氧化应激对肺泡Ⅱ型上皮细胞中促凋亡基因细胞周期和凋亡调控蛋白1表达的影响

Effects of Oxidative Stress on Expression of Cell Cycle and Apoptosis-Regulatory Protein-1 in Lung Epithe-lial Type Ⅱ Cells

目的探讨氧化应激对肺泡Ⅱ型上皮细胞中促凋亡基因细胞周期和凋亡调控蛋白1(CCAR1)表达的影响。方法常规培养人肺泡Ⅱ型上皮细胞系A549细胞。分为正常对照组(无过氧化氢处理的A549细胞)、0.1mmol/L过氧化氢处理的A549细胞组、0.5mmol/L过氧化氢处理的A549细胞组和1.0mmol/L过氧化氢处理的A549细胞组。利用DNA梯状电泳检测细胞凋亡。免疫荧光细胞化学检测CCAR1蛋白在A549细胞中的表达和分布。Western blot检测过氧化氢对A549细胞中CCAR1蛋白表达的影响。比色法测定半胱氨酸蛋白水解酶(Caspase-3)的活性。结果DNA梯状电泳检测显示,1.0mmol/L过氧化氢处理A549细胞24h,电泳出现反映细胞凋亡的梯形条带。免疫荧光细胞化学检测显示CCAR1主要定位在细胞核,但胞质中也有分布。Western blot结果显示过氧化氢呈剂量依赖性地诱导A549细胞中CCAR1蛋白表达上调(Pa<0.05)。CCAR1蛋白的表达与促凋亡的Caspase-3的活性呈显著正相关(r=0.7212P<0.05)。结论氧化应激诱导肺泡Ⅱ型上皮细胞中促凋亡基因CCAR1蛋白表达上调。CCAR1可能参与了氧化应激诱导肺泡Ⅱ型上皮细胞凋亡的调控,其机制可能涉及到Caspase-3活性改变。

Objective To explore the effects of oxidative stress on the expression of cell cycle and apoptosis - regulatory protein - 1 （ CCAR1 ） in lung epithelial type Ⅱ cells. Methods Human lung epithelial type Ⅱ cell line A549 cells were cultured in the presence or absence of hydrogen peroxide （ H2O2 ）. And they were divided into normal control group （ A549 cells untreated with hydrogen peroxide ）, 0. 1 mmol/L hydrogen peroxide - treated group （A549 cells were treated with 0.1 mmol/L hydrogen peroxide for 24 h） ,0.5 mmol/L hydro- gen peroxide -treated group （A549 cells were treated with 0.5 mmol/L hydrogen peroxide for 24 h） and 1.0 mmal/L hydrogen peroxide - treated group （A549 cells were treated with 1.0 mmol/L hydrogen peroxide for 24 h）. Apoptotic DNA Ladder was applied to detect the cell apoptosis. Double - fluorescense immuncytochemisty staining method was used to determine subcellar localization of CCAR1 protein in A549 cell. The expression of CCAR1 protein in A549 cells treated with or without hydrogen peroxide was determined with Western blot. Caspase - 3 relative activity was detected by colorimetric assay. Results Apoptotic DNA Ladder was found in A549 cells treated with 1.0 mmol/L hy- drogen peroxide for 24 h. The CCAR1 protein was mainly localized in nuclei of A549 cells,but which was found in cytoplasm as well. Hydro- gen peroxide induced a dose - dependent increase of CCAR1 protein expression in A549 cells （ P 〈 0.05 ）. Moreover, the CCAR1 protein le vel was significantly correlated with Caspase - 3 activity in A549 cells exposed to Hydrogen peroxide （ r = 0. 721 2 P 〈 0.05 ）. Conclusions Oxidative stress may induce elevation of CCAR1 protein expression in lung epithelial type Ⅱ cells. Caspase - 3 may be implicated in the molecular mechanisms responsible for CCAR1 - regulated apoptosis in lung epithelial type Ⅱ cells exposed to oxidative stress.