【作者】 孟庆玉；

【导师】 段化伟；

【作者基本信息】 中国医科大学 ， 公共卫生（专业学位）， 2023， 硕士

【摘要】 目的:金属(Metal)和多环芳烃(Polycyclic aromatic hydrocarbons,PAHs)是颗粒物(Particulate matter)的主要有害组分。长期暴露于环境颗粒物已被认为是呼吸系统疾病发病和死亡的主要危险因素。生物节律基因的转录调控与呼吸系统疾病的进展之间有着密切联系。然而生物节律基因表达与大气颗粒物主要组分暴露的分子流行病学研究还比较有限。研究方法:本研究以中国两个城市的学龄儿童作为研究对象,通过对两地区全年大气颗粒物污染物浓度、校区定点采样浓度、儿童人群尿液中的多环芳烃羟基代谢物、金属代谢物进行检测获得儿童人群的内、外暴露特征。采用索氏提取法提取采样膜固相颗粒物中的有机组分,检测颗粒物上附着的金属和苯并[a]芘(Benzo[a]pyrene,Ba P)等组分。检测尿多环芳烃羟基代谢物和尿金属浓度,评估颗粒物主要组分的内暴露水平。使用RT-qPCR法测量儿童外周血白细胞CLOCK、BMAL1、PER2及CRY1基因的mRNA表达水平。严格按照美国胸科学会和欧洲呼吸学会的指南对受检儿童进行肺功能测试。检测肺功能指标、血清CC16水平作为肺损伤标志物。检测丙二醛(Malondialdehyde,MDA)评估机体氧化应激水平。采用线性回归模型分析多环芳烃和金属与生物节律基因、血清CC16和肺功能参数的剂量-效应关系。贝叶斯核机器回归模型(Bayesian Kernel Machine Regression,BKMR)评估多环芳烃和金属混合物对生物节律基因及氧化应激的影响,进一步利用中介效应分析生物节律基因、氧化应激在颗粒物主要组分与肺损伤标志物改变中的作用关系。结果:1、颗粒物及其组分与儿童生物节律基因、肺损伤标志物、氧化应激的改变随着颗粒物暴露水平的升高,儿童血清CC16水平升高(P=0.014),白细胞CLOCK、BMAL1、PER2、CRY1表达水平升高(P<0.001),一秒率、第一秒用力呼气容积实测值占预测值百分比、用力肺活量实测值占预测值百分比、最大呼气中段流量实测值占预测值百分比、一秒率实测值占预测值百分比降低(P<0.001)。分层分析发现,铅导致男生CRY1表达升高;镉导致正常体重儿童BMAL1表达升高;2-OHFlu导致初中儿童CRY1表达升高;2-OHNap导致被动吸烟儿童CLOCK表达升高。BKMR模型分析显示,2-OHFlu在CLOCK、CRY1中起重要作用,2-OHNap在BMAL1、PER2中起重要作用。混合暴露物与CLOCK、BMAL1、CRY1呈正向关联。当其他暴露物固定于中位数时,与单暴露线性回归结果大致相符,2-OHNap与CLOCK呈正相关,镉与BMAL1呈正相关,2-OHFlu、铅与CRY1呈正相关。钒与肺活量、用力肺活量、第一秒用力呼气容积呈负相关(P<0.05),与一秒率、一秒率实测值占预测值百分比呈正相关(P<0.05);镉与一秒率、一秒率实测值占预测值百分比呈正相关(P<0.05);铅与肺活量、用力肺活量呈负相关(P<0.05)。校正协变量后,2-OHNap、2-OHFlu每增加一个IQR,肺活量、一秒率实测值占预测值百分比存在不同程度降低(P<0.05)。分层分析发现,2-OHNap、2-OHFlu、铜、锶、铅、铁是肺功能降低的主要组分;钒、钴、镉是肺功能升高的主要组分。钒、铜、钴、锶、镉与尿MDA呈正相关(P<0.05)。MDA随2-OHNap、2-OHFlu、铜、钴、铁、锶水平升高呈剂量依赖性升高(P<0.05)。肺活量、用力肺活量、第一秒用力呼气容积、最大呼气中段流量、第一秒用力呼气容积实测值占预测值百分比、最大呼气中段流量实测值占预测值百分比随MDA升高呈剂量依赖性升高(P<0.05)。BKMR模型分析显示,钴、2-OHNap在MDA中起重要作用。整体混合物高于第70个百分位数时,混合暴露物与MDA呈正相关趋势,差异具有统计学意义,当其他颗粒物固定于中位数时,与单暴露线性回归结果相符,铜、钴、镉与尿MDA呈正相关。2、生物节律基因与儿童肺损伤标志物、氧化应激的关系白细胞BMAL1与一秒率、最大呼气中段流量实测值占预测值百分比、一秒率实测值占预测值百分比呈负相关(P<0.05)。校正协变量后,白细胞BMAL1每改变一个IQR,肺活量、用力肺活量、第一秒用力呼气容积实测值占预测值百分比呈不同程度降低(P<0.05)。分层分析发现,白细胞CLOCK、BMAL1、CRY1升高与肺功能降低密切相关。尿MDA每改变一个IQR,正常体重儿童PER2表达降低12.61%(P=0.009)。3、生物节律基因、氧化应激MDA的中介作用分析MDA在钴与用力肺活量实测值占预测值百分比改变之间存在中介效应。MDA与CRY1在钒致肺活量改变中存在链式中介作用。结论:1、分子流行病学研究发现,颗粒物组分暴露与儿童外周血生物节律基因表达改变有关,且具有一定的暴露-反应关系。2、氧化应激水平改变在金属组分暴露致儿童肺活量改变中具有中介作用。生物节律基因CRY1及氧化应激水平改变在颗粒物组分致儿童肺功能改变中具有链式中介作用。更多还原

【Abstract】 Objective:Metals and Polycyclic aromatic hydrocarbons（PAHs）are the main harmful components of Particulate matter.Long-term exposure to ambient particulate matter has been recognized as a major risk factor for respiratory disease morbidity and mortality.The transcriptional regulation of circadian rhythm genes is closely related to the progression of respiratory diseases.However,molecular epidemiological studies on circadian rhythm gene expression and exposure to major components of atmospheric particulate matter are limited.Methods:In this study,school-age children in two cities in China were selected as the research objects,and the internal and external exposure characteristics of children were obtained through the detection