复方血栓通对人视网膜血管内皮细胞抗氧化损伤的保护作用及其机制  

Protective effects and mechanism of fufang xueshuantong on tert-butyl hydroperoxide-induced injury of human retinal vascular endothelial cells

作　　者：陈晓云[1] 李建桥[2] 朱晓波[1] 肖伟[1] 黄娟[1] 李涛[1] 唐仕波[1] 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]山东大学齐鲁医院眼科,济南252000

出　　处：《中华实验眼科杂志》2011年第10期872-878,共7页Chinese Journal Of Experimental Ophthalmology

基　　金：国家自然科学基金项目（30872819）、广东省科技计划基金项目（2009B030801024）

摘　　要：背景氧化损伤是引起内皮细胞功能障碍和细胞死亡的重要因素之一，它还可导致视网膜血管性疾病的发生。复方血栓通对多种视网膜血管性疾病有一定的疗效，但分子机制尚不明确。目的研究复方血栓通对叔丁基过氧化氢（t—BHP）诱导的人视网膜血管内皮细胞（RVECs）氧化损伤的保护作用及其机制。方法分离健康人供体眼的视网膜，用组织块培养法进行人RVECs的原代培养，并用FITC-vwF染色流式细胞仪对培养细胞进行鉴定。取3～5代的细胞用于实验，在含有5×10^4个／L细胞密度的培养孔中分别加入质量浓度为0．0625、0．1250、0．2500、0．5000、1．000g／L复方血栓通溶液，各质量浓度复方血栓通组和t-BHP模型组均加入终浓度为100μmol／Lt-BHP，干预24h后用MTT法检测各组人RVECs的吸光度（A490）值以评估复方血栓通的细胞毒性。t-BHP模型组分别加入终浓度为75、100、200和300μmol／L的t-BHP，相应的复方血栓通组在不同浓度t-BHP造成细胞氧化损伤的同时均加入终质量浓度为0．2500g／L复方血栓通溶液，干预6h后，用MTT法检测复方血栓通对t-BHP引起氧化损伤的作用。用Annexin V—FITC／PI双染流式细胞仪检测各组细胞的凋亡和坏死率；用倒置显微镜和Hoechst33258细胞核染色观察各组细胞的形态学，利用免疫荧光法检测人RVECs中蛋白氧化损伤和DNA氧化损伤的生物标记物硝基酪氨酸（NT）和8-羟基脱氧鸟苷（8-OHdG）的表达；Western blot法检测t-BHP损伤6、12、24h各组细胞核转录因子．KB（NF—KB）、p53、bcl-2和bax的蛋白表达水平。结果正常对照组、0．0625、0．1250、0．2500、0．5000、1．000g／L复方血栓通组人RVECs的A490值总体比较差异无统计学意义（F=1．989，P〉0．05），75、100、200和300μmol／L的t-BHP作用后细胞存活率下降，而相应的复方血栓通组细胞存活率均较t-BHP模型组升高，差�[Abstract] Background Oxidative damage may cause the functional dysfunction and death of retinal vascular endothelial cells （RVECs） , and further leads to the development of retinal vascular diseases. Fufang xueshuantong has a therapeutic effect on retinal vascular diseases,but little is known about its molecular mechanism. Objective The goal of this study was to investigate the protective effects and mechanism of fufang xueshuantong on injury of human RVECs induced by tert-butyl hydroperoxide （ t-BHP）. Methods Human RVECs were isolated from healthy donor eyes and primarily cultured and then identified by flow cytometry. The third to fifth generations of cells were used in this experiments. The fufang xueshuantong solution of 0. 0625,0. 1250,0. 2500,0. 5000 and 1. 0000 g/L were added in the culture plate with 5×10^4/L cells respectively in the experimental groups, and t-BHP of 75,100,200 and 300 μmol/L were added in the model control groups. MTT was used to detect the A490and survival rate of RVECs. The apoptotic rate and death rate of the cells were evaluated by double staining of Annexin V-FITC/PI. Morphology of human RVECs were examined using invert microscopy and Hoechst33258 staining. The expressions of nitro tyrosine （a marker of oxidative damage of protein） and 8-OHdG （a marker of oxidative damage of DNA） in human RVECs were assessed by the immunofluorescence staining. Western blot was used to detect the expressions of nuclear factorkappa B （ NF-κB ） , p53, bcl-2 and bax after 6,12,24 hours t-BHP action. This study was approved by the Ethic Committee of Zhongshan Ophthalmic Center. Results No significant difference was found in A490value among the normal control group, 0. 0625,0. 1250,0. 2500,0. 5000 and 1. 0000 g/L fufang xueshuantong groups （ F = 1. 989, P〉O. 05）. The survival rates of the cells were lower in 75,100,200 and 300 μmol/L t-BHP groups compared with corresponding fufang xueshuantong groups （ t = 14.57,13.82,21.51 , 32.64, P 〈 0. 01 ）. The percentages of nor

关 键 词：复方血栓通 视网膜血管内皮细胞 氧化损伤 核转录因子-ΚB 凋亡 

分 类 号：R77[医药卫生—眼科]