过氧化氢对FRTL细胞线粒体膜电位和超氧化物生成的影响

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

目的:探讨外源性过氧化氢(H2O2)对Fisher大鼠甲状腺细胞系(FRTL)线粒体膜电位(Δψ)和超氧化物生成的影响。方法:用1mmol/LH2O2处理FRTL细胞10min、30min、24h后,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(rh123),通过荧光分光光度计和荧光显微镜检测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(AO)染色检测细胞凋亡。结果:与对照组相比,1mmol/LH2O2处理的FRTL细胞10min、30min、24h,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、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/LH2O2急性处理(10min,30min)和慢性处理(24h)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。

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