黄曲霉毒素M_1与赭曲霉毒素A联合作用诱导分化Caco-2细胞凋亡的机制  

Combined Effects of Aflatoxin M_1 and Ochratoxin A on the Apoptosis and Mechanism in Differentiated Caco-2 Cells

作　　者：张焕 高亚男[2,3] 郑楠[2,3] 王加启[2,3] 任辉 Zhang Huan;Gao Yanan;Zheng Nan;Wang Jiaq;Ren Hui(Jilin University y College of Food Science and Engineering, Changchun 130022;Staie Key Laboratory of Animal Nutrition, Institute of A nimal Science , Chinese Academy of Agriculture Sciences , Beijing 100193;Minis try of Agricultural Milk and Dairy Inspection and Supervision Center, Beijing 100193)

机构地区：[1]吉林大学食品科学与工程学院,长春130022 [2]中国农业科学院北京畜牧兽医研究所动物营养学国家重点实验室,北京100193 [3]中国农业科学院北京畜牧兽医研究所农业部奶及奶制品质量监督检验测试中心(北京),北京100193

出　　处：《中国食品学报》2019年第3期93-101,共9页Journal of Chinese Institute Of Food Science and Technology

基　　金：国家重点基础研究发展计划(2011CB100805);现代农业产业技术体系专项资金(nycytx-04-01);国家自然科学基金项目(31501399)

摘　　要：目的:研究了黄曲霉毒素M_1(Aflatoxin M_1,AFM_1)和赭曲霉毒素(ochratoxin A,OTA)交互作用诱导分化人结肠癌Caco-2细胞凋亡及其作用机制。方法:AFM_1和OTA单独及联合作用于分化的Caco-2细胞后,通过阿尔玛蓝(Alamar-Blue)法测量细胞存活率,研究AFM_1和OTA对细胞增殖的抑制作用;通过流式细胞术检测细胞凋亡率,活性氧荧光探针检测细胞活性氧(ROS)释放量,JC-1线粒体膜电位荧光探针检测细胞线粒体膜电位变化。蛋白免疫印迹法检测凋亡蛋白Caspase-3和Caspase-9的表达水平。结果:AFM_1和OTA可抑制分化的Caco-2细胞增殖并诱导其凋亡,单独使用12μmol/L AFM_1和6μmol/L OTA作用于细胞存活率分别为95.59%和93.14%,联合作用下细胞存活率为81.57%,提示联合作用具有协同作用;AFM_1和OTA单独处理细胞24 h后,细胞的凋亡率分别为25.68%和33.7%,联合作用细胞的凋亡率为37.4%;AFM_1和OTA单独作用下细胞ROS的释放量分别是空白对照组的1.37倍和1.79倍,交互作用是空白对照组的6.21倍,交互作用对活性氧的释放量明显增加。结论:线粒体膜电位和线粒体相关凋亡蛋白的表达水平变化,说明AFM_1和OTA可能通过线粒体途径诱导分化的Caco-2细胞凋亡。Purpose: To study the combined mechanism of AFM1(aflatoxin M1, AFM1) and OTA(ochratoxin A,OTA) on the apoptosis and mechanism. Method: differentiated Caco-2(human colorectal adenocarcinoma) cells were cultured and treated in the following groups: AFM1(12 μmol/L), OTA(6 μmol/L), AFM1(12 μmol/L)+OTA(6 μmol/L). Cell viability and proliferation were detected by Alamar-Blue method. Cell apoptotic rate was quantified by AnnexinV-FITC/PI staining through flow cytometry, intracellular ROS(reactive oxygen species, ROS) release was detected by active oxygen fluorescent probe, and the mitochondrial membrane potential was detected by a JC-1 probe. Additionally, the protein of Caspase-3 and Caspase-9 were detected by Western blotting. Results: The results showed that AFM1 and OTA have a synergistic effect in inhibiting the proliferation of differentiated Caco-2 cells, and inducing cell apoptosis. Cell viability of the three group data was 95.59%, 93.14% and 81.57%, respectively. Apoptotic rate of the three group was 25.68%,33.7% and 37.4%, respectively. What’s more, the ROS release in AFM1 group was 1.37-fold of control group level, in OTA group was 1.79-fold of control group level, and the one in combined group was 6.21-fold of control group level.Conclusion: Expression levels of mitochondrial membrane potential and apoptotic proteins suggested apoptosis of differentiated Caco-2 cells was induced through mitochondrial pathways, moreover, this induction seemed to be more obvious in the combination group.

关 键 词：黄曲霉毒素M1 赭曲霉毒素A 凋亡 分化的Caco-2细胞 

分 类 号：TS252.5[轻工技术与工程—农产品加工及贮藏工程] TS201.6[轻工技术与工程—食品科学与工程]