表皮生长因子对氧化损伤的人眼Müller细胞增生及迁移的保护作用  

Protective effect of epidermal growth factor on proliferation and migration of the human Miiller cell from oxidative damage

作　　者：陈春丽[1] 周仲楼[1] 闫东升[1] 郑景伟[1] 宋宗明[1] 

机构地区：[1]温州医学院附属眼视光医院,325027

出　　处：《中华实验眼科杂志》2012年第8期715-720,共6页Chinese Journal Of Experimental Ophthalmology

基　　金：浙江省自然科学基金项目（Y206706）

摘　　要：背景目前认为氧化损伤机制在年龄相关性黄斑变性（AMD）的发病中起着重要作用，主要与血一视网膜屏障的破坏有关，而Mailer细胞是稳定血一视网膜内屏障功能的主要细胞成分。研究表明，表皮生长因子（EGF）可促进实验动物视网膜Muller细胞的增生和迁移，但其对人Muller细胞的作用研究较少。目的探讨EGF对体外氧化损伤的人眼Muller细胞增生和迁移的影响及其作用机制。方法将人眼Maller细胞系MIO—M1细胞进行培养并用胶质纤维酸性蛋白（GFAP）、第Ⅷ因子、a-平滑肌肌动蛋白（Ot—SMA）、人角蛋白及S-100免疫组织化学法进行鉴定。在无血清DMEM中加入不同质量浓度（0、1、10、30、100mg／L）EGF，用5-溴脱氧尿嘧啶核苷（BrdU）标记法测定MIO—M1细胞的阳性率。根据干预方式的不同将培养细胞分为EGF组、H2O2损伤组、EGF＋H2O2组、葡萄糖氧化酶（GO）组、GO＋EGF组、EGF＋LY294002＋H2O2组，采用MTT比色法测定各组培养细胞的增生情况（A590）。对培养的人眼Muller细胞采用划痕实验观察H2O2损伤条件下0、1、10、30、100mg／LEGF作用24、48、72h后对Miiller细胞增生、迁移的影响；采用Westernblot技术检测EGF对体外不同培养条件下MUller细胞Akt信号传导通路的作用。结果正常培养条件下10、30、100mg／LEGF作用后Muller细胞的增生率分别为28．0％、32．9％、39．O％，明显高于0mg／L、1mg／LEGF组（24．5％、26．2％）。在H2O2和GO分别培养细胞的条件下，高质量浓度的EGF组Muller细胞的吸光度（A570）值明显大于低质量浓度组，各组总体差异有统计学意义（F=23．582，P=0．000）。与EGF＋H2O2组比较，EGF＋LY294002＋H2O2组Muller细胞的吸光度（A570）值明显下降。10mg／LEGF促进Muller细胞迁移的作用最强。0．08mmol／L H2O2作用Mfiller细胞后Akt信号通路激活，提前2h加入外源性EGF后，100mg／LEGF对抗氧化所�Background Oxidative damage plays an important role in pathogenesis of age-related macular degeneration（AMD） , and its mechanism is the destroy of blood-retinal barrier. Mtiller cells is a primary component to stabilize the inner barrier of the blood-retina. Researches showed that epidermal growth factor（EGF） can promote the proliferation and migration of animal Muller cells, but less study was found in the effect of EGF on human Muller cells. Objective The present study was to investigate the effects of EGF on the proliferation and migration of human Muller cells and its molecular mechanism. Methods Human Mailer cell line MIO-M1 cells were cultured and incubated, and cultured cells were identified using glial fibrillory acidic protein （GFAP） , factor Vm, a-smooth muscle actin （ a-SMA ） , keratin and S-100. Different concentrations of E GF （0,1,10,30,100 mg/L） was added in free- serum DMEM, and the positive rate of the ceils was calculated using 5-bromo-2-deoxyuridine （BrdU） method. The cells were divided into EGF group, H2O2 group, EGF ＋ H2O2 group, glucose oxidase （ GO ） group, GO ＋ EGF group, EGF ＋LY294002＋H2O2 group according to the different intervention, and the effects of LY294002 on the proliferation of Mailer cells（A590）were detected by colorimetric assay for cellular growth and survival（ MTT assay）. The scratch test of Muller cells was used to assess the influence of EGF（0,1,10,30,100 rag/L）on H2O2-induced damage of human Muller cell. Western blot was used to detect the cell proliferation under the protection of EGF on co-cultured cells using LY294002 and H2O2 and the activation of Akt signal pathways. Results The proliferative rates of the cells were 28.0% ,32.9% ,39.0% in 10,30,100 mg/L EGF groups respectively and obviously higher than those in 0, 1 mg/L EGF groups （24.5 % ,26.2% ）. Under the H202 culture, GO culture, respectively, the A570 value of the Mailer cell in high concentrations of EGF groups was significantly increased in comparison with lo

关 键 词：表皮生长因子 MÜLLER细胞 氧化损伤 年龄相关性黄斑变性 

分 类 号：R774.5[医药卫生—眼科]