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香烟烟雾提取物(cigarette smoke extract,CSE)中含有丰富的氧化剂和自由基,由它所引起的氧化应激可导致肺泡壁的损伤进而发展为肺气肿。近年来,围绕CSE损伤肺泡壁作用机制的研究较为活跃,但其结果却一直存在着分歧。本实验的目的是观察CSE对肺泡Ⅱ型上皮细胞的损伤作用并探讨与其相关的分子机制。MTT比色法的结果显示,CSE以时间和剂量依赖性的方式降低细胞的增殖活力,流式细胞术的分析结果表明细胞增殖周期被阻滞在G_1/S期。Hoechst 33258染色以及透射电镜观察从形态上确认CSE诱导细胞凋亡的发生,DNA梯的出现和Annexin V-FITC/碘化丙啶双染色的结果从分子水平得到进一步的证实。同时,运用流式细胞术检测到CSE诱导的凋亡伴随着Fas受体的高表达和caspase-3的显著活化。另外,使用H_2DCFDA染色,经激光共聚焦显微镜术测得细胞内氧自由基在细胞受到CSE刺激以后大量快速积累。结果表明CSE能够抑制肺泡Ⅱ型上皮细胞来源的A549细胞的生长和增殖,并诱导细胞凋亡,由Fas受体所介导的死亡受体途径参与此凋亡过程,而CSE所引起的氧化应激则可能是阻止肺泡上皮细胞生长增殖并诱导其凋亡的始动因素。
Cigarette smoke extract (CSE) is rich in oxidants and free radicals. Oxidative stress caused by it can lead to damage of the alveolar wall and the development of emphysema. In recent years, studies on the mechanism of injury of alveolar walls around CSE have been more active, but the results have been divided. The purpose of this experiment was to observe the effect of CSE on alveolar type Ⅱ epithelial cells and to explore the molecular mechanisms involved. Results of MTT assay showed that CSE decreased the viability of cells in a time-and dose-dependent manner. The results of flow cytometry showed that the cell cycle was arrested in G_1 / S phase. Hoechst 33258 staining and transmission electron microscopy confirmed morphologically CSE-induced apoptosis. The appearance of DNA ladder and the results of Annexin V-FITC / propidium iodide double staining were further confirmed at the molecular level. Meanwhile, CSE-induced apoptosis was detected by flow cytometry accompanied by high expression of Fas receptor and significant activation of caspase-3. In addition, using H 2 DCFDA staining, intracellular oxygen free radicals, as measured by laser scanning confocal microscopy, accumulated rapidly in large numbers after cells were stimulated by CSE. The results showed that CSE could inhibit the growth and proliferation of alveolar type II epithelial cell-derived A549 cells and induce apoptosis, and the death receptor pathway mediated by Fas receptor was involved in this apoptosis process. CSE induced oxidative stress The stimulus may be to prevent alveolar epithelial cell growth and proliferation and induce apoptosis of the initiating factor.