of the annual concentration of atmospheric particulate matter pollutants in the two regions,the concentration of fixed sampling points in school districts,and the PAHs hydroxyl metabolites and metal metabolites in the urine of children.Soxhlet extraction method was used to extract organic components from solid particles in the sampling film,and the metal and Benzo[a]pyrene（Ba P）components attached to the particles were detected.Urinary polycyclic aromatic hydroxyl metabolites and urinary metal concentrations were measured to assess the internal exposure levels of major components of particulate matter.The mRNA expression levels of CLOCK,BMAL1,PER2 and CRY1 genes in children’s peripheral blood cells were measured by RT-q PCR.Lung function tests were performed strictly in accordance with the guidelines of the American Thoracic Society and the European Respiratory Society.Lung function index and serum CC16 level were detected as markers of lung injury.Malondialdehyde（MDA）was used to assess oxidative stress in organism.Linear regression model was used to analyze the dose-response relationship between PAHs and metals and circadian rhythm genes,serum CC16 and lung function parameters.Bayesian Kernel Machine Regression（BKMR）was used to evaluate the effects of PAHs and metal mixtures on circadian rhythm genes and oxidative stress.The relationship between circadian rhythm genes and oxidative stress on the changes of main components of particulate matter and markers of lung injury was further analyzed by using mediating effects.Results:1. Changes of particulate matter and its components in circadian rhythm genes,markers of lung injury and oxidative stress in children.With the increase of particulate matter exposure level,the serum CC16 level was increased（P=0.014）,and the expressions of CLOCK,BMAL1,PER2 and CRY1 in white blood cells were increased（P<0.001）.FEV1/FVC,the percent of predicted FEV1,the percent of predicted FVC,the percent of predicted FEF_25-75%,and the percent of predicted FEV1/FVC decreased（P<0.001）.Stratified analysis showed that Plumbum caused increased CRY1 expression in male students.Cadmium increased the expression of BMAL1 in normal weight children.2-OHFlu increased CRY1 expression in junior middle school children.2-OHNap induced increased CLOCK expression in passive smoking children.BKMR model analysis showed that 2-OHFlu plays an important role in CLOCK and CRY1,and 2-OHNap plays an important role in BMAL1 and PER2.The mixed exposures were positively correlated with CLOCK,BMAL1 and CRY1.When other exposures were fixed at the median,2-OHNap was positively correlated with CLOCK,Cadmium was positively correlated with BMAL1,and 2-OHFlu and Plumbum were positively correlated with CRY1.Vanadium was negatively correlated with vital capacity,forced vital capacity and forced expiratory volume in the first second（P<0.05）,was positively correlated with FEV1/FVC and the percent of predicted