香烟烟雾提取物对人呼吸道上皮细胞DNA损伤和凋亡的影响

DNA Damage and Apoptosis of Human Airway Epithelial Cell Lines Caused by Cigarette Smoke Extract

背景与目的:香烟烟雾可以致多种细胞发生DNA损伤已有报道证实。本研究旨在进一步阐明香烟烟雾提取物(cigarettesmokeextract,CSE)作用于正常人类支气管上皮细胞系(normalhumanbronchialepithelialcells,NHBE)和肺腺癌细胞系SPC-A1后引起的DNA损伤和凋亡情况。方法:不同浓度的CSE作用于NHBE和SPC-A1细胞,用四甲基偶氮唑盐比色法(MTT)检测两种细胞活性。荧光标记的磷酸化H2AX组蛋白(γ-H2AX)抗体特异性标记细胞核内DNA双链断裂(DNAdouble-strandbreaks,DSBs)处的γ-H2AX,然后用流式细胞仪定量检测并分析DNA损伤。用免疫印迹检测γ-H2AX表达。同时,亚G1峰法(SubG1peak)和AnnexinⅤ-FITC/碘化丙啶双染色流式细胞术检测CSE诱导的细胞凋亡。用激光共聚焦显微镜观察细胞损伤和凋亡形态学变化。结果:MTT结果显示,随CSE浓度和作用时间增加,细胞活性均逐渐降低。CSE可引起细胞DNA双链断裂,γ-H2AX最大值在作用后4h左右,然后逐渐降低。DNA损伤后平均12h可以出现凋亡。另外,激光共聚焦显微镜观察到两种细胞内的γ-H2AX量在细胞受到CSE刺激后快速大量的积聚,呈现典型细胞损伤的形态学变化,较为明显的凋亡形态学特征4h后方可出现。结论:CSE能直接引起NHBE和SPC-A1细胞的DNA损伤和凋亡,且存在着浓度和时间依赖关系。

BACKGROUND ＆ OBJECTIVE： Cigarette smoke exposure has been reported to induce DNA damage in a variety of cell types. This study was to investigate DNA damage and apoptosis in normal human bronchial epithelial cell line NHBE and lung carcinoma cell line SPC-A1 caused by cigarette smoke extract. METHODS： NHBE and SPC-A1 cells were incubated with different concentrations of cigarette smoke extract. Cell viability was evaluated by M-FI assay. Fluorescence-labeled anti-histone γ- H2AX polyclonal antibody was used to detect DNA double-strand breaks （DSBS） in chromatin. DNA damage was analyzed by flow cytometry. The expression of γ-H2AX was detected by Western blot. Cigarette smoke extractinduced cell apoptosis was detected by sub G1 peak method and annexin VFITC/propidium iodide （PI） staining assay. Cell morphology of DNA damage and apoptosis was observed by confocal laser microscopy. RESULTS： MTT assay results showed that cigarette smoke extract decreased the viability of NHBE and SPC-A1 cells in time- and concentration-dependent manners. Cigarette smoke extract induced DSBS in NHBE cells and SPC-A1 cells, and led to H2AX phosphorylation （denoted γ-H2AX） in time- and concentration- dependent manners. The maximal value of γ-H2AX was seen about 4 h after the treatment, and the value decreased subsequently, but did-not reduce to a normal level. Cell apoptosis appeared about 12 h after DNA damage. Cigarette smoke extract also initiated the accumulation of γ-H2AX in these cells, and cell apoptosis morphology was observed 4 h later. CONCLUSION： Cigarette smoke extract can induce DSBS and apoptosis in NHBE and SPC- A1 cells in time- and concentration- dependent manners.