摘要目的 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.

abstractsObjective 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.