补体受体1对补体激活的氧化应激状态下人视网膜色素上皮单层细胞屏障的保护作用  

Protective effect of complement receptor 1 on barrier of cultured human retinal epithelial cells under complement-activated oxidative stress

作　　者：王菁[1] 蔡萌[1] 李静[1] 田蓉[1] 林少芬[1] 田景毅[1] 李佳 俞德超 罗燕[1] 

机构地区：[1]中山大学中山眼科中心眼科学国家重点实验室,广州510060 [2]信达生物制药(苏州)有限公司

出　　处：《中华眼底病杂志》2014年第4期399-403,共5页Chinese Journal of Ocular Fundus Diseases

基　　金：国家自然科学基金（81170864、81371020）

摘　　要：目的 观察补体受体1（CR1）对补体激活的氧化应激状态下人视网膜色素上皮（hRPE）单层细胞屏障的保护作用.方法 原代培养3～5代hRPE细胞,建立稳定的hRPE单层细胞屏障模型.500 μmol/L叔丁基过氧化氢和10％正常人血清处理hRPE细胞,建立hRPE单层细胞屏障补体激活的氧化损伤模型.将补体激活的氧化应激状态下hRPE单层细胞屏障分为模型组和CR1治疗组.模型组加入1 μl的磷酸盐缓冲液,CR1治疗组加入1 μl的CR1溶液使其终浓度为1μg/ml,继续培养4h.以正常hRPE单层细胞屏障作为正常对照组,检测模型组和CR1治疗组细胞的跨表皮细胞膜电阻（TER）值.酶联免疫吸附试验检测模型组和CR1治疗组血管内皮生长因子（VEGF）、趋化因子配体2（CCL2）、C3a、C5a、膜攻击复合物（MAC）的蛋白量.结果 hRPE单层细胞屏障于接种后3周稳定形成.模型组、CR1治疗组补体激活的氧化损伤hRPE细胞TER值分别为正常hRPE细胞的54.01％、63.48％.模型组与CR1治疗组补体激活的氧化损伤hRPE细胞TER值比较,差异有统计学意义（t=21.60,P＜0.05）.CR1治疗组VEGF、CCL2蛋白量分别较模型组下降了11.48％、23.47％;两组VEGF、CCL2蛋白量比较,差异均有统计学意义（t=3.26、2.43,P＜0.05）.CR1治疗组C3a、C5a、MAC蛋白量较模型组分别下降了24.00％、27.87％、22.44％;两组C3a、C5a、MAC蛋白量比较,差异均有统计学意义（t=9.86、2.63、6.94,P＜0.05）.结论 CR1对补体激活的氧化应激状态下hRPE单层细胞屏障具有保护作用,抑制补体激活、下调CCL2和VEGF表达可能是其机制.Objective To observe the effect of complement receptor 1 （CR1） on barrier of cultured human retinal epithelial cells （hRPE） under complement-activated oxidative stress.Methods The third to fifth passage of hRPE cultured on Transwell insert were used to establish a stable hRPE monolayer barrier.The hRPE monolayer barrier was exposed to 500 μmol/L ten-butyl hydroperoxide and 10％ normal human serum to establish the hRPE monolayer barrier model of complement-activated oxidative stress in vitro.hRPE monolayer barriers under complement-activated oxidative stress were divided into two groups including model group and CR1 treatment （1 μg/ml） group.Model group and CR1 treatment group were treated with 1 μl phosphate buffer solution （PBS） or CR1 for 4 hours.Normal hRPE monolayer barrier were used as control in transepithelial resistance （TER） measurement experiment.TER was measured to evaluate the barrier function of hRPE.The hRPE-secreted vascular endothelial growth factor （VEGF） and chemokine （C-C Motif） Ligand 2 （CCL2）,together with complement bioactive fragments （C3a,C5a） and membrane-attack complex （MAC） in the supernatant were detected by enzyme-linked immune sorbent assay.Results Stable hRPE monolayer barrier was established 3 weeks after hRPE seeded on Transwell insert.Complement-activated oxidative stress resulted in a sharp decrease of TER to 54.51％ compared with normal hRPE barrier.CR1 treatment could significantly improve TER of barrier under complement-activated oxidative stress to 63.48％ compared with normal hRPE barrier（t =21.60,P＜0.05）.Compared with model group,CR1 treatment could significantly decrease the concentration of VEGF and CCL2 by 11.48％ and 23.47 ％ secreted by hRPE under complement-activated oxidative stress （t =3.26,2.43; P＜0.05）.Compared with model group,CR1 treatment could also decreased the concentration of C3a,C5a and MAC by 24.00 ％,27.87 ％,22.44 ％.The difference were statistically significant （t =9.86,2.63,6.94 ; P＜0

关 键 词：视网膜色素上皮 受体 补体3b 拮抗剂和抑制剂 补体激活 动物实验 

分 类 号：R774.1[医药卫生—眼科]