典型室内空气复合污染物对多靶器官毒性评价研究

发布时间：2018-03-19 05:22

  本文选题：室内空气　切入点：挥发性有机物　出处：《大连理工大学》2013年博士论文　论文类型：学位论文

【摘要】：由于多种复合型建筑和装饰材料以及日常有机生活用品的使用,使得室内挥发性有机物(VOCs)为主的化学性污染越来越严重,而目前针对室内空气污染的健康评价,主要采用室内空气污染物的浓度、居住人员的健康问卷调查及单一污染物的毒性研究,缺乏能够反映复合污染健康风险的综合性评价指标。本项目通过建立典型室内空气污染物的复合暴露染毒小鼠的实验动物模型,研究吸入室内空气典型污染物致小鼠的呼吸系统毒性、免疫毒性、遗传毒性及其分子机理,为人群健康检测提供灵敏的效应生物标志物,同时也为修订和完善室内空气污染相关标准提供科学依据。主要内容包括： (1)建立室内挥发性有机物短期复合染毒模型(甲醛+苯+甲苯+二甲苯),设置对照组和4个不同剂量复合VOCs暴露组,依次是室内空气质量标准浓度的10、30、50、100倍。对小鼠进行连续10天,每天2小时的呼吸暴露染毒,研究VOCs对小鼠的氧化应激、免疫、呼吸和血清生化毒性的影响。结果发现,VOCs暴露对小鼠的肺和肝脏产生了氧化损伤、引起了脾的T细胞免疫功能异常、影响了肺灌洗液(BALF)中白细胞介素-6(IL-6)、神经营养因子-3(NT-3)、P物质水平和炎症细胞数量,并且也对血清生化和血常规方面产生了影响。小鼠肝、肺脏中活性氧(ROS)、丙二醛(MDA)水平、脾细胞CD4+CD3+(%)、CD4+CD3+/CD8+CD3+的比率与VOCs浓度之间存在剂量-效应的正相关关系,肝脏组织的谷胱甘肽过氧化物酶(GSH-Px)、肺脏组织的谷胱甘肽(GSH)、脾细胞CD8+CD3+(%)比率与VOCs浓度之间存在剂量-效应的负相关关系。结果提示：小鼠肝和肺脏中ROS水平、肝脏组织的GSH-Px、脾细胞的淋巴细胞亚群比率可作为复合VOCs短期暴露的敏感效应生物学标志物；复合VOCs短期暴露气道炎症机制主要可能是通过NO信号路径和ROS来诱导的,同时NT-3可以介导保护机制来抑制这种炎症。 (2)利用miRNA芯片技术来获得VOCs复合暴露引起的肺组织的高通量生物信息变化,分析比较了miRNA差异表达,探讨特异性生物标志物,并对差异表达的miRNA预测靶基因,进行其生物学功能和影响路径(Pathway)分析,同时用ELISA来检测BALF中白细胞介素-8(IL-8)的含量。实验设置对照组和3个不同剂量的复合VOCs(甲醛+苯+甲苯+二甲苯)暴露组,依次是室内空气质量标准浓度的30、50、100倍,对小鼠进行连续10天,每天2小时的呼吸暴露染毒。结果发现,与对照组比较,染毒组1,2,3分别有96、68、18个miRNA上调表达,有662、592、11个miRNA下调表达。在表达差异的miRNA中,染毒组1,2,3同时上调表达的miRNA有6个,均下调表达的只有1个；有1个miRNA在染毒组1,2上调表达,在染毒组3下调表达。另外,IL-8在暴露组也显著升高。结果提示：可以优先考虑mmu-miR-5105作为小鼠肺脏VOCs复合暴露的敏感miRNA分子标志物；mmu-miR-494、mmu-miR-744、mmu-miR-335-5p和VOCs浓度间存在负相关关系；通过计算机网络分析预测miRNA靶基因发现,VOCs复合暴露可能潜在导致癌症和气道炎症的发生,IL-8的ELISA检测结果证实了VOCs复合暴露引起了气道炎症。 (3)建立长期暴露、较低浓度室内VOCs复合染毒模型(甲醛+苯+甲苯+二甲苯),设置对照组和4个不同剂量复合VOCs暴露组,依次是室内空气质量标准浓度的1/2、1、5、10倍。对小鼠进行连续90天,每天2小时的呼吸暴露染毒,观察亚慢性的VOCs复合暴露对小鼠的氧化应激、免疫、呼吸、遗传和血清生化毒性。结果发现,复合VOCs的亚慢性暴露对小鼠肺产生了氧化应激作用、导致了Thl和Th2细胞因子的紊乱,提高了BALF中嗜酸粒细胞趋化因子(Eotaxin)和神经生长因子(NGF)水平以及炎症细胞数量,激发了血清IgE抗体的产生,抑制了脾细胞CD8+T细胞亚群比例,对肝细胞产生了遗传毒性,影响了血清和全血部分指标。小鼠肺脏组织总抗氧化能力(T-AOC)、 GSH-Px与VOCs浓度之间存在剂量-效应的负相关关系；小鼠脾细胞CD4+CD3+/CD8+CD3+的比率、BALF的白介素-4(IL-4)水平、彗星实验的肝脏细胞DNA损伤的标志物尾距(TM)以及DNA-蛋白质交联的标志物TM与复合VOCs浓度之间存在剂量-效应的正相关关系。结果提示：小鼠肺脏组织T-AOC.脾细胞CD4+CD3+/CD8+CD3+的比率、BALF的IL-4水平、彗星实验肝脏细胞DNA损伤标志物TM以及DNA-蛋白质交联标志物TM可作为较低浓度复合VOCs亚慢性暴露的敏感效应生物学标志物;VOCs亚慢性暴露肺部炎症的部分机制可能是由于ROS和来自于被激活的炎症细胞释放的炎症因子来介导的。 (4)建立颗粒物+金黄色葡萄球菌+VOCs(甲醛+苯+甲苯+二甲苯)短期暴露复合染毒模型,设置对照组和6个染毒组,依次为颗粒物组、金黄色葡萄球菌组、颗粒物+金黄色葡萄球菌组、颗粒物+金黄色葡萄球菌+VOCs(10倍室内空气质量标准)组、颗粒物+金黄色葡萄球菌+VOCs(50倍室内空气质量标准)组、颗粒物+金黄色葡萄球菌+VOCs(100倍室内空气质量标准)组,对小鼠进行连续10天,每天2小时的呼吸暴露染毒,观察VOCs+颗粒物+金黄色葡萄球菌的复合暴露对小鼠的氧化应激、炎症因子及8羟基鸟嘌呤(8-OHdG)的影响。结果发现,三种物质的复合暴露影响了小鼠的正常生长发育、对小鼠肺产生了氧化性损伤；肺组织匀浆中MDA、T-AOC、NO以及GSH/GSSG比率在复合暴露下表现较敏感,其中T-AOC与复合VOCs浓度之间存在一定的剂量-效应关系;三种物质的复合暴露也对小鼠呼吸道产生了炎症效应,且其毒性效应大于单一物质的毒性效应；同时复合暴露也显著影响了小鼠血清8-OHdG水平。结果提示：小鼠肺脏的T-AOC、BALF中IL-4和IL-8水平可作为颗粒物、金黄色葡萄球菌与VOCs三者复合暴露情况下的敏感效应生物学标志物。
[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 缁囧寑娴嗕腑MDA,T-AOC,NO浠ュ強GSH/GSSG姣旂巼鍦ㄥ�嶅悎鏆撮湶涓嬭〃鐜拌緝鏁忔劊�

本文编号：1633004