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目的建立人肺癌A549细胞直接暴露可吸入物质体外试验方法并进行初步的应用。方法将二氧化氮气体用洁净空气稀释后分别配制成5.1、10.7、15.0、20.7 mg/m3,在多孔膜上生长的A549细胞放置在水平扩散染毒仓内,通过气液界面技术(ALI)暴露于不同浓度的二氧化氮气体,15 ml/min,37℃染毒1 h,利用MTT和NUR试验检测细胞毒性;采集不同工况下的汽油发电机尾气,10 ml/min,37℃染毒A549细胞15 min,利用CCK-8和MTT试验检测细胞毒性。结果细胞存活率随着NO2气体浓度的升高(5.1~20.7 mg/m3)显著降低(P<0.05),并且呈剂量-效应关系(MTT拟合曲线:yMTT=0.989-0.021xNO2R2=0.84,P<0.01;NUR拟合曲线:yNUR=1.032-0.026xNO2R2=0.88,P<0.01)。怠速(冷启动)、53%和100%负荷下,A549细胞MTT和CCK-8存活率显著低于空气暴露组(P<0.05)。结论体外细胞直接暴露可吸入物质方法可以应用于有害物质的体外吸入毒性研究,对工业和环境场所中的有害物质的毒性评价提供了新的技术和方法。
Objective To establish an in vitro test method for directly exposing human lung adenocarcinoma A549 cells to inhalable substances and to conduct preliminary application. Methods After the nitrogen dioxide gas was diluted with clean air, the cells were prepared into 5.1,10.7,15.0,20.7 mg / m3 respectively. The A549 cells grown on the porous membrane were placed in the horizontal diffusion and poisoning chamber. Through the gas-liquid interface technique (ALI) Exposed to different concentrations of nitrogen dioxide gas, 15 ml / min, 37 ℃ for 1 h, the use of MTT and NUR test cytotoxicity; collection under different conditions, gasoline generator exhaust, 10 ml / min, 37 ℃ dye Toxic A549 cells for 15 min, using CCK-8 and MTT assay cytotoxicity. Results The cell viability was significantly decreased with the increase of NO2 concentration (5.1-20.7 mg / m3) (P <0.05), and the dose-response relationship was observed (MTT curve: yMTT = 0.989-0.021xNO2R2 = 0.84, P <0.01; NUR fitting curve: yNUR = 1.032-0.026xNO2R2 = 0.88, P <0.01). The survival rates of MTT and CCK-8 in A549 cells were significantly lower than those in the air exposure group (P <0.05) at idle (cold start), 53% and 100% load. Conclusion The direct exposure of inhalable substances to cells in vitro could be applied to the in vitro toxicity study of harmful substances and provided new techniques and methods for the toxicity evaluation of harmful substances in industrial and environmental sites.