摘要
目的 探讨氧化应激状态下肺泡Ⅱ型上皮细胞（ATⅡ型细胞）修复存活、凋亡及其细胞外信号调节激酶（ERK）对凋亡的调控作用。方法 采用过氧化氢（H2O2）处理体外分离纯化培养的原代大鼠ATⅡ型细胞；用蛋白质免疫印迹法（Western blotting）检测H2O2刺激后ATⅡ型细胞磷酸化ERK1／2（p—ERK1／2）的表达，用四甲基偶氮唑盐微量酶反应比色法（MTT）检测细胞存活率；用流式细胞仪检测细胞凋亡率，并观察ERK特异性抑制剂PD98059干预后细胞凋亡的变化。结果 与对照组比较，10μmol／L和100μmol／L的H2O2处理对ATⅡ型细胞存活率和凋亡率无明显影响；500μmol／L和1000μmol／L的H2O2处理可导致ATⅡ型细胞的存活率明显降低，凋亡率显著升高（P均＜0．05），呈剂量依赖性。500μmol／LH2O2处理ATⅡ型细胞30min，细胞存活率和凋亡率均无显著变化；处理180min细胞存活率明显降低，凋亡率明显增高（P均＜0．05），呈时间依赖性。500μmol／LH2O2可刺激P—ERK1／2阳性表达，以刺激30min表达最强；使用ERK抑制剂（PD98059）干预后，ATⅡ型细胞凋亡率明显增高。结论 H2O2可以剂量和时间依赖的方式诱导ATⅡ型细胞凋亡，ERK信号转导途径参与了ATⅡ型细胞的凋亡调控，对氧化应激状态下的ATⅡ型细胞可能起到保护作用。
Objective To investigate survival and apoptotic responses of alveolar type Ⅱ epithelial cells （AT Ⅱ cells） under oxidative stress and the regulation mechanism mediated by extracellular signal - regulated kinase （ERK）. Methods Primary passage of cultured rat AT I cells were challenged with hydrogen peroxide （H2O2）, and the cells were pretreated with specific inhibitor of ERK （PD98059） in another group. Cell viability, apoptotic rate and the expression of phosphorylated ERK1/2 （p -ERK1/2） were measured by 3 -（4, 5 - dimethylthiazol - 2 - yl）- 2, 5 - diphenyhetrazolium bromide （MTT） assay, flow cytometry and Western blotting analysis, respectively. Results Compared with control group, decreased cell viability and increased apoptotic rate in AT Ⅱ cells occurred in dose -dependent manner when treated with H2O2 500 and 1 000 μmol/L （all P（0.05）, but no differences were found when H2O2 were 10 and 100 μmol/L in concentrations. When the cells were treated with 500 μmol/L H202 for 30 minutes, no differences in cell viability and apoptotic rate were found compared with control group, but reduced cell viability and increased apoptotic rate were found when the duration was 180 minutes, and it was in time - dependent manner （both P〈0. 05）. The expression of p -ERK peaked at 30 minutes after stimulation by 500 μmol/L H2O2. When PD98059 was added, it enhanced apoptotic rate after H2O2 -exposure. Conclusion Apoptosis can be induced by H2O2 in AT Ⅱ cells in dose -and time -dependent manners. ERK signaling pathway plays a role in the regulation of apoptosis and may be protective for AT Ⅱ cells under oxidative stress.
出处
《中国危重病急救医学》
CAS
CSCD
北大核心
2007年第4期193-196,共4页
Chinese Critical Care Medicine
基金
国家自然科学基金资助项目（30370618）
关键词
肺泡Ⅱ型上皮细胞
细胞凋亡
细胞外信号调节激酶
活性氧
alveolar type Ⅱ epithelial cell apoptosis
extracellular signal -regulated kinase reactive oxygen species
