气-液界面暴露汽车尾气致BEAS-2B细胞的毒性效应

Toxicity of vehicle exhaust on BEAS-2B cells in vitro by air-liquid interface

目的应用新型的气-液界面细胞染毒手段,探讨汽车尾气对人肺细胞毒性效应的影响程度,以及采用0.2μm滤器过滤尾气干预后的细胞毒性变化情况。方法用20 L Tedlar气袋收集汽车尾气,并通过粒径谱仪分析0.2μm滤器过滤前后的汽车尾气颗粒物数量、表面积和质量浓度。实验分为空白对照组、洁净空气组、汽车尾气过滤组和汽车尾气组。空白对照组未作任何处理;洁净空气组为21%O_2和79%N_2;汽车尾气过滤组为用0.2μm颗粒物过滤器过滤后的汽车尾气;汽车尾气组为用气袋直接收集的汽车尾气。除了空白对照组,其余3组利用气-液界面体外染毒技术,在染毒流量为25 mL/min,37℃水浴条件下,对生长在气-液界面插件多孔膜上的BEAS-2B细胞持续染毒60 min。利用CCK-8(cell counting Kit-8)检测法评价细胞的存活率。采用2',7'-二氯荧光黄双乙酸盐(2',7'-dichloro-dihydro-fluorescein diacetate,DCFH-DA)探针检测细胞内活性氧(reactive oxygen species,ROS)水平。Annexin V-FITC/PI细胞凋亡检测试剂盒检测汽车尾气对BEAS-2B细胞凋亡及坏死率的影响。结果汽车尾气组中0.5~10μm颗粒物数量浓度、表面积浓度和质量浓度分别为0.24×10~3个/cm^3、0.29×10~3μm^2/cm^3和0.19μg/m^3,10~500 nm颗粒物数量浓度、表面积浓度和质量浓度分别为56×10~3个/cm^3、34.53×10~8nm^2/cm^3和95 ng/m^3。汽车尾气过滤组0.5~10μm颗粒物数量浓度、表面积浓度和质量浓度分别低于1个/cm^3、1μm^2/cm^3和0.001μg/m^3,10~500 nm颗粒物数量浓度、表面积浓度和质量浓度与汽车尾气组相比分别降低了89.79%、93.57%和90.55%。洁净空气组的细胞存活率、胞内ROS、早期凋亡率和晚期凋亡坏死率分别为(90.15±4.25)%、(1.92±0.34)×10~5、(1.09±0.48)%、(8.93±3.31)%。与洁净空气组比较,两组汽车尾气组的细胞存活率、胞内ROS和晚期凋亡及坏死率差异均有统计学意义(P<0.05)。汽车尾气过滤组与汽车尾气组之间的比较发现,汽车尾气组细胞存活率[(58.09±4.06)%]低于汽车尾气过滤组[(66.85±3.49)%],差异有统计学意义(t=6.331,P<0.001),胞内ROS[汽车尾气过滤组∶汽车尾气组=(2.94±0.21)×10~5∶(3.32±0.49)×10~5,t=-1.252,P=0.279]、早期凋亡率[汽车尾气过滤组∶汽车尾气组=(1.09±0.30)%∶(0.99±0.10)%,t=0.708,P=0.497]和晚期凋亡坏死率[汽车尾气过滤组∶汽车尾气组=(21.75±10.37)%∶(15.32±2.74)%,t=1.347,P=0.242]差异均无统计学意义(P>0.05)。结论人肺细胞气-液界面暴露于汽车尾气可产生显著的毒性效应,0.2μm过滤器过滤尾气干预后,细胞毒性会明显降低。

Objective To evaluate the toxic effect of vehicle exhaust（ VE） on lung epithelial cells by air-liquid interface（ ALI） method in vitro,and analyze the different toxicity of VE after being treated with 0. 2 μm filter. Methods VE were collected using20 liter Tedlar bags and their particulate matter（ PM） number, surface and mass concentration were measured by particle size spectrometer for the interference of 0. 2 μm filter or non-filter. Four groups were included,which divided into blank control group,clean air group,filtered VE exposure group,non-filtered VE exposure group. The blank control group did not do any treatment; the clean air group was an artificial gas containing21% O_2 and 79% N_2; the filtered VE group（ marked as f VE） was filtered using a 0. 2μm particle filter for VE. The VE group was used VE directly collected by air bag and marked as non-f VE. Except the blank control group,BEAS-2B cells were treated with clean air or VE by ALI method at a flow rate of 25 mL/min,37 ℃ for 60 min in vitro.Cell relative viability was evaluated by CCK-8 assay. The reactive oxygen species（ ROS）generation was determined via flow cytometry with 2＇,7＇-dichloro-dihydro-fluorescein diacetate（ DCFH-DA） probe. Apoptosis and necrosis rate were measured using the commercial kit of Annexin V-FITC/PI by flow cytometry. Results In the non-f VE group,the PM of number,surface and mass concentration for 0. 5-10 μm diameters were 0. 24×10~3N/cm^3,0. 29 ×10~3μm^2/cm^3 and 0. 19 μg/m^3,respectively,and for the PM of 10-500 nm diameters,they were 56 ×10~3N/cm^3、34. 53 ×10~8nm^2/cm^3 and 95ng/m^3,respectively. The PM of 0. 5-10 μm diameters in f VE group,their number,surface and mass concentration were less than 1 N/cm^3,1 μm^2/cm^3和 0. 001 μg/m^3,respectively.After filtration,the number,surface and mass concentration of PM in 10-500 nm diameters reduced by 89. 79%,93. 57% and 90. 55%,respectively,as compared with non-f VE. In the clean air group, the cell relative viability, ROS generation, early apoptosis rate and late apoptosis and necrosis rate were（ 90. 15 ± 4. 25） %,（ 1. 92 ± 0. 34）×10~5,（ 1. 09 ± 0. 48） % and（ 8. 93 ± 3. 31） %,respectively. Compared with the clean air group,the cell relative viability,the ROS generation and the late apoptosis and necrosis rate of the two VE exposure groups were significantly different（ all P 0. 05）.The cell relative viability of f VE exposure group were significantly higher than that in the non-f VE exposure group（ t = 6. 331,P 0. 001）,and had no significant difference about the ROS generation[f VE ∶ non-VE =（ 2. 94 ± 0. 21） ×10~5∶（ 3. 32 ± 0. 49） ×10~5,t =-1. 252,P = 0. 279],early apoptosis rate [f VE∶ non-VE =（ 1. 09 ± 0. 30） % ∶（ 0. 99 ±0. 10） %,t = 0. 708,P = 0. 497] and late apoptosis and necrosis rate [f VE ∶ non-VE =（ 21. 75 ± 10. 37） % ∶（ 15. 32 ± 2. 74） %,t = 1. 347,P = 0. 242] between f VE and nonf VE exposure group（ all P 0. 05）. Conclusion Increased toxicity of human lung cells（ BEAS-2B） in vitro were observed by ALI method at a flow rate of 25 mL/min,37 ℃ for60 min. After using a 0. 2 μm filter,the toxicity was obviously decreased.