작용;염색질면역침정(ChIP)연합실시정량PCR분석량조세포중HPA여VEGF기인계동자지간적상호작용.결과 (1)면역형광염색결과현시저양유도조HRECs포핵내HPA형광화Pol Ⅱ형광균교정상대조조증강,차저양유도조포핵내HPA분포여PolⅡ상문합.(2)면역침정결과표명재저양유도HRECs중HPA급Pol Ⅱ상호결합공동작용,이대조조중HPA급Pol Ⅱ칙무결합.(3) ChIP연합실시정량PCR실험결과현시HPA급Pol Ⅱ여VEGF계동자결합적고발생구단주요위우VEGF계동자서렬-1165여-984지간,저양유도HRECs세포핵내HPA급Pol Ⅱ결합VEGF기인계동자수평균교정상조승고(t=-13.591,P=0.001;t=-3.188,P =0.049).결론 재저양유도적HRECs중,핵내HPA여VEGF기인계동자직접결합,병삼여료VEGF기인전록활성적조공.
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 hypoxia 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 100μtmol/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-1combined with Pol Ⅱ in HRECs cells treated with Cocl2,while no interaction of two proteins existed in normal HRECs cells.Real-time PCR-based ChIP results showed the high-confidence HPA-binding 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.