人角膜内皮细胞体外氧化损伤及黄酮抗氧化保护作用的机制  

Mechanisms of oxidative damages of human cornea endothelial cells and antioxidative effects of flavone in vitro

作　　者：樊廷俊[1] 赵君[1] 李凌[1,2] 杨秀霞[1] 于苗苗[1] 王晶[1] 

机构地区：[1]中国海洋大学海洋生命学院海洋生物系,中国山东省青岛市266003 [2]中国科学院海洋研究所实验海洋生物学开放实验室,中国山东省青岛市266071

出　　处：《国际眼科杂志》2008年第8期1536-1541,共6页International Eye Science

基　　金：中国国家“十五”863项目资助(No.2001AA625050);“十一五”863项目资助(No.2006AA02A132)~~

摘　　要：目的:揭示黄酮对人角膜内皮细胞(human corneal endothe-lial cells,HCEC)的抗氧化保护作用及其机制。方法:利用HCEC的体外培养体系,采用形态观察,吖啶橙/溴化乙锭荧光双染色和DNA琼脂糖凝胶电泳等技术手段研究了黄酮和/或过氧化氢(H2O2)处理对HCEC细胞凋亡的影响,使用酶联免疫吸附试验分别对70μmol/L黄酮和/或600μmol/L H2O2处理不同时间后HCEC中凋亡诱导因子(AIF)与细胞色素c(CytC)的浓度变化进行了检测。结果:在600μmol/LH2O2处理8h,细胞凋亡率约为60%的HCEC氧化损伤模型中,70μmol/L黄酮预处理30min可使HCEC的细胞凋亡率降至约8%,而仅用黄酮处理组HCEC的细胞凋亡率约为1%。HCEC的总CytC浓度于H2O2处理3h后达到峰值(约为对照组的7.97倍),总AIF浓度于处理6h后达到峰值(约为对照组的1.28倍),而黄酮预处理组和仅用黄酮处理组HCEC的总CytC和AIF浓度与阴性对照组没有显著差异。结论:H2O2诱发HCEC凋亡是促使线粒体中CytC和AIF的外流引起的;70μmol/L黄酮对600μmol/L H2O2所致HCEC细胞凋亡具有很强的抗氧化保护作用。AIM： To lay foundations for prevention and therapy of keratopathy caused by HCEC apoptosis, to study the mechanisms of oxidative damages of human cornea endothelial cells （HCECs） and antioxidative effects of flavone. METHODS： The effects and antioxidative activities of hydrogen peroxide （ H2O2 ） and/or flavone were investigated with in vitro cultured HCECs by techniques of morphological observation, acridine orange/ethidium bromide （AO/EB） double fluorescent staining and DNA agarose gel electrophoresis. And the concentrational variabilities of cytochrome c （CytC） and apoptosiso inducing factor （AIF） of HCECs treated with 600μmol/L H2O2 and/or 70μmol/L flavone after different times were inspected by enzyme-linked immunosorbent, respectively. RESULTS： In the oxidative damage model of HCECs with an apoptotic ratio of 60% after treated with 600μmol/L of H2O2 for 8 hours, HCECs preincubated with 70μmol/L flavone for 30 minutes had a lowed apoptotic ratio of 8% while HCECs treated with 70μmol/L flavone only had an apoptotic ratio of 1%. The overall concentration of CytC from H2O2-treated HCECs reached to a peak at 3 hours which was 7.97 times higher than that of untreated normal HCECs, while that of AIF from H2O2-treated HCECs reached to a peak at 6 hours which was 1.28 times higher than that of untreated normal HCECs. Contrarily, the overall concentrations of CytC and AIF from flavone preincubated HCECs and only flavone treated HCECs showed no significant difference to those of untreated normal HCECs. CONCLUSION： The mechanism of apoptosis-inducing effect of H2O2 is most probably achieved by promoting the release of CytC and AIF from mitochondria to initiate apoptosis of HCECs. On the other hand, 70μmol/L flavone has an intensive antioxidative effect on H2O2- treated HCECs, and its mechanism is most probably realized by preventing HCECs from apoptosis via inhibiting the release of CytC and AIF from mitochondria.

关 键 词：人角膜内皮细胞 过氧化氢 黄酮 细胞凋亡 抗氧化保护作用 

分 类 号：R285.5[医药卫生—中药学]