内质网应激对光照诱导的人视网膜色素上皮细胞损伤的促进作用  

Mediated effects of endoplasmic reticulum stress on light-induced apoptosis and inflammation of human retinal pigment epithelial cell

作　　者：姜文静 张丽娜[2] 于晓 牛膺筠 

机构地区：[1]青岛市海慈医疗集团眼科,266033 [2]青岛大学附属医院眼科,266071 [3]青岛市第三人民医院眼科,266042

出　　处：《中华实验眼科杂志》2017年第9期816-823,共8页Chinese Journal Of Experimental Ophthalmology

基　　金：国家自然科学基金项目（30572010）;山东省优秀中青年科学家科研奖励基金项目（BS2013YY051）;青岛市应用基础研究计划项目（15-9-1-68-jch）

摘　　要：背景视网膜色素上皮（RPE）细胞的光损伤模型是多种视网膜变性类疾病的研究工具，光诱导RPE细胞损伤的主要病理基础是凋亡及炎症反应，但是内质网应激（ERS）反应是否参与其病理机制的研究国内外少有报道。目的探讨ERS对光损伤诱导的人RPE细胞凋亡的作用及其机制。方法体外培养人RPE细胞株（ARPE-19），将培养的细胞分为正常对照组及光照3、6、12和24h组，各光照组在培养箱内以（2000±500）lx的白色荧光灯光照细胞建立光损伤模型，正常对照组细胞在暗环境中培养且不给予光照射，筛选实验最适光照时间。将细胞分为正常对照组、光照组（光照12h）和苯基丁酸（4-PBA）预处理＋光照组，4-PBA预处理＋光照组先用ERS抑制剂4-PBA培养细胞30min，然后光照细胞12h。采用流式细胞仪检测各组人RPE细胞的凋亡率和细胞内活性氧（ROS）的荧光强度；采用ELISA法检测各组细胞上清液中炎性因子白细胞介素-1β（IL-1β）和肿瘤坏死因子-α（TNF-α）质量浓度；分别采用实时荧光定量PCR和Western blot法检测人RPE细胞中ERS标志物活化转录因子-6（ATF-6）、C／增强结合蛋白同源蛋白（CHOP）和细胞凋亡标志物半胱氨酸天冬氨酸蛋白酶-12（caspase-12）mRNA及其蛋白的表达。结果光照后细胞形态呈长梭形改变，边界不清，细胞质脱颗粒，细胞碎片增多，且随光照时间的延长而加重。流式细胞仪检测发现，随光照时间的延长，人RPE细胞内ROS含量逐渐增加，细胞凋亡率逐渐升高，差异均有统计学意义（F=763．00、119．30，均P〈0．01）。ELISA法检测发现，与正常对照组比较，光照后6h细胞上清液中IL-1β和TNF-α质量浓度均明显升高，12h达峰。实时荧光定量PCR和Westernblot检测显示，与正常对照组比较，光照后人RPE细胞中ATF-6、CHOP和caspase．12 mRNA及其蛋白的相对表达量均明显升高，均Background The light damage model of retinal pigment epithelium （RPE） cells is a research direction of retinal degeneration diseases, and RPE cell apoptosis induced by light damage and inflammation is an important pathologic basis of light-induced RPE cell damage. However,whether endoplasmic reticulum stress （ERS） paticipates in light-induced RPE cell damage is rarely reported. Objective This study was to explore the effects of ERS on light-induced RPE cell damage. Methods Human RPE cell line （ARPE-19） was cultured, and light damage models were created by irradiating the cells for 3-,6-, 12- and 24-hours with white fluorescent lamp with the intensity of （2 000±500）lx for the selection of optimal irradiating time, and the cells in the normal control group were cultured in the dark environment. The cells were divided into normal control group,light exposure group and 4-phenylb utyric acid （4-PBA） pretreated＋light exposure group. The cells from 4-PBA pretreated＋light exposure group were cuhued firstly with 4-PBA for 30 minutes and followed by light exposure for 12 hours. The apoptisis rate of the cells and intracellular reactive oxygen species （ROS） content were detected by flow cytometry; the concentrations of interleukin 1β （IL-1β） and tumor necrosis factor-α （TNF-α） in the cell supernatant were assyed by ELISA. The relative expressing levels of activating transcription factor 6 （ATF-6） , C/enhancer binding protein homologous protein （CHOP） and caspase-12 mRNA and protein in the cells were detected by real-time quantitative PCR and Western blot,respectively. Results The cultured cells showed a long spindle shape,the border was not clear,the cytoplasm was degranulation,and the cell fragments increased. Flow cytometry showed that compared with the normal control group, the ROS content in the cells and the apoptosis rate were evidently increased with the lapse of light exposure time （F=763.00,119.30,both at P〈0. 01 ）. ELISA results showed that the concentrations

关 键 词：氧化应激 光/不良作用 内质网/异常 眼色素上皮细胞/异常状态 细胞系 人 凋亡 炎症 

分 类 号：R774.1[医药卫生—眼科]