FEV1/FVC（P<0.05）;Cadmium was positively correlated with FEV1/FVC and the percent of predicted FEV1/FVC（P<0.05）;Plumbum was negatively correlated with vital capacity and forced vital capacity（P<0.05）.After adjusting for covariates,vital capacity and the percent of predicted FEV1/FVC decreased with each IQR increase of 2-OHNap and 2-OHFlu（P<0.05）.Stratified analysis showed that 2-OHNap,2-OHFlu,Cuprum,Strontium,Plumbum and Ferrum were the main components of reduced lung function.Vanadium,Cobalt and Cadmium are the main components of elevated lung function.Vanadium,Cuprum,Cobalt,Strontium and Cadmium were positively correlated with urinary MDA（P<0.05）.MDA increased dose-dependent with the levels of 2-OHNap,2-OHFlu,Cuprum,Cobalt,Ferrum and Strontium（P<0.05）.Vital capacity,forced vital capacity,forced expiratory volume in the first second,mean forced expiratory flow between 25%and 75%of FVC,the percent of predicted FEV1,and the percent of predicted FEF_25-75%increased dose-dependent with the increase of MDA（P<0.05）.BKMR model analysis shows that Cobalt and 2-OHNap play an important role in MDA.When the total mixture was above the 70th percentile,the mixed exposure was positively correlated with MDA,and the difference was statistically significant.When other particulate matter was fixed at the median,it was consistent with the linear regression result of single exposure,and Cuprum,Cobalt and Cadmium were positively correlated with urinary MDA.2. Relationship between circadian rhythm genes and markers of lung injury and oxidative stress in children.Leukocyte BMAL1 was negatively correlated with FEV1/FVC,the percent of predicted FEF_25-75%and the percent of predicted FEV1/FVC（P<0.05）.After correcting for covariates,vital capacity,forced vital capacity and the percent of predicted FEV1decreased to different degrees with each change of IQR of BMAL1（P<0.05）.Stratified analysis showed that the increase of Leukocyte CLOCK,BMAL1 and CRY1 was closely related to the decrease of lung function.The expression of PER2 in normal weight children decreased by 12.61%（P=0.009）for each change of IQR in urinary MDA.3. Analysis of mediating role of circadian rhythm gene and oxidative stress MDA.The mediating effect of MDA on the change of cobalt and the percent of predicted FVC was found.MDA and CRY1 have a chain mediated effect on vanadium-induced lung capacity.Conclusion:1.Molecular epidemiological studies have found that exposure to particulate matter components is associated with changes in the expression of circadian rhythm genes in children’s peripheral blood,and there is a certain expotion-response relationship.2.The changes of oxidative stress levels play a mediating role in the changes of lung capacity in children induced by exposure to metal components.The circadian rhythm gene CRY1 and the change of oxidative stress level play a chain mediating role in the changes of pulmonary function induced by particulate matter.更多还原