缺氧诱导人视网膜色素上皮细胞19中血管内皮生长因子mRNA表达及金雀异黄素的抑制作用  

Hypoxia-induced vascular endothelial growth factor mRNA expression in human retinal pigment epithelial-19 cells and the inhibitive effects of genistein

作　　者：袁志兰[1] 周青[1] 王斌[2] 戈应滨[3] 袁孝如[3] 

机构地区：[1]南京医科大学第一附属医院眼科,江苏省南京市210029 [2]南京医科大学药理学系,江苏省南京市210029 [3]南京医科大学生理学系,江苏省南京市210029

出　　处：《中国临床康复》2005年第18期163-165,共3页Chinese Journal of Clinical Rehabilitation

基　　金：国家自然科学基金资助项目(30000207)~~

摘　　要：目的:观察缺氧对人视网膜色素上皮细胞19中血管内皮生长因子mR-NA表达的诱导作用,以及金雀异黄素对其血管内皮生长因子mRNA和蛋白表达的抑制作用。方法:实验于2004-02/12在南京医科大学生理学实验室完成。视网膜色素上皮细胞缺氧(体积分数为0.05的CO2/体积分数为0.95的N2)2,12,24,36h后,用反转录聚合酶链反应检测该区域血管内皮生长因子mRNA表达;细胞用50,100,200μmol/L金雀异黄素和50μmol/LPD98059预处理后缺氧2和24h,用反转录聚合酶链反应检测人视网膜色素上皮细胞19细胞中血管内皮生长因子mRNA表达,用酶联免疫吸附分析检测细胞培养液上清中血管内皮生长因子蛋白表达。结果:①缺氧2,12,24,36h人视网膜色素上皮细胞19细胞血管内皮生长因子mRNA表达是正常组的2.6,3.1,8.4,2.9倍(P<0.05,n=8)。②缺氧2h:50,100,200μmol/L金雀异黄素和50μmol/LPD98059可以抑制缺氧组血管内皮生长因子mRNA表达的51.1%,71.6%,79.7%和55%(P=0.01,n=10),蛋白表达分别为(97.00±12.79),(58.85±20.21),(37.93±14.41),(57.83±9.66)ng/L。③缺氧24h:50,100,200μmol/L金雀异黄素和50μmol/LPD98059可以抑制缺氧组血管内皮生长因子mRNA表达的55.0%,78.7%,90.7%和67.2%(P=0.000,n=10),蛋白表达分别为(189.60±20.20),(117.70±21.97),(49.7±13.17),(86.33±12.47)ng/L。结论:缺氧对血管内皮生长因子mRNA表达的诱导呈时间依赖性;金雀异黄素可以在转录及转录后水平抑制缺氧诱导的人视网膜色素上皮细胞19细胞中血管内皮生长因子表达的上调。金雀异黄素对血管内皮生长因子mRNA和蛋白表达的抑制作用也呈浓度依赖性。AIM:To investigate the induction of vascular endothelial growth factor(VEGF) mRNA expression by hypoxia in human retinal pigment epithelial(PRE) cells,ARPE- 19 cells,and probe into the inhibitive effects of genistein on the VEGF mRNA an d protein expression. METHODS:The experiment was finished in the Laboratory for Physiology of Nanji ng Medical University from February to December 2004.RPE cells were exposed to h ypoxia(50 mL/L CO2 or 95 mL/L N2) for 2,12,24 and 36 hours,then the expression o f VEGF mRNA was examined by reverse transcript ploymerase chain reaction(RT- PC R).The RPE cells pretreated with 50,100,200 μ mol/L genistein and 50 μ mol/L P D98059 were exposed to hypoxia for 2 and 24 hours,then RT- PCR was used to exam ine the VEGF mRNA expression in ARPE- 19 cells,and enzyme linked immunosorbent assay(ELISA) to detect the VEGF protein expression in the culture medium. RESULTS:① Hypoxia could increase the VEGF mRNA expression in ARPE- 19 cells 2.6,3.1,8.4,2.9 times in hypoxia group as much as the normal control group at 2 ,12,24 and 36 hours(P< 0.05,n=8).② Hypoxia for 2 hours:the VEGF mRNA expression could be inhibited by 51.1% ,71.6% ,79.7% and 55% by 50,100,200 μ mol/L genistein and 50 μ mol/L PD98059 of the hypoxia group(P= 0.01,n=10),and the pro tein level in the culture medium was (97.00± 12.79),(58.85 ± 20.21),( 37.93± 14.41) and (57.83± 9.66) ng/L.③ Hypoxia for 24 hours:the VEGF mRNA exp ression could be inhibited by 55.0% ,78.7% ,90.7% and 67.2% by 50,100,200 μ mol/L genistein and 50 μ mol/L PD98059 of the hypoxia group(P=0.000,n=10),th e protein level in the culture medium was(189.60± 20.20),(117.70± 21.97),(49.7 ± 13.17) and (86.33± 12.47) ng/L. CONCLUSION:Hypoxia can induce the VEGF mRNA expression in a time- dependent manner.Genistein can suppress the upregulation of hypoxia- induced VEGF express ion in AREP- 19 cells flatly during and after RT- PCR,and inhibit the expressi on of VEGF mRNA and protein concentration- dependently.

关 键 词：视网膜 色素上皮 眼 上皮细胞 内皮生长因子 

分 类 号：R774.1[医药卫生—眼科]