线粒体损伤在镉诱导肝细胞凋亡和DNA损伤中的作用  

Role of mitochondrial damage in cadmium-induced cell apoptosis and DNA damage of hepatocytes

作　　者：张玉静 蒋妮 柳强[1] 朱勇飞[1] 黄昕[1] Zhang Yujing;Jiang Ni;Liu Qiang;Zhu Yongfei;Huang Xin(Key Laboratory of Molecular Epidemiology of Hunan Province School of Medicine,Hunan Normal University,Changsha 410081,China)

机构地区：[1]湖南师范大学医学院分子流行病学湖南省重点实验室,长沙410081

出　　处：《卫生研究》2020年第2期290-297,共8页Journal of Hygiene Research

基　　金：国家自然科学基金青年基金(No.81803272);湖南省大学生研究性学习和创新性实验计划(No.2018017)。

摘　　要：目的探讨活性氧(reactive oxidative species, ROS)介导的线粒体损伤在镉(cadmium, Cd)暴露诱导肝L02细胞凋亡和DNA损伤中的作用。方法以肝L02细胞为研究对象,利用0~90μmol/L的Cd处理细胞24 h,采用噻唑兰法检测Cd暴露对细胞生存率的影响;以0、20和40μmol/L的Cd染毒细胞24 h,分别采用克隆形成实验、流式细胞术、彗星实验、2′,7′-二氯荧光黄双乙酸盐非标记性氧化敏感的荧光探针、线粒体红色荧光探针(Mitotracker Red CMXRos)和10-N-壬基-吖啶橙等荧光探针标记、线粒体膜电位检测试剂盒(JC-1)、ATP测定试剂盒以及Western Blot等方法,检测Cd暴露对细胞克隆形成能力、细胞凋亡、DNA损伤、ROS水平、线粒体形态、线粒体膜电位(mitochondrial membrane potential, MMP/Δψm)、线粒体质量、ATP含量及相关蛋白的影响;利用90μmol/L抗氧化剂维生素C预处理细胞1 h后给予40μmol/L Cd处理细胞24 h,检测ROS水平、Δψm、线粒体质量、ATP含量、细胞凋亡、DNA损伤及相关蛋白的改变。结果随Cd处理浓度的增高和处理时间的延长,肝L02细胞存活率显著降低,克隆形成实验结果显示,与对照组相比,20和40μmol/L Cd处理组的克隆形成率分别为8.23%和6.17%,细胞凋亡率分别为15.85%和26.26%,且B细胞淋巴瘤/白血病-2(B cell lymphoma/lewkmia-2, Bcl-2)基因的蛋白水平显著降低,Bcl-2相关X蛋白(Bcl-2 associated X protein, Bax)和激活型半胱天冬蛋白酶-3(cleaved cysteine aspastic acid-specific protease 3, cleaved-caspase-3)蛋白水平呈剂量依赖性升高,Cd处理还可诱导细胞DNA损伤的发生以及细胞内ROS的大量蓄积,伴随线粒体颗粒状改变、Δψm、线粒体质量、ATP含量和线粒体细胞色素C(cyt c)的显著降低及胞浆cyt c的表达增高(P<0.05);此外,与单独Cd处理组相比,维生素C预处理不仅能够显著增高Δψm、线粒体质量、ATP含量和线粒体cyt c,降低胞浆cyt c的表达(P<0.05),还能够减轻Cd诱导的�OBJECTIVE To investigate the role of mitochondrial damage mediated by reactive oxidative species(ROS) in cadmium-induced cell apoptosis and DNA damage of L02 hepatocytes, so as to provide experimental basis for the subsequent study and protection of people exposed to Cd. METHODS The L02 hepatocytes were cultured in vitro treated with 0-90 μmol/L Cd for 24 h, and the methylthiazolyldiphenyltetrazoliumbromide assay was used to detect the cell viability. The colony formation assay, flow cytometry, comet assay, 2′,7′-dichlorofluorescein diacetate, MitoTracker Red CMXRos and 10-N-nonyl-acridine-orange, mitochondrial membrane potential detection kit(JC-1) and adenosine triphosphate(ATP) assay kits and Western Blot were used to investigate cell growth and proliferation, cell apoptosis, DNA damage, ROS levels, mitochondrial morphology, mitochondrial membrane potential, mitochondrial mass, ATP content and related proteins after the cells exposed to 0, 20, 40 μmol/L Cd for 24 h. The cells were pretreated with vitamin C before adding Cd exposure, and ROS levels, mitochondrial function, cell apoptosis, DNA damage and proteins were measured. RESULTS The cell viability was significantly inhibited with the increase of Cd concentration and treatment time. The cells were treated with Cd for 24 h for further study according to the result of MTT assay. Compared with control group, the colony formation rate were 8.23% and 6.17% respectively in 20 and 40 μmol/L Cd treatment and the apoptosis rate were 15.85% and 26.26%, respectively. We also found that the B cell lymphoma/leukemia(Bcl-2) gene protein was significantly reduced, while the levels of Bcl-2 associated X protein(Bax) and cleaved cysteine aspastic acid-specific protease 3(cleaved-caspase-3) were increased in a dose-dependent manner. Cd treatment also induced DNA damage and accumulation of intracellular ROS, accompanied by a mitochondrial morphological change, significant decrease in Δψm, mitochondrial mass, ATP content, mitochondrial cytochrome C(cyt c) and an incr

关 键 词：镉 肝L02细胞 细胞凋亡 活性氧 线粒体损伤 

分 类 号：Q593.1[生物学—生物化学] Q26