镉对人肝细胞WRL-68离体线粒体结构及功能的影响

Influence of cadmium on structure and functions of mitochondria in hepatic cells

目的 研究氯化镉（CdCl2）对离体肝细胞线粒体结构及功能的影响。方法从培养肝细胞（WRL-68）中提取线粒体，各组分别用0、1、5、10μmol／LCdCl2进行处理。采用分光光度法检测线粒体膜通透性转运孔（MPTP）的开放程度；电镜下观察线粒体的形态改变；罗丹明123检测线粒体膜电位（MMP）的变化；测定线粒体内ATP酶LDH、谷胱甘肽过氧化物酶（GSH-Px）、超氧化物歧化酶（SOD）的活性以及丙二醛（MDA）的含量。结果随着CdCl2浓度的增加MPTP开放程度增加，并且MPTP的开放可被环孢霉素A（CsA）所抑制；电镜观察发现，线粒体经CdCl2处理后出现不同程度的肿胀变形；CdCl2诱导MMP显著降低；同时引起SOD、GSH-Px、Na^＋．K^＋-ATP酶、Ca^＋-Mg^＋-ATP酶LDH活性趋于下降，5、10μmol／L CdCl2显著下调了各种酶的活性（P〈0．05）；CdCl2处理组MDA含量升高，10μmol／LCdCl2处理组与对照组比较有统计学意义（P〈0．05）。结论CdCl2能引起离体肝细胞线粒体结构的破坏，并致MPTP开放、MMP下降、线粒体酶活性的改变等与细胞凋亡相关过程的发生。

Objective To study the effects of cadmium on the structure and functions of mitochondria in hepatocytes. Methods Mitochondria were isolated from cultured human hepatocytes of the line WRL-68 and co-cultured with cadmium chloride （ CdC12 ） of the concentration of 1,5, and 10 μmol/L, and WRL-68 cells not treated with CdC12 （0 μmol/L） was used as control group. Cytosporin A （CsA） was added into the culture medium. Mitochondrial permeability transition pore （MPTP） opening degree was tested by spectrophotometer. Morphologic changes of mitochondria were observed under transmission electron microscope. The activities of Na^＋ -K^＋ -ATPase, Ca^2＋ -Mg^2＋ -ATPase, LDH, superoxide dismutase （ SOD）, glutathione peroxidase （GSH-Px）, and the contents of malondialdehyde （MDA） were measured. Mitochondria membrane potential （MMP） was monitored by spectrofluorimeter with fluorescence dye Rh- 123. Results CdC12 reduced the absorbance of mitochondria, signifying the opening of MPTP, concentration-dependently. The absorbance of mitochondria co-cultured with CsA and CdC12 10 μmol/L was higher than that of the . CdC12 10 μmol/L group. Mild swelling was seen in the mitochondria treated with CdC12. The MMP values of the CdC12 5 and 10 μmol/L groups were significantly lower than that of the control group （ P 〈 0. 05, P 〈 0. 01 ） . The activity levels of ATPase, LDH, SOD, and GSH-Px in mitochondria decreased in the CdC12 groups （ all P 〈 0.05 ） , and the contents of MDA increased in the CdC12 groups compared with the control group. Conclusion CdC12 causes destruction of mitochondria structure, opening of MPTP, decrease of MMP, and changes of vitality of mitochondria enzymes that all play important roles in apoptosis of hepatocytes.