【摘要】：焦?fàn)t逸散物(Coke oven emissions,COE)是焦化生產(chǎn)過(guò)程中的主要排放污染物,其中含有大量的顆粒物(Particulate matter,PM)和多環(huán)芳烴(Polycyclic aromatic hydrocarbons,PAHs)。流行病學(xué)研究表明,高濃度的顆粒物或多環(huán)芳烴暴露是呼吸系統(tǒng)疾病發(fā)生發(fā)展的主要原因之一,其中COE所致肺癌被列為明確的職業(yè)性腫瘤。細(xì)顆粒物(Fine particulate matter,PM2.5)由于具有小于2.5μμm的空氣動(dòng)力學(xué)直徑和易于富集有毒有害物質(zhì)的表面,對(duì)呼吸系統(tǒng)的損害效應(yīng)也更加明確。但在COE暴露與效應(yīng)標(biāo)志物的劑量-反應(yīng)關(guān)系研究中,以致癌性比較明確的PAHs為主要目標(biāo)污染物開(kāi)展的較多,還缺少對(duì)于其中PM2.5健康效應(yīng)的暴露-反應(yīng)關(guān)系的分析。雖然,流行病學(xué)研究已經(jīng)證實(shí)職業(yè)COE暴露與焦?fàn)t工人罹患肺癌間的因果聯(lián)系,但從暴露到肺癌發(fā)生過(guò)程中的重要生物學(xué)改變和早期不良健康效應(yīng)的標(biāo)志物研究還很有限。研究發(fā)現(xiàn),循環(huán)系統(tǒng)中多種蛋白質(zhì)表達(dá)的變化與維持呼吸系統(tǒng)穩(wěn)態(tài)密切相關(guān),包括肺表面活性蛋白 A(Surfactant protein A,SP-A)、肺表面活性蛋白 D(Surfactant protein D,SP-D)和克拉拉細(xì)胞蛋白(Club cell protein,CC16)等。研究人員已經(jīng)在臨床科研工作中開(kāi)展了多項(xiàng)肺損傷蛋白與慢性阻塞性肺病等呼吸疾病發(fā)生和進(jìn)展的研究。但是,關(guān)于COE致血液中肺損傷相關(guān)標(biāo)志物改變的研究還未見(jiàn)報(bào)道。顆粒物和PAHs暴露可導(dǎo)致多種疾病的發(fā)病風(fēng)險(xiǎn)增加,尤其是在致癌過(guò)程中,表觀遺傳學(xué)改變可能發(fā)揮了重要作用。表觀遺傳不涉及DNA序列的改變,具有可逆性等特點(diǎn)。DNA甲基化是重要的表觀遺傳修飾之一。我們以及其他研究團(tuán)隊(duì)在早期研究中發(fā)現(xiàn)PAHs暴露可以導(dǎo)致多個(gè)DNA修復(fù)基因和抑癌基因的甲基化異常改變。但對(duì)基因組整體水平的甲基化研究報(bào)道較少,目前也缺乏PM2.5致機(jī)體基因組DNA甲基化與DNA甲基轉(zhuǎn)移酶(DNA methyltransferase,DNMTs)關(guān)系的研究。為了解釋上述問(wèn)題,我們?cè)诒狈侥辰够瘡S人群中開(kāi)展了 COE暴露與早期損害標(biāo)志物的暴露-反應(yīng)關(guān)系的分子流行病學(xué)研究。首先對(duì)工作場(chǎng)所多個(gè)工種進(jìn)行顆粒物和多環(huán)芳烴等污染物的暴露評(píng)價(jià);然后招募558名焦?fàn)t作業(yè)工人作為暴露人群,210名氧氣廠和冷軋廠工人作為對(duì)照。檢測(cè)血清中機(jī)體炎癥水平(hs-CRP)、肺損傷標(biāo)志物(CC16,SP-A和SP-D)水平,結(jié)合肺功能變化,探討焦?fàn)t逸散物中PM2.5和PAHs對(duì)肺部的損傷效應(yīng)。觀察研究對(duì)象白細(xì)胞基因組DNA甲基化水平和DNA甲基化轉(zhuǎn)移酶的表達(dá)水平改變,并結(jié)合中介效應(yīng)探討DNA甲基化轉(zhuǎn)移酶在COE主要成分致基因組DNA甲基化和肺損傷改變中的作用。一、焦?fàn)t逸散物致血清呼吸系統(tǒng)損傷標(biāo)志物的改變通過(guò)稱(chēng)重法計(jì)算COE中的PM2.5含量,超高效液相色譜法檢測(cè)顆粒相中多環(huán)芳烴的濃度。超高效液相色譜串聯(lián)質(zhì)譜法檢測(cè)尿中1-羥基芘(1-hydroxyrene,1-OHP)的濃度,ELISA法檢測(cè)血清中SP-A、SP-D和CC16的濃度,同時(shí)檢測(cè)研究對(duì)象的肺功能。研究發(fā)現(xiàn)隨著COE中PM2.5和PAHs暴露程度的增高,血清CC16呈降低趨勢(shì)(P=0.046)。在全人群為基礎(chǔ)的暴露-反應(yīng)關(guān)系分析中發(fā)現(xiàn),COE中PM2.5增加1個(gè)IQR(121.98 μg/m3)導(dǎo)致血清 CC16 降低 5.24%(P= 0.004);COE 中總 PAHs 增加 1 個(gè)IQR(3.81μg/μm3),會(huì)導(dǎo)致血清 CC16 降低 5.69%(P = 0.027);尿 1-OHP 每增加 1個(gè)IQR(1.06μmol/mol肌酐),會(huì)導(dǎo)致血清CC16降低4.67%(P= 0.041)。在男性工人中,還發(fā)現(xiàn)血清 CC16 每降低 1 個(gè) IQR(3.95 ng/mL),FEVl/FVC 降低 0.86%(P=0.045)。二、焦?fàn)t逸散物致基因組DNA甲基化改變采用ELISA法檢測(cè)基因組DNA甲基化(5-mC%)的含量,實(shí)時(shí)熒光定量PCR(RT-qPCR)法進(jìn)行DNMTs的mRNA定量分析,同時(shí)檢測(cè)研究對(duì)象血清中的葉酸和維生素B12水平。研究發(fā)現(xiàn),全人群中,隨著COE暴露水平的增加,基因組DNA甲基化(5-mC%)水平呈顯著的下降趨勢(shì)(P = 0.001)。