镉致hek293细胞离体线粒体损伤的研究生物化学与分子生物学专业论文.docxVIP

镉致HEK293细胞离体线粒体损伤的研究摘 镉致HEK293细胞离体线粒体损伤的研究 摘 要 镉可以诱导HEK 293细胞发生凋亡并且其凋亡依赖于线粒体途径。研究镉 诱导的线粒体损伤对于镉的分子毒理学机制的阐明和镉所致的损伤与致癌机理 的研究具有重要意义。本文对镉诱导的I-IEK 293细胞离体线粒体损伤做了如下 研究工作： l镉对HEK 293细胞离体线粒体膜的损伤 建立镉致HEK 293细胞离体线粒体膜损伤模型，采用分光光度法检测CdCl2 诱导的线粒体渗透性转变孔的开放情况；通过透射电镜鉴定CdCl2诱导的线粒体 形态的改变；采用荧光分光光度计检测线粒体膜电位的变化。结果显示：CdCl2 能够诱导HEK293细胞离体线粒体的功能损伤，镉严重损伤了线粒体的膜系统， 导致线粒体膜电位的急剧下降，CdCl2能诱导线粒体渗透性转变孔的开放，并且 这种开放能够被环胞素(CsA)所抑制；电镜结果显示CdCl2诱导下线粒体肿 胀变形。 2镉致HEK 293细胞离体线粒体活性氧及抗氧化系统的变化 本实验利用荧光探针DCFH．DA检测了线粒体内ROS含量的变化；测定 了线粒体内两种主要的抗氧化酶SOD和GSH．Px的活力；同时测定了线粒体内 MDA的含量变化。结果表明：镉能够引起RoS含量的显著增加；SOD和 GSH．Px酶活力在镉的作用下降低明显。说明CdCh诱导的离体线粒体氧化损伤， 氧化应激可能在镉引发的细胞凋亡过程中起重要作用。 3镉对IIEK 293细胞离体线粒体酶活力的影响 检测CdCh对HEK 293细胞离体线粒体酶活力的影响，主要对与细胞呼吸 相关的ATPase和LDH进行了测定。结果表明：CdCl2能诱发HEK 293细胞 离体线粒体内多种酶活力的明显下降，并且随着CdCl2浓度的增加， Na+．K+．ATP酶、Ca2+．Mg+-ATP酶和LDH活力都有的显著降低，说明镉可能 破坏了线粒体的呼吸系统，对细胞的能量代谢造成阻碍。 关键词：镉 线粒体线粒体渗透性转变孔线粒体膜电位活性氧抗氧化酶 镉致HEK293细胞离体线粒体损伤的研究Abstract 镉致HEK293细胞离体线粒体损伤的研究 Abstract Cadmium can induce apoptosis in many kinds of cells．Studying the damage of isolated mitochondria induced by cadmium is necessary for US to CUre lots of diseases caused by cadmium．And it is also very important for the molecular toxicological study of cadmium．This paper studied some aspects of cadmium- induced damage of isolated mitochondria from HEK293 cells： 1 The damage of mitochondria membrane induced by cadmium The opening of the mitochondrial permeability transition pore(MPTP)Was tested by spectrophotometer．Morphological changes of mitochondria were observed under transmission electron microscope and mitochondrial membrane potential(MMP)was monitored by spectrofluorimeter with fluorescence dye Rh-123．The results showed that MPTP of exposed groups decreased compared with that of the control group，and it can be inhibited by Cs A．Mitochondria incubated by CdCl2 were found swell under transmission electron microscope． 2 The change of ROS and antioxidases induced by cadmium The contents of ROS were detected with DCFH·DA．Superoxide dismutase (SOD)，giutat