氧化应激反应对视网膜色素上皮细胞迁移的影响及相关机制的探讨  

The effect of oxidative stress on retinal pigment epithelial cell migration

作　　者：姜岩 何世坤[2] 

机构地区：[1]辽宁省本溪市中心医院眼病防治中心,117000 [2]美国南加州大学Keck医学院Doheny眼科研究所

出　　处：《中华眼科杂志》2005年第2期100-105,共6页Chinese Journal of Ophthalmology

摘　　要：目的探讨氧化剂tertbutylhydroperoxide(TBH)诱导的氧化应激反应对视网膜色素上皮(RPE)细胞迁移的影响。方法将人RPE细胞内氧化反应产物以CarboxyH2DCFDA染色标记;细胞的迁移采用改良的Boyden腔测定;其存活及黏附通过MTT比色法测定。肌动蛋白,局部黏附激酶及纽带蛋白的表达采用免疫荧光染色观察。通过蛋白电泳观察分裂原激活的蛋白激酶和局部黏附激酶的表达。结果TBH处理后RPE细胞内氧化反应产物增加。TBH对RPE细胞迁移的影响呈双向反应,即低浓度促进,高浓度抑制。高浓度TBH降低RPE细胞黏附力,也能减少局部黏附激酶和纽带蛋白的表达,同时TBH可活化分裂原激活的蛋白激酶。结论由TBH诱导的氧化应激反应既能刺激也能抑制RPE细胞的迁移。Objective This study sought to determine if the migration and adhesion of retinal pigment epithelial (RPE) cell could be modulated by oxidative stress induced by t-butyl hydroperoxide (TBH). Methods Intracellular RPE reactive oxygen species (ROS) was labeled by Carboxy-H_2DCFDA staining. Cell migration was measured by a modified Boyden Chamber Assay. After RPE cells were treated with different concentrations of TBH (1-200 μmol/L) for one hour, chemotaxis toward PDGF (30 ng/ml) was measured after additional 5 hours incubation. The actin cytoskeleton was evaluated by phalloidin (F-actin) staining. Focal adhesion kinase (FAK) and vinculin expression were evaluated using immunofluorescence staining. The expression of MAP Kinase and FAK were studied by western blot. Cell survival and attachment of treated RPE cells were determined using MTT assay. Results ROS production was elevated in the RPE cells pretreated with TBH. The PDGF-independent RPE cell migration was enhanced by TBH at low concentration up to 30 μmol/L, and the PDGF-induced RPE cell migration was significantly inhibited by TBH at concentration higher than 50 μmol/L. F-actin was aggregated in the periphery of the RPE cells after treatment with TBH at a dose higher than 100 μmol/L. FAK and vinculin expression in RPE cells decreased and their distribution were changed after the treatment of higher concentrations of TBH. MAP Kinase level was up-regulated in response to 10-200 μmol/L TBH exposure. The cell attachment was declined upon addition of TBH above 50 μmol/L. Conclusions Oxidant stress induced by TBH can inhibit or promote RPE cell migration. (Chin J Ophthalmol,2005,41:100-105)

关 键 词：氧化应激反应 视网膜色素 上皮细胞 细胞运动 

分 类 号：R774.1[医药卫生—眼科]