暴露-反應(yīng)關(guān)系分析發(fā)現(xiàn),COE中PM2.5增加一個(gè)IQR(121.98 μg/m3),會(huì)引起基因組 DNA 甲基化(5-mC%)降低 5.78%(P0.001);總PAHs每增加一個(gè)IQR(3.81 μg/m3),導(dǎo)致基因組DNA甲基化降低6.09%(P=0.007)。研究發(fā)現(xiàn)尿1-OHP與基因組DNA的5-mC含量呈顯著的負(fù)相關(guān)(β=-0.025,P0.001)。DNA甲基化轉(zhuǎn)移酶的定量研究發(fā)現(xiàn),隨著COE暴露程度的增加,DNMT3A基因的mRNA表達(dá)水平呈下降趨勢(shì)(P = 0.047);其中總PAHs每增加一個(gè)IQR(3.81 μg/m3),DNMT3A表達(dá)量降低13.25%(P= 0.027),沒(méi)有發(fā)現(xiàn)DNMT1表達(dá)量的顯著改變�；貧w分析顯示基因組DNA甲基化(5-mC%)與DNMT1和DNMT3A均存在顯著的正相關(guān)。中介效應(yīng)分析發(fā)現(xiàn)PAHs暴露對(duì)基因組DNA甲基化效應(yīng)的5.95%可以被DNMT3A所介導(dǎo)。三、焦?fàn)t逸散物致肺損傷與DNA甲基化標(biāo)志物的關(guān)系研究采用相關(guān)分析和多元線性回歸分析探討了肺損傷標(biāo)志物(血清CC16、SP-A、SP-D)、肺功能與DNA甲基化指標(biāo)(5-mC%、DNMT1、DNMT3A)間的相互關(guān)系。發(fā)現(xiàn)血清CC16的濃度與DNMT1表達(dá)量呈顯著的正相關(guān)(r = 0.075,P=0.040)。經(jīng)年齡分層后發(fā)現(xiàn),在小于45歲年齡組中血清CC16與DNMT1和DNMT3A均存在顯著的正相關(guān)。中介效應(yīng)分析沒(méi)有發(fā)現(xiàn)DNA甲基化在COE致肺損傷中的中介作用。研究結(jié)論1.研究發(fā)現(xiàn)焦?fàn)t逸散物中PM2.5和PAHs都可以導(dǎo)致呼吸系統(tǒng)損傷和DNA甲基化改變,且其作用模式不同。2.焦?fàn)t逸散物暴露與血清CC16存在明確的暴露-反應(yīng)關(guān)系,血清CC16降低可作為焦?fàn)t逸散物致肺損傷的早期效應(yīng)標(biāo)志物。3.焦?fàn)t逸散物暴露可引起基因組DNA低甲基化改變,DNMT3A是介導(dǎo)基因組DNA甲基化降低的重要因素。
[Abstract]:Coke oven emissions (COE) are the main pollutants discharged during the coking process, which contain a large number of particulate matter (PM) and polycyclic aromatic hydrocarbons (PAHs). Epidemiological studies have shown that exposure to high concentrations of particulate matter or polycyclic aromatic hydrocarbons (PAHs) is the occurrence and development of respiratory diseases. Fine particulate matter (PM2.5) has an aerodynamic diameter of less than 2.5 microns and is prone to enrich toxic and harmful substances on the surface. The damage effect on respiratory system is also more clear. However, the exposure and effect markers of COE are agents. Among the dose-response relationships, PAHs with more definite carcinogenicity are the main target pollutants, and there is a lack of analysis on the exposure-response relationship of PM2.5 health effects. Although epidemiological studies have confirmed the causal link between occupational COE exposure and lung cancer in coke oven workers, the process from exposure to lung cancer is still lacking. Significant biological changes and biomarkers of early adverse health effects are still limited. Studies have found that changes in the expression of various proteins in the circulatory system are closely related to the maintenance of respiratory homeostasis, including lung surfactant protein A (SP-A), lung surfactant protein D (SP-D) and clara. Club cell protein (CC16) and so on. Researchers have carried out a number of clinical