本文選題：室內(nèi)空氣　切入點：揮發(fā)性有機物　出處：《大連理工大學(xué)》2013年博士論文　論文類型：學(xué)位論文

【摘要】：由于多種復(fù)合型建筑和裝飾材料以及日常有機生活用品的使用,使得室內(nèi)揮發(fā)性有機物(VOCs)為主的化學(xué)性污染越來越嚴(yán)重,而目前針對室內(nèi)空氣污染的健康評價,主要采用室內(nèi)空氣污染物的濃度、居住人員的健康問卷調(diào)查及單一污染物的毒性研究,缺乏能夠反映復(fù)合污染健康風(fēng)險的綜合性評價指標(biāo)。本項目通過建立典型室內(nèi)空氣污染物的復(fù)合暴露染毒小鼠的實驗動物模型,研究吸入室內(nèi)空氣典型污染物致小鼠的呼吸系統(tǒng)毒性、免疫毒性、遺傳毒性及其分子機理,為人群健康檢測提供靈敏的效應(yīng)生物標(biāo)志物,同時也為修訂和完善室內(nèi)空氣污染相關(guān)標(biāo)準(zhǔn)提供科學(xué)依據(jù)。主要內(nèi)容包括： (1)建立室內(nèi)揮發(fā)性有機物短期復(fù)合染毒模型(甲醛+苯+甲苯+二甲苯),設(shè)置對照組和4個不同劑量復(fù)合VOCs暴露組,依次是室內(nèi)空氣質(zhì)量標(biāo)準(zhǔn)濃度的10、30、50、100倍。對小鼠進(jìn)行連續(xù)10天,每天2小時的呼吸暴露染毒,研究VOCs對小鼠的氧化應(yīng)激、免疫、呼吸和血清生化毒性的影響。結(jié)果發(fā)現(xiàn),VOCs暴露對小鼠的肺和肝臟產(chǎn)生了氧化損傷、引起了脾的T細(xì)胞免疫功能異常、影響了肺灌洗液(BALF)中白細(xì)胞介素-6(IL-6)、神經(jīng)營養(yǎng)因子-3(NT-3)、P物質(zhì)水平和炎癥細(xì)胞數(shù)量,并且也對血清生化和血常規(guī)方面產(chǎn)生了影響。小鼠肝、肺臟中活性氧(ROS)、丙二醛(MDA)水平、脾細(xì)胞CD4+CD3+(%)、CD4+CD3+/CD8+CD3+的比率與VOCs濃度之間存在劑量-效應(yīng)的正相關(guān)關(guān)系,肝臟組織的谷胱甘肽過氧化物酶(GSH-Px)、肺臟組織的谷胱甘肽(GSH)、脾細(xì)胞CD8+CD3+(%)比率與VOCs濃度之間存在劑量-效應(yīng)的負(fù)相關(guān)關(guān)系。結(jié)果提示：小鼠肝和肺臟中ROS水平、肝臟組織的GSH-Px、脾細(xì)胞的淋巴細(xì)胞亞群比率可作為復(fù)合VOCs短期暴露的敏感效應(yīng)生物學(xué)標(biāo)志物；復(fù)合VOCs短期暴露氣道炎癥機制主要可能是通過NO信號路徑和ROS來誘導(dǎo)的,同時NT-3可以介導(dǎo)保護(hù)機制來抑制這種炎癥。 (2)利用miRNA芯片技術(shù)來獲得VOCs復(fù)合暴露引起的肺組織的高通量生物信息變化,分析比較了miRNA差異表達(dá),探討特異性生物標(biāo)志物,并對差異表達(dá)的miRNA預(yù)測靶基因,進(jìn)行其生物學(xué)功能和影響路徑(Pathway)分析,同時用ELISA來檢測BALF中白細(xì)胞介素-8(IL-8)的含量。實驗設(shè)置對照組和3個不同劑量的復(fù)合VOCs(甲醛+苯+甲苯+二甲苯)暴露組,依次是室內(nèi)空氣質(zhì)量標(biāo)準(zhǔn)濃度的30、50、100倍,對小鼠進(jìn)行連續(xù)10天,每天2小時的呼吸暴露染毒。結(jié)果發(fā)現(xiàn),與對照組比較,染毒組1,2,3分別有96、68、18個miRNA上調(diào)表達(dá),有662、592、11個miRNA下調(diào)表達(dá)。在表達(dá)差異的miRNA中,染毒組1,2,3同時上調(diào)表達(dá)的miRNA有6個,均下調(diào)表達(dá)的只有1個；有1個miRNA在染毒組1,2上調(diào)表達(dá),在染毒組3下調(diào)表達(dá)。另外,IL-8在暴露組也顯著升高。結(jié)果提示：可以優(yōu)先考慮mmu-miR-5105作為小鼠肺臟VOCs復(fù)合暴露的敏感miRNA分子標(biāo)志物；mmu-miR-494、mmu-miR-744、mmu-miR-335-5p和VOCs濃度間存在負(fù)相關(guān)關(guān)系；通過計算機網(wǎng)絡(luò)分析預(yù)測miRNA靶基因發(fā)現(xiàn),VOCs復(fù)合暴露可能潛在導(dǎo)致癌癥和氣道炎癥的發(fā)生,IL-8的ELISA檢測結(jié)果證實了VOCs復(fù)合暴露引起了氣道炎癥。 (3)建立長期暴露、較低濃度室內(nèi)VOCs復(fù)合染毒模型(甲醛+苯+甲苯+二甲苯),設(shè)置對照組和4個不同劑量復(fù)合VOCs暴露組,依次是室內(nèi)空氣質(zhì)量標(biāo)準(zhǔn)濃度的1/2、1、5、10倍。對小鼠進(jìn)行連續(xù)90天,每天2小時的呼吸暴露染毒,觀察亞慢性的VOCs復(fù)合暴露對小鼠的氧化應(yīng)激、免疫、呼吸、遺傳和血清生化毒性。結(jié)果發(fā)現(xiàn),復(fù)合VOCs的亞慢性暴露對小鼠肺產(chǎn)生了氧化應(yīng)激作用、導(dǎo)致了Thl和Th2細(xì)胞因子的紊亂,提高了BALF中嗜酸粒細(xì)胞趨化因子(Eotaxin)和神經(jīng)生長因子(NGF)水平以及炎癥細(xì)胞數(shù)量,激發(fā)了血清IgE抗體的產(chǎn)生,抑制了脾細(xì)胞CD8+T細(xì)胞亞群比例,對肝細(xì)胞產(chǎn)生了遺傳毒性,影響了血清和全血部分指標(biāo)。小鼠肺臟組織總抗氧化能力(T-AOC)、 GSH-Px與VOCs濃度之間存在劑量-效應(yīng)的負(fù)相關(guān)關(guān)系；小鼠脾細(xì)胞CD4+CD3+/CD8+CD3+的比率、BALF的白介素-4(IL-4)水平、彗星實驗的肝臟細(xì)胞DNA損傷的標(biāo)志物尾距(TM)以及DNA-蛋白質(zhì)交聯(lián)的標(biāo)志物TM與復(fù)合VOCs濃度之間存在劑量-效應(yīng)的正相關(guān)關(guān)系。結(jié)果提示：小鼠肺臟組織T-AOC.脾細(xì)胞CD4+CD3+/CD8+CD3+的比率、BALF的IL-4水平、彗星實驗肝臟細(xì)胞DNA損傷標(biāo)志物TM以及DNA-蛋白質(zhì)交聯(lián)標(biāo)志物TM可作為較低濃度復(fù)合VOCs亞慢性暴露的敏感效應(yīng)生物學(xué)標(biāo)志物;VOCs亞慢性暴露肺部炎癥的部分機制可能是由于ROS和來自于被激活的炎癥細(xì)胞釋放的炎癥因子來介導(dǎo)的。 (4)建立顆粒物+金黃色葡萄球菌+VOCs(甲醛+苯+甲苯+二甲苯)短期暴露復(fù)合染毒模型,設(shè)置對照組和6個染毒組,依次為顆粒物組、金黃色葡萄球菌組、顆粒物+金黃色葡萄球菌組、顆粒物+金黃色葡萄球菌+VOCs(10倍室內(nèi)空氣質(zhì)量標(biāo)準(zhǔn))組、顆粒物+金黃色葡萄球菌+VOCs(50倍室內(nèi)空氣質(zhì)量標(biāo)準(zhǔn))組、顆粒物+金黃色葡萄球菌+VOCs(100倍室內(nèi)空氣質(zhì)量標(biāo)準(zhǔn))組,對小鼠進(jìn)行連續(xù)10天,每天2小時的呼吸暴露染毒,觀察VOCs+顆粒物+金黃色葡萄球菌的復(fù)合暴露對小鼠的氧化應(yīng)激、炎癥因子及8羥基鳥嘌呤(8-OHdG)的影響。結(jié)果發(fā)現(xiàn),三種物質(zhì)的復(fù)合暴露影響了小鼠的正常生長發(fā)育、對小鼠肺產(chǎn)生了氧化性損傷；肺組織勻漿中MDA、T-AOC、NO以及GSH/GSSG比率在復(fù)合暴露下表現(xiàn)較敏感,其中T-AOC與復(fù)合VOCs濃度之間存在一定的劑量-效應(yīng)關(guān)系;三種物質(zhì)的復(fù)合暴露也對小鼠呼吸道產(chǎn)生了炎癥效應(yīng),且其毒性效應(yīng)大于單一物質(zhì)的毒性效應(yīng)；同時復(fù)合暴露也顯著影響了小鼠血清8-OHdG水平。結(jié)果提示：小鼠肺臟的T-AOC、BALF中IL-4和IL-8水平可作為顆粒物、金黃色葡萄球菌與VOCs三者復(fù)合暴露情況下的敏感效應(yīng)生物學(xué)標(biāo)志物。
[Abstract]:Due to the use of various types of composite construction and decoration materials and daily necessities of the organic, the volatile organic compounds (VOCs) as chemical pollution is more and more serious, and the indoor air pollution and health assessment, mainly by the concentration of indoor air pollutants, toxicity of health survey personnel living and single pollutant. The lack of comprehensive evaluation index can reflect the composite