Yu Tao, Zhang Xueyan, Wang Yanhua, Meng Tao, Wang Zhongxu, Li Bin, Zheng Yuxin, Bin Ping
National Institute of Occupational Health and Poison Control, Chinese Center for Disease Control and Prevention, Beijing 100050, China.
Wei Sheng Yan Jiu. 2017 Sep;46(5):689-694.
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.
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.
In the non-f VE group, the PM of number, surface and mass concentration for 0. 5-10 μm diameters were 0. 24×103N/cm3, 0. 29 ×103μm2/cm3 and 0. 19 μg/m3, respectively, and for the PM of 10-500 nm diameters, they were 56 ×103N/cm3、34. 53 ×108nm2/cm3 and 95ng/m3, respectively. The PM of 0. 5-10 μm diameters in f VE group, their number, surface and mass concentration were less than 1 N/cm3, 1 μm2/cm3 0. 001 μg/m3, 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)×105, ( 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) ×105∶( 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).
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.
采用气液界面（ALI）法体外评价汽车尾气（VE）对肺上皮细胞的毒性作用，并分析经0.2μm过滤器处理后VE的不同毒性。
用20升泰德拉袋收集VE，通过粒径谱仪测量其颗粒物（PM）数量、表面积和质量浓度，以考察0.2μm过滤器或无过滤器的干扰情况。实验分为四组，即空白对照组、清洁空气组、过滤后VE暴露组、未过滤VE暴露组。空白对照组不做任何处理；清洁空气组为含21% O₂和79% N₂的人工气体；过滤后VE组（标记为f VE）使用0.2μm颗粒过滤器对VE进行过滤。未过滤VE组直接使用气袋收集的VE，标记为non-f VE。除空白对照组外，采用ALI法将BEAS-2B细胞在体外以25 mL/min的流速、37℃处理60 min，分别给予清洁空气或VE。采用CCK-8法评估细胞相对活力。用2',7'-二氯二氢荧光素二乙酸酯（DCFH-DA）探针通过流式细胞术测定活性氧（ROS）生成。采用Annexin V-FITC/PI商业试剂盒通过流式细胞术检测凋亡和坏死率。
在未过滤VE组中，直径0.5 - 10μm的PM数量、表面积和质量浓度分别为0.24×10³N/cm³、0.29×10³μm²/cm³和0.19μg/m³，直径10 - 500nm的PM数量、表面积和质量浓度分别为56×10³N/cm³、34.53×10⁸nm²/cm³和95ng/m³。在过滤后VE组中，直径0.5 - 10μm的PM数量、表面积和质量浓度分别小于1 N/cm³、1μm²/cm³和0.001μg/m³。过滤后，直径10 - 500nm的PM数量、表面积和质量浓度与未过滤VE相比分别降低了89.79%、93.57%和90.55%。在清洁空气组中，细胞相对活力、ROS生成、早期凋亡率和晚期凋亡及坏死率分别为（90.15±4.25）%、（1.92±0.34）×10⁵、（1.09±0.48）%和（8.93±3.31）%。与清洁空气组相比，两个VE暴露组的细胞相对活力、ROS生成和晚期凋亡及坏死率均有显著差异（均P < 0.05）。过滤后VE暴露组的细胞相对活力显著高于未过滤VE暴露组（t = 6.331，P < 0.001），而过滤后VE与未过滤VE暴露组之间的ROS生成[f VE∶non-VE =（2.94±0.21）×10⁵∶（3.32±0.49）×10⁵，t = -1.252，P = 0.279]、早期凋亡率[f VE∶non-VE =（1.09±0.30）%∶（0.99±0.10）%，t = 0.708，P = 0.497]和晚期凋亡及坏死率[f VE∶non-VE =（21.75±10.37）%∶（15.32±2.74）%，t = 1.347，P = 0.242]均无显著差异（均P > 0.05）。
采用ALI法在体外以25 mL/min的流速、37℃处理60 min时，观察到对人肺细胞（BEAS-2B）毒性增加。使用0.2μm过滤器后，毒性明显降低。