research work in lung injury protein and chronic obstructive pulmonary disease and other respiratory disease occurrence and progress of research. However, the study on COE-induced lung injury related markers in the blood has not been reported. Particulate matter and PAHs exposure can lead to a variety of. Epigenetic changes may play an important role in increasing the risk of disease, especially in carcinogenesis. Epigenetics does not involve changes in DNA sequences and is reversible. DNA methylation is one of the most important epigenetic modifications. We and other research teams found that PAHs exposure can lead to more in early studies. The methylation of DNA repair genes and tumor suppressor genes was abnormal. However, there were few reports on the methylation at the whole genome level. At present, there was no study on the relationship between DNA methyltransferase (DNMTs) and DNA methyltransferase (PM2.5). In order to explain the above problems, we carried out a study in a northern coking plant population. Molecular epidemiological study on the relationship between COE exposure and exposure-response of early damage markers was carried out. Firstly, exposure to particulate matter and polycyclic aromatic hydrocarbons (PAHs) was assessed in several workplaces. Then 558 coke oven workers were recruited as exposed population and 210 workers in oxygen plant and cold rolling plant as controls. The levels of somatic inflammation (hs-CRP), lung injury markers (CC16, SP-A and SP-D) and the effects of PM2.5 and PAHs in coke oven effluents on lung function were investigated. The changes of genomic DNA methylation and expression of DNA methylation transferase in leukocytes were observed, and the mediating effects of DNA methylation transferase were discussed. The role of coke oven fugitives in the alteration of genomic DNA methylation and lung injury induced by COE. 1. Changes of biomarkers in serum respiratory system injury induced by coke oven fugitives. PM2.5 content in COE was calculated by weighing method. Polycyclic aromatic hydrocarbons (PAHs) concentration in granular phase was detected by ultrahigh performance liquid chromatography-tandem mass spectrometry. Urine 1-Hydroxyl group was detected by ultrahigh performance liquid chromatography-tandem mass spectrometry. The concentration of pyrene (1-hydroxyrene, 1-OHP) in serum was detected by ELISA. The pulmonary function of the subjects was also measured. The study found that the serum CC16 decreased with the increase of exposure to PM2.5 and P AHs in COE (P = 0.046). In a population-based exposure-response relationship analysis, one IQR (12) was increased by PM2.5 in COE. 1.98 ug/m3 decreased serum CC16 by 5.24% (P = 0.004); total PAHs increased by 1 IQR (3.81 ug/m3) in COE, decreased serum CC16 by 5.69% (P = 0.027); urinary 1-OHP increased by 1 IQR (1.06 ugol/mol creatinine) and decreased serum CC16 by 4.67% (P = 0.041) in male workers. Ng / mL, FEVl / FVC decreased by 0.86% (P = 0.045). 