p66^(Shc)对亚砷酸钠诱导心肌细胞氧化应激的影响  

Effect of p66^(Shc) on sodium arsenite-mediated oxidative stress in H9C2 cells

作　　者：朱海英[1,2] 高小博 骆海燕[2] 马旭 陆彩玲[1,2] 

机构地区：[1]北京协和医学院研究生院,北京100005 [2]国家卫生计生委科学技术研究所遗传优生中心,北京100081

出　　处：《生殖医学杂志》2017年第6期578-584,共7页Journal of Reproductive Medicine

基　　金：国家自然科学基金项目(81470525);国家科技重大专项(2014ZX09304307-002);国家重点研发计划(2016YFC1000501)

摘　　要：目的探讨p66^(Shc)在亚砷酸钠介导的心肌细胞氧化应激中的作用。方法利用亚砷酸钠诱导的H9C2心肌细胞氧化应激模型,分别用CCK-8比色法检测细胞活力;双氯荧光素探针检测细胞内活性氧(ROS)水平;Western Blot法检测p66^(Shc)、磷酸化p66^(Shc)的表达变化;免疫荧光共聚焦检测p66^(Shc)的亚细胞定位。结果与对照组相比,5μM亚砷酸钠处理组细胞活力显著降低(P<0.05),细胞内ROS水平显著升高(P<0.001);砷引起了p66^(Shc)表达量增加,而且磷酸化p66^(Shc)与p66^(Shc)蛋白总量的比值也显著升高(P<0.05),p66^(Shc)在砷刺激下从细胞浆向线粒体转位。过表达野生型p66^(Shc)质粒增加砷介导的心肌细胞内ROS水平,而过表达突变型p66^(Shc)质粒和敲低p66^(Shc)的表达都降低砷处理组细胞内ROS水平。结论研究显示亚砷酸钠可能通过上调p66^(Shc)的表达和磷酸化水平诱导心肌细胞ROS水平的升高。Objective： To explore the role of p66^Shc in sodium arsenite-mediated oxidative stress in cardiomyocytes. Methods： The oxidative stress model of H9C2 cells was induced by sodium arsenite. The cell viability was measured by CCK 8 colorimetry. The intracellular reactive oxygen species （ROS） level was measured by dichlorofluorescein probe. Western blot was used to detect the expression of p66^Shc and phosphorylated p66^Shc. Sub-cellular localization of p66^Shc was detected by immunofluorescence confocal detection. Results： Compared to the control,sodium arsenite treatment significantly decreased cell viability,but increased intracellular ROS level （P〈0.05）. Western blotting showed that the ratio of phosphorylated- p66^Shc/p66^Shc was significantly increased, as well as the total expression of p66^Shc （P 〈 0.05） . Immunohistochemistry showed that p66^Shc was translocated from cytoplasm to mitochondria. Over- expression of wild-type p66She increased arsenic-mediated ROS levels in H9C2 cells, whereas over- expression of mutant p66^Shc and knockdown p66^Shc decreased the levels of ROS in arsenic-exposed H9C2 cells. Conclusions： The study results suggested that sodium arsenite induces elevation of ROS level in H9C2 cells by upregulating p66^Shc expression and phosphorylation levels.

关 键 词：亚砷酸钠 心肌细胞 p66^Shc 活性氧 

分 类 号：R[医药卫生]