温石棉诱导人支气管上皮细胞凋亡及氧化的作用

目的 研究温石棉诱导永生化人支气管上皮细胞(BEAS-2B)的凋亡和活件氧(ROS)的生成,并观察自南基清除剂对其诱导作用的拮抗作用.方法 选用BEAS-2B,用0、5、10、20、100、200μg/cm2浓度的温石棉悬液及0、100、200、400、800、1600 μg/ml的温石棉浸出液,设立正常空白对照、阴性对照(标准硅灰石)和阳性对照(标准石英),用锥虫蓝排斥法检测细胞存活率,琼脂糖凝胶电泳检测凋亡细胞凋亡片段,流式细胞术检测细胞凋亡率及反转录-聚合酶链反应(RT-PCR)技术检测凋亡相关基因caspase-3的表达水平.采用荧光探针2',7'-二氯双氢荧光素二乙酸酯(DCFH-DA)测定温石棉刺激后细胞产生的ROS水平,并观察自由基清除剂过氧化氢酶、二甲业砜和甘露醇对ROS生成和基因表述水平的影响.结果 温石棉刺激BEAS-2B 24 h后,0、5、10 μg/cm2温石棉悬液和0、100、200μg/ml温石棉浸出液的细胞存活率大于90%.并且检测到凋亡细胞DNA凋亡片段.随着温石棉悬液和浸出液浓度的增加,BEAS-2B凋亡率上升,与阴性对照组比较,差异有统计学意义(P<0.05).温石棉悬液和浸出液可明显增强细胞caspase-3 mRNA的表达和诱导ROS产生.过氧化氢酶、二甲亚砜和甘露醇等自由基清除剂则可减轻caspase-3表达和阻断ROS生成,对温石棉悬液组细胞的ROS阻断率分别为23.7%、21.6%和11.2%,对温石棉浸出液组ROS阻断率分别为37.9%、40.3%和10.6%.结论 温石棉可诱导BEAS-2B发生凋亡,ROS在BEAS-2B的凋亡中起重要作用.

Chrysotile fibers induced apoptosis of human bronchial epithelial cells and effect of oxidation

Objective To investigate the apoptosis rate and the reactive oxygen species(ROS) level induced by chrysotile fibers in BEAS-2B cells and the blockage effect of free radical scavengers on the induc-tion of chrysotlie fiber.Methods The cell survival rate,the morphological variation of BEAS-2B cells,the apoptosis rate,the expression levels of gene caspase-3 and the ROS generation level were measured by using trypan blue phagocytosis,hematoxylin and eosin staining,oligonucleosomal DNA fragmentation assay,FCM,RT-PCR and fluorescent probe DCFH-DA in the suspension(0,5,10,20,100 and 200 μg/cm2) and the filtrate (0,100,200,400,800 and 1600 μg/ml) of chrysotile fibers.Addition of free radical scavengers such as cata-lase,dimethyl sulfoxide and mannitol prevented the radical generation and gene expression.Results Survival rates of BEAS-2B cells treated by the suspension( 0,5 and 10 μg/cm2) and the fihrate (0,100 and 200 μg/ml ) of chrysotile fibers for 24 hours were above 90%.The apoptotic rates of BEAS-2B were increased with the con-centration of suspension and filtrate from chrysotile fibers (P<0.05).Otherwise,caspase-3 mRNA and ROS were stimulated by chrysotile fibers.Free radical scavengers such as CAT,DMSO and mannitol couht reduce these stimulations.The ROS blocking rate of suspension of chrysotile fibers was 23.7%,21.6% and 11.2% re-spectively,and that of filtrate was 37.9% ,40.3% and 10.6% respectively.Conclusion Apoptosis is induced in BEAS-2B cells exposed to chrysotile fibers suspension and filtrate.Generation of ROS plays an important role in chrysotile fibers -induced BEAS-2B cell apoptosis.