高峰, 李园园, 牛勇, 宾萍, 鱼涛, 张荣, 陈雯, 郑玉新. 应用气-液界面染毒技术研究柴油机尾气对16HBE细胞的毒性作用[J]. 环境与职业医学, 2015, 32(3): 193-198. DOI: 10.13213/j.cnki.jeom.2015.14600
引用本文: 高峰, 李园园, 牛勇, 宾萍, 鱼涛, 张荣, 陈雯, 郑玉新. 应用气-液界面染毒技术研究柴油机尾气对16HBE细胞的毒性作用[J]. 环境与职业医学, 2015, 32(3): 193-198. DOI: 10.13213/j.cnki.jeom.2015.14600
GAO Feng , LI Yuan-yuan , NIU Yong , BIN Ping , YU Tao , ZHANG Rong , CHEN Wen , ZHENG Yu-xin . Acute Toxicity of Diesel Engine Exhaust on 16HBE Cells by Air-Liquid Interface Exposure Method[J]. Journal of Environmental and Occupational Medicine, 2015, 32(3): 193-198. DOI: 10.13213/j.cnki.jeom.2015.14600
Citation: GAO Feng , LI Yuan-yuan , NIU Yong , BIN Ping , YU Tao , ZHANG Rong , CHEN Wen , ZHENG Yu-xin . Acute Toxicity of Diesel Engine Exhaust on 16HBE Cells by Air-Liquid Interface Exposure Method[J]. Journal of Environmental and Occupational Medicine, 2015, 32(3): 193-198. DOI: 10.13213/j.cnki.jeom.2015.14600

应用气-液界面染毒技术研究柴油机尾气对16HBE细胞的毒性作用

Acute Toxicity of Diesel Engine Exhaust on 16HBE Cells by Air-Liquid Interface Exposure Method

  • 摘要: 目的 应用气-液界面染毒技术探索柴油机尾气对人支气管上皮(16HBE)细胞急性毒性作用。

    方法 在流速为20 mL/min, 37℃的条件下,用柴油机尾气分别对生长在多孔膜上的16HBE细胞持续染毒5、10、15、20、30 min,分别使用CCK-8(Cell Counting Kit-8)检测法、乳酸脱氢酶(LDH)释放法、中性红摄入法(NRU)检测柴油机尾气对细胞存活率的影响;对细胞存活率约50%以上的染毒组用流式细胞术检测柴油机尾气对细胞凋亡率的影响;同时对每个染毒组分别设置用滤膜过滤的洁净空气作为对照组,暴露时间和检测方法均与染毒组一致。

    结果 3种存活率检测方法的结果表明,染毒组与对照组相比,柴油机尾气持续染毒15 min及更长时间以后,细胞存活率降低,差异有统计学意义(P < 0.05),且呈随染毒时间延长而逐渐降低的趋势。3种方法比较表明,低流速短时间柴油机尾气对16HBE细胞的损伤作用以细胞膜损伤最为显著,其次是线粒体损伤。用柴油机尾气持续染毒10、15 min,染毒组细胞早期凋亡率、晚期凋亡及坏死率较对照组均升高,差异均有统计学意义(P < 0.05)。10、15 min染毒组的晚期凋亡及坏死细胞多于早期凋亡细胞(P < 0.05)。

    结论 气-液界面染毒技术可以应用于柴油机尾气等气溶胶有害物质的体外毒性研究;同时发现柴油机尾气导致的16HBE细胞急性毒性为细胞膜损伤、线粒体损伤及细胞凋亡。

     

    Abstract: Objective To evaluate acute toxic effects of diesel engine exhaust (DEE) in human bronchial epithelial (16HBE) cells by an air liquid interface aerosol dynamic direct exposure method.

    Methods The 16HBE cells seeded on porous membranes were exposed to DEE for 5, 10, 15, 20, 30 minutes at a flow rate of 20 mL/min, 37℃. Cell viabilities were evaluated by CCK-8 assay, lactic dehydrogenase (LDH) release assay, and neutral red uptake (NRU) assay, respectively. Apoptosis rates of cells with viability > 50% were determined by flow cytometry assay. Cells exposed to filtered clean air served as the control group and were applied the same exposure protocol as the treatment group.

    Results The data of three assays determining cellular viability showed that cellular viabilities of the treatment groups after continuous exposure to DEE for 15, 20, 30 minutes were significantly reduced compared with the control group (P < 0.05), and possessed good time-response relationships. The damage of 16HBE cells after short-term exposure to DEE at a low flow rate was mainly cell membrane injury, followed by mitochondrial injury. Both the early apoptosis rates and the late apoptosis and necrosis rates of the treatment groups after exposure to DEE for 10, 15 minutes were significantly higher than those of the control group (P < 0.05). Of the apoptosis cells in the 10 and 15 min treatment groups, the late apoptosis and the necrosis cell counts were more than the early apoptosis cells (P < 0.05).

    Conclusion Air liquid interface exposure method can be applied to in vitro toxicity studies of DEE and other harmful aerosols. The findings also indicate that DEE could lead to acute toxicity such as cell membrane damage, mitochondrial injury and apoptosis.

     

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