摘要
【目的】探讨氧化应激在镉诱导大鼠肝细胞凋亡中的作用。【方法】采用两步灌流法获得大鼠肝细胞,经过24 h培养,用醋酸镉处理细胞,或者Z-VAD-fmk、NAC和镉共同处理细胞。应用MTT法检测细胞存活率,流式细胞仪检测细胞凋亡率、细胞内ROS水平和线粒体膜电位,分光光度法检测caspase-3活性、GSH和MDA含量。【结果】结果表明,肝细胞暴露于浓度为2.5、5、10μmol.L-1的镉后,细胞相对存活率显著下降(P<0.01),凋亡率显著或极显著升高(P<0.05或P<0.01),呈剂量-效应关系;2.5和5μmol.L-1镉组在1.5 h之前导致细胞内ROS水平显著或极显著升高(P<0.05或P<0.01);镉暴露可使肝细胞Δψm显著或极显著降低(P<0.05或P<0.01);NAC能够显著减少镉引起的凋亡细胞数量和极显著降低凋亡率(P<0.01),可以显著降低单独镉暴露组ROS水平升高和阻止ΔΨm降低(P<0.05);细胞内GSH含量12 h时随镉剂量增高而降低,部分剂量组极显著低于对照组(P<0.01),24 h时随剂量增高而升高,部分剂量组显著或极显著高于对照组(P<0.05或P<0.01);细胞内MDA含量随着镉浓度的增大而升高,10μmol.L-1剂量组MDA的含量显著高于对照组(P<0.05);未见caspase-3活性升高,caspase抑制剂Z-VAD-fmk对镉致细胞凋亡无影响。【结论】醋酸镉致肝细胞凋亡与其引起ROS产生并导致氧化损伤的非caspase途径有关。
【Objective】The role of oxidative stress on the apoptosis of rat hepatocytes induced by cadmium was studied.【Method】Rat hepatocytes were isolated by a two-step perfusion technique.After 24 h planting,hepatocytes were treated with cadmium acetate,or treated with Cd in the presence or absence of Z-VAD-fmk or NAC.Cell viability was measured with MTT assay,the apoptosis,reactive oxygen species（ROS） generation,mitochondrial membrane potential（ΔΨm） collapse were measured by flow cytometry.The hepatocytes homogenate was prepared to detect the levels of caspase-3,malondialdehyde（MDA） and reduced glutathione hormone（GSH） in spectro photometric assay.【Result】 The results showed that cellular viability decreased and apoptosis rate increased significantly or very significantly（P0.05 or P0.01） in a dose-dependent manner after exposure to 2.5,5 and 10 μmol？L-1 Cd.Cd（2.5 and 5 μmol？L-1） induced ROS generation significently in 1.5 hours（P0.05 or P0.01）.Cd induced ΔΨm collapse.NAC effectively protected hepatocytes against apoptosis induced by Cd.Cd induced ROS generation and ΔΨm collapse were blocked by NAC（P0.05）.The GSH content decreased with the increase of the content of Cd.There was very significant difference in partial groups than the control group at 12 h（P0.01）,but GSH content increased with the increase of the dose of Cd at 24 h.The activities of MDA level increased.The caspase-3 activity didn＇t increase.No effect of Z-VAD-fmk on apoptosis was observed.【Conclusion】These results showed that ROS generation and oxidative stress by Cd triggers apoptosis via caspase-independent pathway.
出处
《中国农业科学》
CAS
CSCD
北大核心
2011年第18期3895-3902,共8页
Scientia Agricultura Sinica
基金
江苏省自然科学基金(BK2008214)
江苏省高校"青蓝工程"中青年学术带头人培养对象资助项目(2006)
关键词
镉
大鼠肝细胞
细胞凋亡
氧化应激
cadmium
rat hepatocytes
apoptosis
oxidative stress
