氯乙酸致人支气管上皮细胞16HBE的凋亡机制研究  

Apoptosis Mechanism of Human Bronchial Epithelial 16HBE Cells Induced by Chloroacetic Acid

作　　者：孟涛[1] 苗盼盼[1] 杨墨[1,2] 贾强[3] 戴宇飞[1] 

机构地区：[1]中国疾病预防控制中心职业卫生与中毒控制所,化学污染与健康安全重点实验室,北京100050 [2]山西医科大学公共卫生学院,山西太原030001 [3]山东省医学科学院,山东省职业卫生与职业病防治研究院,山东济南250062

出　　处：《环境与职业医学》2016年第9期858-864,共7页Journal of Environmental and Occupational Medicine

基　　金：公益性行业科研专项(编号:201402021);十二五国家科技支撑计划资助项目(编号:2014BAI12B02)

摘　　要：[目的]探讨氯乙酸对人支气管上皮细胞16HBE氧化应激以及线粒体凋亡通路的影响。[方法]以16HBE细胞为研究对象,将实验分为对照组和0.5、1.0、1.5、2.0、2.5 mmol/L的氯乙酸染毒组,染毒24 h后检测细胞存活率、凋亡率及超氧化物歧化酶(SOD)活力;染毒0.25、0.5、1、8、24 h后检测细胞内活性氧(ROS)水平;染毒8、24 h后检测线粒体膜电位以及Bcl-2和Bax m RNA表达水平。用1.5、2.5 mmol/L氯乙酸染毒24 h后,检测Bcl-2、Bax、细胞色素C、Caspase-9、Caspase-3、PARP-1凋亡相关蛋白的表达水平。[结果]随氯乙酸染毒浓度升高,染毒24 h后细胞存活率、SOD活力和线粒体膜电位呈剂量依赖性下降(r=-0.902、-0.732和-0.863,P<0.05),而细胞凋亡率呈剂量依赖性上升(r=0.914,P<0.05)。与对照组相比,1.5、2.0、2.5 mmol/L剂量组细胞存活率分别降低了12%、20%和30%,SOD活力分别降低了9%、21%和30%,线粒体膜电位分别降低了11%、18%和24%,而细胞凋亡率分别是对照组的3、4和7倍。各剂量组细胞内ROS水平呈先升高后降低的趋势,在染毒0.5 h达到峰值,与对照组相比分别增加了15%、35%、48%和55%(P<0.05),染毒1 h后反而降低。染毒各时间点染毒浓度与ROS水平均存在剂量-效应关系(r=0.756、0.893、0.735、0.667和0.653,均P<0.05);相关性分析表明染毒24 h后ROS水平与细胞凋亡率呈明显正相关(r=0.826,P<0.05)。染毒8、24 h两个时间点,染毒浓度与Bcl-2、Bax m RNA表达量存在剂量效应关系(8 h:r=-0.634和0.754,24 h:r=-0.773和0.823,P<0.05);与对照组相比,染毒24 h后染毒浓度≥1.5 mmol/L时Bcl-2 m RNA和蛋白表达明显下调,而Bax m RNA和蛋白表达明显上调(P<0.05);相关性分析表明Bax m RNA表达与ROS水平具有正相关(r=0.886和0.824,P<0.05),而Bcl-2m RNA表达与ROS水平具有负相关(r=-0.862和-0.815,P<0.05)。与对照组相比,染毒24 h后染毒浓度2.5 mmol/L时细胞色素C、活化的Caspase-3和Caspase-9蛋白表达量显著增加,�[Objective] To examine the chloroacetic acid induced oxidative stress and the related effect on mitochondrial pathway related apoptosis in human normal bronchial epithelial 16HBE cells. [Methods] Cell viability, apoptosis, and superoxide dismutase （SOD） activity were determined in 16HBE cells exposed to 0, 0.5, 1.0, 1.5, 2.0, and 2.5 mmol/L chloroacetic acid for 24 h in vitro. Reactive oxygen species （ROS） were assayed after 16HBE cells were treated with the above-mentioned concentrations for 0.25, 0.5, 1, 8, 24 h, respectively. Mitochondrial membrane potential and expression levels of Bcl-2 and Bax mRNA were measured after 8 and 24 h of treatment. Expression levels of apoptosis-related proteins including Bcl-2, Bax, cytochrome C, Caspase-9, Caspase-3, and PARP-1 were measured after 16HBE cells treated with 1.5 and 2.5 mmol/L chloroacetic acid for 24 h.[Results] The cell viability rate, SOD activity, and mitochondrial membrane potential decreased in a dose-dependent manner with increasing concentrations of chloroacetic acid （r=-0.902, -0.732, and -0.863, respectively, P 〈 0.05）; but the cell apoptosis rate increased in a dose-dependent manner （r=0.914, P 〈 0.05）. In comparison with the control group, remarkable reductions of 12%, 20%, and 30% for cell viability, 9%, 21%, and 30% for SOD activity, and 11%, 18%, and 24% for mitochondrial membrane potential were found in the 1.5, 2.0, and 2.5 mmol/L chloroacetic acid treated groups （P 〈 0.05） respectively; moreover, the cell apoptosis rates were 3, 4, and 7 times of the control group respectively. The intracellular ROS levels increased first and then decreased; peak values shown after 0.5 h of exposure in the 1.0, 1.5, 2.0, 2.5 mmol/L treatment groups, and significantly increased by 15%, 35%, 48%, and 55% respectively in comparison with the controls （P 〈 0.05）; the ROS levels in various concentration groups gradually dropped after 1h. There were significant dose-response relationships between ROS levels and concentrations of chloroace

关 键 词：氯乙酸 氧化应激 活性氧 凋亡 线粒体通路 

分 类 号：R114[医药卫生—卫生毒理学]