摘要
目的应用气-液界面(air-liquid interface,ALI)染毒方式研究汽油发动机尾气(gasoline engine exhaust,GEE)对大鼠原代肺泡巨噬细胞(primary alveolar macrophage,PAM)吞噬功能和炎症细胞因子分泌的影响。方法利用原位肺泡灌洗技术获取大鼠PAM,通过免疫荧光和瑞氏染色鉴定细胞。以摩托车尾气作为GEE染毒物质,实验分为空白对照组、洁净空气组、1∶10稀释尾气组(1∶10 GEE组)、1∶5稀释尾气组(1∶5 GEE组)和纯尾气组(纯GEE组)。PAM接种在Transwell 6孔板小室内,利用ALI细胞暴露系统以10 mL/min为暴露流量持续染毒1 h。染毒结束后,用瑞氏染色法在显微镜下观察细胞状态;CCK-8法(Cell Counting Kit-8,CCK-8)检测PAM相对存活率;流式细胞术和荧光酶标仪检测PAM吞噬功能;酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)检测细胞上清中肿瘤坏死因子-α(tumor necrosis factor,TNF-α)、白细胞介素-1β(interleukin-1β,IL-1β)、白细胞介素-4(interleukin-4,IL-4)、白细胞介素-10(interleukin-10,IL-10)浓度。结果通过原位肺泡灌洗提取到大鼠PAM数量为1.1×10^(6)~1.6×10^(6)个/只大鼠,纯度可达到96.40%;镜下观察瑞氏染色后的PAM具有明显的巨噬细胞样特征。ALI暴露GEE后,瑞氏染色镜下观察PAM,与洁净空气组相比,1∶5 GEE组和纯GEE组细胞碎片和脱核细胞明显增多;PAM的相对存活率随着GEE稀释比例减小而下降,呈现良好的剂量依赖效应(F=18.84,P<0.001);流式细胞术检测发现PAM吞噬率随着尾气浓度升高而显著增加,组间差异具有统计学意义(F=103.80,P<0.001);使用荧光酶标仪检测的结果发现,与洁净空气组相比,1∶5 GEE组和纯GEE组的吞噬功能显著增强(P<0.05)。PAM染毒后培养上清液中炎症因子TNF-α、IL-1β分泌水平在1∶10 GEE组和1∶5 GEE组出现促进作用,在纯GEE组则分泌水平均显著降低,且组间差异具有统计学意义(F值分别为6.18和3.55,P<0.05);纯GEE组PAM分泌抗炎因子IL-4和IL-10增加,其中IL-4显著高于空白对照组和1∶10 GEE组,且组间差异有统计学意义(F=5.72,P=0.002)。结论ALI暴露GEE整体组分可显著影响PAM的吞噬功能,可能与PAM促炎/抗炎细胞因子分泌有关。
Objective To study the effects of gasoline engine exhaust(GEE)on phagocytosis and inflammatory cytokine secretion in rat primary alveolar macrophages(PAMs)by using the air-liquid interface(ALI)exposure method.Methods Rat PAMs were obtained using the in situ alveolar lavage technique,and the cells were identified with immunofluorescence and Wright’s staining.Using motorcycle exhaust for GEE exposure,five groups were set up:blank control group,clean air group,1∶10 diluted GEE group(1∶10 GEE group),1∶5 diluted GEE group(1∶5 GEE group),and pure GEE group(pure GEE group).PAMs were inoculated in a Transwell 6-well plate chamber to be exposed to clean air or GEE at an exposure flow rate of 10 mL/min for 1 hour through the ALI cell exposure system.Subsequently,cell morphology was observed with Wright’s staining under a microscope;the relative survival rate of PAMs was measured using Cell Counting Kit-8;the phagocytic function of PAMs was examined using flow cytometry and a fluorescence microplate reader;and the concentrations of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),interleukin-4(IL-4),and interleukin-10(IL-10)in the cell culture supernatant were determined by enzyme-linked immunosorbent assay(ELISA).Results The number of PAMs extracted by in situ alveolar lavage was 1.1×10^(6)to 1.6×10^(6)cells per rat,with the purity reaching 96.40%,and the Wright’s stained-PAMs showed distinct macrophage-like features.After ALI exposure to GEE,cellular debris and denucleation were more common in the 1∶5 GEE group and the pure GEE group than in the clean air group;the relative survival rate of PAMs decreased with a decreasing dilution ratio of GEE,showing a significant dose-dependent effect(F=18.84,P<0.001);the flow cytometry-detected phagocytic rate of PAMs was increased significantly with the increasing concentration of GEE(F=103.80,P<0.001);the fluorescence microplate reader-detected phagocytic function was significantly enhanced in the 1∶5 GEE and pure GEE groups compared with that in the clean air group(P<0.05).The levels of pro-inflammatory TNF-αand IL-1βwere slightly higher in the 1∶10 GEE group and 1∶5 GEE group while significantly reduced in the pure GEE group,and there were statistically significant differences among different groups(F=6.18,3.55,respectively;P<0.05).Anti-inflammatory IL-4 and IL-10 levels were increased in the pure GEE group,and the level of IL-4 was significantly higher compared with that in the blank control and 1∶10 diluted GEE group,and there were statistically significant differences among different groups(F=5.72,P=0.002).Conclusion ALI exposure to GEE can significantly affect the phagocytic function of PAMs,which may be related to the secretion of pro-inflammatory/anti-inflammatory cytokines by PAMs.
作者
晋瑜
裴梦娟
李国梁
鱼涛
张雪艳
赵玮
郑敏
李斌
宾萍
JIN Yu;PEI Meng-juan;LI Guo-liang;YU Tao;ZHANG Xue-yan;ZHAO Wei;ZHENG Min;LI Bin;BIN Ping(National Institute for Occupational Health and Poison Control,Chinese Center forDisease Control and Prevention,Beijing 100050,China)
出处
《环境卫生学杂志》
2025年第5期380-388,421,共10页
JOURNAL OF ENVIRONMENTAL HYGIENE
基金
国家自然科学基金面上项目(82073596,81472955)。
关键词
汽油发动机尾气
原代肺泡巨噬细胞
吞噬功能
细胞因子
gasoline engine exhaust
primary alveolar macrophage
phagocytic function
inflammatory cytokine
作者简介
晋瑜,硕士研究生,从事卫生毒理学研究,Email:15935320163@163.com;通信作者:宾萍,研究员,从事卫生毒理学研究,Email:binping@niohp.chinacdc.cn。
