天津医药

• 实验研究 •    

过氧化氢对FRTL细胞线粒体膜电位和超氧化物生成的影响

李敏1,李兰英2,姚小梅2,陈祖培2   

  1. 1. 天津医科大学病理生理学教研室
    2.
  • 收稿日期:2009-12-07 修回日期:2009-12-17 出版日期:2010-03-15 发布日期:2010-03-15
  • 通讯作者: 李敏

Influence of Mitochondrial Membrane Potential and Superoxide Production in FRTL Cell Induced by Hydrogen Peroxide

  • Received:2009-12-07 Revised:2009-12-17 Published:2010-03-15 Online:2010-03-15

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摘要： 摘要 目的：探讨外源性过氧化氢（hydrogen peroxide，H2O2）对FRTL细胞线粒体膜电位和超氧化物生成的影响。方法：用１mmol/L H2O2处理FRTL细胞10min，30min，24h，利用MitoSOX，通过活细胞影像法、流式细胞术检测线粒体超氧化物生成；利用罗丹明123（rhodamine123，rh123），通过荧光分光光度计和荧光显微镜检测线粒体膜电位（mitochondrial membrane potential，Δψ）；MTT比色法检测细胞活力；光镜观察细胞形态学变化；吖啶橙(acridine orange，AO)染色检测细胞凋亡。结果：与对照组相比，1mmol/L H2O2处理10min，30min，24h FRTL细胞内MitoSOX荧光强度明显增强，rh123荧光强度和MTT吸光度明显下降（p＜0.01），光镜下可见细胞脱壁，细胞破碎，AO染色可见核变小，变圆，染色质浓缩、边集，核碎裂改变。结论：１mmol/L H2O2急性处理（10 min，30 min）和慢性处理（24 h）均能明显增加FRTL细胞线粒体超氧化物生成，降低线粒体膜电位，造成细胞坏死和凋亡。

关键词: 过氧化氢, FRTL细胞, 线粒体, 超氧化物, 线粒体膜电位

Abstract: Abstract Objective：To investigate the effects of exogenous hydrogen peroxide (H2O2) expose on mitochondrial superoxide production and mitochondrial membrane potential（Δψ）changes in Fisher rat thyroid cell line (FRTL) cells. Methods：Following exposed in 1 mmol/L H2O2 for 10 min, 30 min and 24 h respectively mitochondrial superoxide production in FRTL cells were measured by living cell imaging and flow cytometry using MitoSOX. After exposed in 1 mmol/L H2O2 for 30 min and 24h mitochondrial membrane potential (Δψ) was assayed by spectrofluorimeter and fluorescent microscopy using rhodamine 123(rh123). Cell viability was detected by MTT colorimetric method. Morphological changes were observed by invert microscope. Apoptosis assay was performed by acridine orange staining. Results：Quantitative measurements of the mean intensities of MitoSOX from the samples demonstrated significantly increased with 1mmol/L H2O2 following 10 min,30 min and 24 h treatment. Fluorescence intensity of rh123 in FRTL cells and optical density of MTT colorimetric method markedly decreased with 1mmol/L H2O2 following 30 min and 24 h treatment（p＜0.01）. Under light microscope and fluorescence microscope after acridine orange staining, the characteristic morphological features of programmed cell death, picknosis, karyorrhexis, and cell shrinkage were observed. Conclusion：Acute and chronic exogenous H2O2 expose cause oxide stress in FRTL cells which result in mitochondrial superoxide production increase , Δψ decline, cell necrosis and apoptosis .

Key words: hydrogen peroxide, FRTL cells, mitochondrion, superoxide, mitochondrial membrane potential