促红细胞生成素抑制氧化损伤诱导的人眼Müller细胞凋亡  

Erythropoietin inhibits Müller cell apoptosis induced by oxidative damage

作　　者：陈春丽[1] 宋宗明[2] 贾新国[3] 周仲楼[2] 汪朝阳[3] 

机构地区：[1]胜利油田中心医院眼科,山东东营257034 [2]温州医科大学附属眼视光医院,浙江温州325027 [3]上海交通大学医学院附属新华医院,上海200092

出　　处：《山东大学耳鼻喉眼学报》2015年第3期65-71,75,共8页Journal of Otolaryngology and Ophthalmology of Shandong University

基　　金：国家自然科学基金(81100677);国家自然科学基金(面上项目)(81371040);上海市青年科技启明星计划(12QA1402200)

摘　　要：目的探讨促红细胞生成素(EPO)抑制氧化损伤导致的Müller凋亡的可能性及其分子机制。方法对培养的人眼Müller细胞系MIO-M1细胞采用Brd U标记法和MTT比色法观察正常及过氧化氢(H2O2)或葡萄糖氧化酶(GO)氧化损伤条件下0、0.01、0.1、1、10、30、100 U/m L EPO作用24、48、72 h后对Müller细胞增殖、迁移的影响;采用MTT比色法观察加PI3K/PDK1/PKB(Akt)信号传导通路阻断剂LY294002后Müller细胞增殖的变化;采用ELISA实验观察Müller细胞对EPO的表达和分泌;通过Western-blotting技术检测体外不同条件培养下EPO对ERK1/2及Akt信号传导通路的作用。结果正常培养条件下,EPO有轻度促进Müller细胞增殖迁移的作用,但差异无统计学意义;正常培养条件下,Müller细胞自身不分泌EPO,0.4 mmol/L H2O2致Müller细胞损伤80%时,其培养液内EPO的表达量为正常培养液下的1.42倍;氧化损伤状态下,0.08 mmol/L H2O2或8 U/L GO作用Müller细胞24 h后导致其半数死亡且Akt信号通路激活;提前2 h加入外源性EPO后,发现30 U/m L EPO对抗氧化所致Müller细胞的损伤作用最明显;同时提前2 h加入Akt信号通路阻断剂LY294002后,Akt信号传导通路的保护作用被阻断减弱。结论 EPO对体外正常培养的Müller细胞不具有促进增殖迁移作用;正常培养条件下Müller细胞自身不表达并且不分泌EPO,氧化损伤条件下Müller细胞自身可低分泌EPO;加入外源性EPO后,EPO可能通过Akt信号传导通路对H2O2损伤的Müller细胞发挥保护作用。Objective To demonstrate whether EPO can inhibit Muller cell apoptosis induced by oxidative damage and to investigate the molecular mechanism.Methods The proliferation effect of EPO on cultured human Muller cell-MIO-M1 was determined by BrdU marking and MTT assay both under normal condition and exposed to H2 O2 or GO with differ-ent concentration （0 U/mL,0.01 U/mL,0.1 U/mL,1 U/mL,10 U/mL,30 U/mL and 100 U/mL）at 24 h,48 h and 72 h after exposure.The Muller cell proliferation changes after the use of PI3K/PDK1 /PKB（Akt）signal pathway blocker-LY294002 was surveyed by MTT assay.The Muller cell function on EPO expression and secretion was ob-served by ELISA.The EPO effect on ERK1 /2 and Akt signal pathway was detected under different cultured condition in vitro by Western-blotting.Results EPO had no effect on Muller cell proliferation under normal culture condition.The 80% damaged Muller cell caused by 0.4 mmol/L H2 O2 can secrete 1.42 times EPO as that under normal culture condi-tion.Under oxidative damage condition,0.08 mmol/L H2 O2 or 8 U/L GO could cause half of Muller cells to be dead and activate Akt signal pathway.While adding EPO 2 h earlier,30 U/mL of EPO can reduce the oxidative damage to the minimum.The protection effect of Akt signal pathway could be reduced by its blocker LY294002 spontaneously. Conclusion EPO has no proliferation and migration effects on Muller cells cultured in vitro.Under normal culture con-dition,Muller cells did not express and secrete EPO.While under oxidative damage,Muller cells can secret EPO which can protect Muller cells by Akt signal pathway.

关 键 词：促红细胞生成素 MÜLLER细胞 增殖 年龄相关性黄斑变性 

分 类 号：R774.1[医药卫生—眼科]