应激状态下肠上皮细胞凋亡水平的变化及其机制

Change in apoptosis and its mechanism in intestinal epithelial cells under oxidative stress

目的探讨在氧化应激状态下肠上皮细胞凋亡水平的变化以及凋亡异常发生的分子机制。方法使用过氧化氢(H2O2)处理培养的HT-29细胞模拟机体活性氧(ROS)损伤肠上皮细胞的体内状况,采用四甲基偶氮唑盐(MTT)微量酶反应比色法进行细胞生存力的检测;采用流式细胞术进行细胞凋亡的检测;采用蛋白质免疫印迹法(Westernblot)检测凋亡相关蛋白的表达。结果H2O2可降低HT-29细胞生存率,且呈现剂量依赖性和时间依赖性(P均<0.05);与空白对照组相比,随着H2O2浓度的增高,细胞凋亡率增加(P均<0.05),随着作用时间的延长,细胞凋亡率也增加(P<0.05);以不同浓度H2O2刺激HT-29细胞24h后发现,与空白对照组相比,Bax的表达随着H2O2浓度的增高而增加,Bcl-2的表达随着H2O2浓度的增高而降低。以500μmol/L,浓度的H2O2刺激HT-29细胞发现,Bax表达随着H2O2作用时间延长而增加,Bcl-2表达随着H2O2作用时间延长而降低。结论应激状态下,肠上皮细胞氧化应激水平与其凋亡程度相关,凋亡调控蛋白Bcl-2/Bax比值失调可能是肠上皮细胞凋亡过度的机制之一。

Objective To investigate change in apoptosis level and its mechanism of intestinal epithelial cells under oxidative stress. Methods HT - 29 cells were cultured in vitro, which were treated with hydrogen peroxide(H2O2), to simulate the intestinal epithelial cells injured by reactive oxidative species. The cells viability was observed by 3 -(4, 5 - dimethylthiazol - 2 - yl)- 2, 5- diphenyltetrazolium bromide (MTT) assay. Moreover, cell apoptosis and apoptosis associated proteins were evaluated by flow cytometry and Western blot. Results Cells viability of HT - 29 was decreased by H2O2 which showed dose -dependent and time -dependent patterns (all P<0. 05). The cell apoptotic ratios were increased with the concentration of H2O2 increased and the time of the stimulation prolonged compared with the controls (all P<0. 05). Although the expression of the Bax was increased when HT - 29 cells were stimulated with different concentrations of H2O2 for 24 hours, the expression of Bcl - 2 was decreased. While HT - 29 cells were stimulated with 500 μmol/L H2O2, the expression of the Bax was increased and that of Bcl - 2 was decreased overtime. Conclusion These data suggest that oxidative stress appears to be related to the apoptosis in intestinal epithelial cells under stress. The imbalance of Bcl - 2/Bax expression might result in intestinal epithelial cell apoptosis in oxidative stress.