pollution and health risk. The project through the establishment of typical indoor air pollutants composite exposed mice exposed to the experimental animal model of immune toxicity method, respiratory air suction burglary of typical pollutants in mice, genetic toxicity and its molecular mechanism, provide a sensitive biomarker of effect for human health detection, but also for the revision and improvement of indoor air pollution standards provide a scientific basis. The main contents include:
(1) the establishment of short-term exposure to volatile organic compounds (formaldehyde composite model + Benzene + toluene + xylene), the control group and 4 different doses of compound VOCs exposure group, followed by 10,30,50100 times the standard of indoor air quality concentration. The mice were exposed for 10 days, 2 hours a day, breathing, oxidative stress. Study of VOCs on mice immune, respiration and serum biochemical toxicity. The results showed that VOCs exposure on mice liver and lung have caused oxidative damage, T cell immune function of splenic abnormalities, effect of bronchoalveolar lavage fluid (BALF) of interleukin -6 (IL-6), -3 (neurotrophic factor NT-3), the number of levels of substance P and inflammatory cells, and also has an influence on serum biochemical and blood routine. Liver, active oxygen in the lung (ROS), malondialdehyde (MDA) levels, spleen cell CD4+CD3+ (%), the ratio between CD4+CD3+/CD8+CD3+ and VOCs concentration. The positive correlation between dose effect, liver glutathione peroxidase (GSH-Px), glutathione (GSH) in lung tissue, spleen cells (CD8+CD3+%) negative correlation exists between dose effect ratio and VOCs concentration. The results showed that: the level of ROS in liver and lung of mice, liver tissue GSH-Px ratio lymphocyte subsets of spleen cells can be used as a composite VOCs short-term exposure of sensitive effect biomarkers; composite VOCs short-term exposure may be the main mechanism of airway inflammation induced by NO signal pathway and ROS, while NT-3 can mediate protection mechanism to inhibit the inflammation.
