过氧化氢诱导体外培养的人脾细胞凋亡

Hydrogen peroxide induces apoptosis of human spleen cells in vitro

目的:研究过氧化氢(H2O2)对人脾细胞影响的时效和量效关系,以获得稳定的人脾细胞凋亡模型. 方法:采用研磨法获得悬浮的人脾细胞,将细胞分组后给予相应的生理盐水或不同浓度的H2O2处理.运用四唑盐(MTT)比色法检测线粒体功能,将细胞与Annexin V—FITC/PI混合孵育后使用流式细胞仪检测早期凋亡细胞. 结果:人脾细胞线粒体功能及凋亡率与H2O2呈剂量一效应关系,随着H2O2浓度的增高,线粒体功能逐渐降低,而凋亡率则随H2O2浓度的增高而增加.同时它们与H2O2处理时间也存在时间-效应的关系.各处理组间细胞总凋亡率分别为55.01±9.11%(100μmol/L),44.07±9.00%(50μmol/L),30.20±6.75%(25 μmol/L)和9.97±1.68% (对照组)存在显著差异(P<0.05),以H2O2 100 μmol/L处理的细胞组于6 h达到凋亡高峰(69.28±3.01)%. 结论:H2O2可以诱导体外培养的人脾细胞凋亡,建立人脾细胞凋亡模型.

AIM: To investigate the dose- and time-effect of hydrogen peroxide (H2O2) on human spleen cells, and to establish a stable apoptotic model of human spleen cells. METHODS: The human spleen cells, obtained by the method of grinding, were divided into four groups and treated with saline solution or various concentrations of H2O2 respectively. The function of mitochondrions was assessed by the 3-(4,5- dimethylthiazol-2-yl)-2,5-diphe-nyl tetrazolium bromide (MTT) assay and the early apoptotic cells were detected by flow cytometry with a combination of Annexin V-FITC/PI. RESULTS: H2O2 affected the mitochondrial function (negatively) and apoptosis (positively) of the human spleen cells in a dose- and time-dependent manner The apoptotic rates were significantly different between different groups (Total: 55.01±9.11%, 44.07±9.00%, 30.20±6.75% and 9.97±1.68% for 100, 50, 25 μmol/L and control group respectively, P<0.05). The apoptotic rate of cells reached the highest value (69.28±3.01)% at the concentration of 100 μmol/L 6 hours after treatment. CONCLUSION: H2O2 can induce the apoptosis of human spleen cells in vitro, which can be used to establish apoptotic model.