活性氧诱导线粒体损伤导致晶体上皮细胞凋亡

REACTIVE OXIDANT SPECIES CAUSE APOPTOSIS OF LENS EPITHELIAL CELLS BY INDUCING MITOCHONDRIA DAMAGE

目的探讨线粒体损伤在活性氧诱导晶体上皮细胞凋亡中的作用。方法以过氧化氢为处理因素,MTT方法测定过氧化氢对晶体上皮细胞的半数致死浓度(IC50),使用确定的IC50处理培养的人晶体上皮细胞,琼脂糖凝胶电泳检测DNA片段化降解,流式细胞术检测细胞线粒体跨膜电位(Δψm)变化、透射电镜观察细胞线粒体形态,定量免疫印迹检测胞质溶胶中细胞色素c含量的变化及caspase-3的活化。结果过氧化氢对晶体上皮细胞的IC50是32.24μmol/L。32.24μmol/L的过氧化氢处理12h可以检测到晶体上皮细胞染色体DNA发生片段化降解;6h可以检测到线粒体跨膜电位去极化,且随时间延长逐渐加强;18h透射电镜观察可见明显的线粒体膜损伤。定量免疫印迹分析显示细胞色素c在胞质溶胶中的表达逐渐提高及caspase-3活化加强。结论活性氧可能是通过诱导线粒体结构和功能损伤导致晶体上皮细胞凋亡。

Objective To investigate the role of mitochondria damage in the apoptosis of lens epithelial cells （LECs） induced by reactive oxidant species. Methods Peroxide hydrogen was used to treat LECs and its IC50 to LECs was determined by MTT method. After 6, 12, 18 and 24 treated with peroxide hydrogen in the determined IC50 concentration, the degradation of LEC chromatin DNA and the change of LEC mitochondria membrane potential （△ψm） were analyzed by agarose gel eletrophoresis and flow cytometry respectively. The structure of mitochondria in treated cells were observed by transmission electron microscopy. The expression level of cytochrome c and the activation of caspase-3 in cytosole were detected by quantitive Western blotting. Results The IC50 of peroxide hydrogen to LECs was 32. 24μmol/L. After 12 hours＇ peroxide hydrogen treatment, the LEC chromosome DNA degraded in a pattern of discrete fragments. After 6 hours, the mitochondria membrane potential depolarized and the effect was enhanced with the time. Damage on the mitochondria membrane was clearly presented by transmission electron microscopy in LECs treated by peroxide hydrogen for 18 hours. Detected by quantitive Western blotting, the expression level of cytochrome c and activation of caspase-3 were raised during the LEC apoptosis process. Conclusion The mitochondria damage may probably underly the apoptosis of lens epithelial cells induced by reactive oxidant species.