(2) to obtain the change of high-throughput biological information VOCs composite exposure lung tissue caused by the use of miRNA chip technology, analysis and comparison of the miRNA differential expression of specific biomarkers, the predicted target gene and the expression of miRNA, its biological functions and effects of path analysis (Pathway), at the same time with ELISA BALF detection of interleukin -8 (IL-8). The contents of experiment control group and 3 different doses of compound VOCs (formaldehyde + benzene toluene xylene + +) exposure group, followed by 30,50100 times the concentration of indoor air quality standards, the mice were exposed for 10 days, 2 hours a day of breathing. The results showed that compared with the control group, exposure group 1,2,3 respectively the expression of 96,68,18 miRNA was up-regulated, down regulated expression of 662592,11 in miRNA. The differential expression of miRNA in group 1,2,3 at the same time the upregulated expression of miRNA 6, were down regulated only There are 1; there are 1 miRNA in group 1,2 were up-regulated in group 3 decreased. In addition, IL-8 also increased significantly in the exposed group. The results suggested that mmu-miR-5105 could be preferred as mice lung VOCs sensitive miRNA molecular composite exposed markers; mmu-miR-494, mmu-miR-744, there was a negative correlation between mmu-miR-335-5p and VOCs concentration through the computer network; analysis and prediction of target genes of miRNA, VOCs composite exposure may cause cancer and potential airway inflammation, the results of ELISA IL-8 demonstrated that VOCs composite exposure induced airway inflammation.
(3) the establishment of long-term exposure to low concentration of indoor VOCs composite exposure model (formaldehyde + Benzene + toluene + xylene), the control group and 4 different doses of compound VOCs exposure group, followed by 1/2,1,5,10 times the standard of indoor air quality concentration. The mice were exposed for 90 days, 2 hours a day to breathe observe, subchronic VOCs compound on mice exposed to oxidative stress, immune, respiratory, genetic and biochemical toxicity. The results showed that the compound VOCs sub chronic exposure to increased oxidative stress in mice lungs, causing Thl and Th2 cytokines in disorder, increased BALF in eosinophil chemotactic factor (Eotaxin) and nerve growth factor (NGF) level and the number of inflammatory cells, stimulate the serum IgE antibodies, inhibited the proportion of spleen cells of CD8+T cell subsets, the genetic toxicity on liver cells, the impact of some blood and blood index in mice. The total antioxidant capacity of lung tissue (T-AOC), a negative correlation between dose effect between GSH-Px and VOCs concentration ratio; CD4+CD3+/CD8+CD3+ mouse spleen cells, BALF and interleukin -4 (IL-4) level, signs of damage liver cells DNA. The tail from the comet assay (TM) positive correlation exists between dose effect and mark DNA- protein crosslinking of TM and composite VOCs concentration. The results showed that: the ratio of lung tissue of T-AOC. mice spleen cells of CD4+CD3+/CD8+CD3+, BALF IL-4, DNA experimental liver cell comet injury markers TM and DNA- protein crosslinks marker TM can be used as a low concentration compound VOCs sub chronic exposure of sensitive biomarkers of effect; VOCs subchronic exposure mechanisms of lung inflammation may be due to ROS and from the release of inflammatory cytokines by activated inflammatory cells mediated.
(4) the establishment of particles of Staphylococcus aureus +VOCs (+ + + + formaldehyde benzene toluene xylene) exposed to short-term exposure composite model, the control group and 6 control group, followed by the particle group, Staphylococcus aureus, Staphylococcus aureus particles + group + particles of Staphylococcus aureus +VOCs (10 times the standard of indoor air quality) group, particles + Staphylococcus aureus +VOCs (50 times the standard of indoor air quality) group, particles + Staphylococcus aureus +VOCs (100 times the standard of indoor air quality) group, the mice were exposed for 10 days, 2 hours a day to breathe, observation VOCs+ particles + Staphylococcus aureus composite mice after exposure to oxidative stress, inflammatory factors and 8 hydroxy guanine (8-OHdG) effect. The results show that the combined exposure of three substances affect the normal growth of the development of the mouse, the oxidative damage of mice lung; Lung Group 緇囧寑嫻嗕腑MDA,T-AOC,NO浠ュ強GSH/GSSG姣旂巼鍦ㄥ鍚堟毚闇蹭笅琛ㄧ幇杈冩晱鎰，

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