2. Genomic DNA methylation induced by coke oven fugitives was detected by ELISA. Genomic DNA methylation (5-mC%) was detected by real-time fluorescence quantitative PCR (RT-qPCR) and the levels of folic acid and vitamin B12 in serum were detected by real-time fluorescence quantitative PCR. Genomic DNA methylation (5-mC%) decreased significantly with the increase of COE exposure (P = 0.001). Exposure-response relationship analysis showed that an IQR (121.98 ug/m3) was added to PM2.5 in COE, resulting in a 5.78% (P 0.001) decrease in genomic DNA methylation (5-mC%) and an IQR (3.81 ug/m3) increase in total PAHs, resulting in genomic DNA methylation. A significant negative correlation was found between urinary 1-OHP and genomic DNA 5-mC content (beta = - 0.025, P 0.001). Quantitative study of DNA methylation transferase showed that the mRNA expression level of DNMT3A gene decreased with the increase of COE exposure (P = 0.047), and the total PAHs increased by an IQR (3.81 ug/m3) and DNMT3A table. Regression analysis showed that genomic DNA methylation (5-mC%) was positively correlated with both DNMT1 and DNMT3A. Intermediate effect analysis showed that 5.95% of genomic DNA methylation induced by PAHs exposure could be mediated by DNMT3A. 3. lung injury induced by coke oven fugitives and DNA A Correlation analysis and multiple linear regression analysis were used to explore the relationship between lung injury markers (serum CC16, SP-A, SP-D), lung function and DNA methylation markers (5-mC, DNMT1, DNMT3A). There was no mediating role of DNA methylation in COE-induced lung injury. Conclusion 1. Both PM2.5 and PAHs in coke oven effluents can cause respiratory system damage and DNA methylation changes, and their modes of action are different. 2. There is a clear exposure-response relationship between coke oven fugitive exposure and serum CC16. Reduction of serum CC16 can be used as an early marker of lung injury induced by coke oven fugitive exposure. 3. Coke oven fugitive exposure can cause hypomethylation of genomic DNA. DNMT3A is an important factor mediating the reduction of genomic DNA methylation.
【學(xué)位授予單位】：中國(guó)疾病預(yù)防控制中心
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R13

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