二甲基甲酰胺诱导H9c2心肌细胞凋亡机制研究  

Study on apoptosis mechanism of H9c2 cardiomyocytes induced by N, N-dimethylformamide

作　　者：荣青秀 杨永坚[1] 张婉婉[1] 王娟 马晓丽[1] 向梅[3] 王璐璐[1] 孙文静[1] 

机构地区：[1]安徽医科大学公共卫生学院劳动卫生与环境卫生学教研室,合肥230032 [2]安徽国际旅行卫生保健中心 [3]安徽医科大学第一附属医院感染管理科

出　　处：《中华劳动卫生职业病杂志》2017年第2期106-110,共5页Chinese Journal of Industrial Hygiene and Occupational Diseases

摘　　要：目的 观察二甲基甲酰胺（DMF）致H9c2心肌细胞凋亡过程中核因子-κB（NF-κB）信号通路相关蛋白胞质p65蛋白、核内p65蛋白、磷酸化核因子-κB抑制（p-IκB）蛋白以及线粒体通路相关蛋白胞质细胞色素C（Cyt-c）、 活化的半胱氨酸天冬氨酸蛋白酶-3（Cleaved caspase-3）、B细胞淋巴瘤/白血病-2（Bcl-2）水平的变化,探讨DMF引起H9c2心肌细胞凋亡的可能机制.方法 给予体外培养的H9c2心肌细胞200 mmol/L DMF染毒,蛋白免疫印迹（Western blotting）法检测染毒0、2、4、6、8、12 h后胞质p65、核内p65、p-IκB的蛋白表达水平以及染毒0、2、4、8、12、24 h后胞质Cyt-c、Cleaved caspase-3、Bcl-2的蛋白表达水平,免疫荧光细胞化学（IFC）法对染毒不同时间后的细胞内Cyt-c进行定位.结果 各组胞质p65、核内p65、p-IκB蛋白表达水平组间差异均有统计学意义（F=7.79、33.11、90.25,均P〈0.01）;与对照组比较,2、4、6 h染毒组胞质p65蛋白表达水平均降低,2、4、6、8 h染毒组核内p65蛋白表达水平均升高,2、4、6 h染毒组p-IκB蛋白表达水平均升高,差异均有统计学意义（均P〈0.01）.各组胞质Cyt-c、Cleaved caspase-3、Bcl-2蛋白表达水平组间差异均有统计学意义（F=51.42、503.68、73.37,均P〈0.01）;与对照组比较,8、12 h染毒组Cyt-c蛋白表达水平均升高,2、4、8、12、24 h染毒组Cleaved caspase-3蛋白表达水平均升高、Bcl-2蛋白表达水平均降低,差异均有统计学意义（均P〈0.01）.IFC法对细胞内Cyt-c检测显示,随着DMF染毒时间的延长,Cyt-c逐渐从线粒体释放到胞质内.结论 NF-κB信号通路与线粒体通路可能参与了DMF诱导的H9c2心肌细胞凋亡.Objective To observe the change levels of nuclear factor-kappa B （NF-κB） p65 protein in cytoplasm and nuclear, phosphorylation of inhibitor of kappa B （p-IκB） protein and cytochrome C （Cyt-c）, cleaved cysteinyl aspartate specific proteinase-3 （Cleaved caspase-3）, B-cell lymphoma/leukemia-2 （Bcl-2） in cytoplasm in the process of N, N-dimethylformamide （DMF）-induced apoptosis in H9c2 cardiomyocytes, and explore the tentative mechanism of apoptosis. Methods H9c2 cardiomyocytes were exposed to 200 mmol/L DMF. Western blotting was used to detect the protein expression levels of p65 in cytoplasm and nuclear, p-IκB after exposure for 0, 2, 4, 6, 8, 12 h, and the protein expression levels of Cyt-c, Cleaved caspase-3, Bcl-2 in cytoplasm after exposure for 0, 2, 4, 8, 12, 24 h. Immunofluorescencecytochemistry （IFC） was used to observe the location of Cyt-c after 200 mmol/L DMF exposure for different times. Results The levels of p65 in cytoplasm and nuclear and p-IκB among groups were statistically significant （F were 7.79, 33.11, 90.25, respectively, all P〈0.01）. Compared with the control group, the levels of p65 in cytoplasm of 2, 4, 6 h group were significantly decreased（all P〈0.01）;the levels of p65 in nuclear of 2, 4, 6, 8 h were significantly increased（all P〈0.01）;the levels of p-IκB of 2, 4, 6 h group were significantly increased（all P〈0.01）. The levels of Cyt-c, Cleaved caspase-3 and Bcl-2 among groups were statistically significant（F were 51.42, 503.68, 73.37, respectively, all P〈0.01）. Compared with the control group, the levels of Cyt-c of 8, 12 h group were significantly increased（both P〈0.01）;the levels of Cleaved caspase-3 of 2, 4, 8, 12, 24 h were significantly increased（all P〈0.01）;the levels of Bcl-2 of 2, 4, 8, 12, 24 h group were significantly decreased（all P〈0.01）. IFC showed that Cyt-c was released from the mitochondria to the cytoplasm gradually as the extension of the exposure time. Conclusion NF-κB signaling pathway and

关 键 词：二甲基甲酰胺 H9C2心肌细胞 细胞凋亡 线粒体 

分 类 号：R114[医药卫生—卫生毒理学]