生产性粉尘致巨噬细胞炎性反应及其与接尘工人健康损害的关系

发布时间：2018-06-23 19:27

本文选题：生产性粉尘 + 标化死亡比　；参考：《华中科技大学》2012年博士论文

【摘要】：在生产过程中,劳动者长期吸入石英含量10%的生产性粉尘可导致以慢性肺纤维化为主要病理改变的职业病——矽肺,其纤维化的病变具有不可逆性,且目前无法治愈,是我国最严重的职业病。因此研究石英粉尘导致矽肺纤维化的发生及发展,对于矽肺的早期诊断、治疗及预防具有十分重要的意义。越来越多的研究表明,石英粉尘引发的氧化活性及肺部炎性反应是导致机体损伤的关键因素。最近,体内外实验研究表明石英粉尘诱导产生高水平的IL-1p,但IL-1β作用模式及其导致的健康损害仍不清楚。为探讨不同粉尘引发炎性反应的程度和诱发炎性反应的机制及影响因素,我们收集了3个企业(江西的1个陶瓷厂和1个钨矿,广西的1个锡矿)作业点的生产性粉尘,以及不同粒径的标准石英粉尘,选取PMA诱导THP-1单核细胞株分化的巨噬细胞作为靶细胞,采用体外细胞实验的方法分析各厂矿粉尘毒性及致炎作用,结合接尘工人的健康状况,综合评价粉尘的致病能力,本研究主分为三个部分：(1)不同厂矿粉尘的化学组分及对接尘工人健康的影响；(2)粉尘致对巨噬细胞损伤和炎性反应及其接尘工人健康损害的关系；(3)IL-1β在石英粉尘导致巨噬细胞炎性反应中的作用。第一部分不同厂矿粉尘的化学组分及对接尘工人健康的影响目的：探讨粉尘的化学组分及其对接尘工人健康的影响。方法：生产性粉尘样本来自上述3个企业,采用焦磷酸质量法测定粉尘中游离二氧化硅的含量,采用原子吸收光谱法和原子荧光光谱法测定粉尘中化学元素的含量。本部分以上述3个企业1960年1月1日至1974年12月31日期间在册且工作一年以上的所有接尘工人为研究对象建立队列,追访至2003年底,以卫生部发布的全国城市居民年龄别平均死亡率(1973-2003年)为对照计算标化死亡比。结果：(1)瓷厂和锡矿粉尘中游离二氧化硅含量接近,分别为43.39%、43.60%；而钨矿粉尘中游离二氧化硅含量高于瓷厂和锡矿粉尘,达到57.51%。 (2)陶瓷厂中除铝元素的含量最高(10.64%)以外,其他元素的含量都很低；钨矿粉尘中铁元素的含量最高,达到3.47%,锌元素的含量最低,为0.09%；锡矿粉尘中钙、锌、砷元素的含量最高,分别为9.62%、0.33%、0.34%,铝元素的含量最低,为2.80%。 (3)陶瓷厂队列人数为907人,尘肺人数为148人,发病率为2.82‰。全死因标化死亡比(SMR)为1.15,尘肺、恶性肿瘤及肺癌标化死亡率比分别为85.26、0.58、0.77；钨矿厂队列人数为2350人,尘肺人数为788人,发病率为8.44‰。全死因标化死亡比(SMR)为1.24,尘肺、恶性肿瘤及肺癌标化死亡率比分别为101.80、0.67、0.73；锡矿队列人数为3108人,尘肺人数为466人,发病率为2.42%o。全死因标化死亡比(SMR)为1.07,尘肺、恶性肿瘤及肺癌标化死亡率比分别为48.67、1.44、2.43。粉尘中游离二氧化硅含量是影响尘肺发病的关键性因素。陶瓷厂粉尘中铝元素含量高于其它2类金属矿山,可能是造成陶瓷厂工人累积接矽尘量高而尘肺发病相对较低的主要原因。锡矿恶性肿瘤的升高与粉尘中砷的含量存在一定关联。第二部分粉尘致巨噬细胞损伤和炎性反应及其接尘工人健康损害的关系第一节石英诱导巨噬细胞产生IL-1β的模型筛选目的：探讨石英粉尘激活巨噬细胞产生IL-1p的作用条件、时间效应及剂量效应。方法：以PMA诱导THP-1单核细胞株分化的巨噬细胞作为靶细胞,选取三个粒径的标准石英DQ12(DQ12-PM1,1μm；DQ12-PM3-5,3～5μm；DQ12-PM5,5μm),配制成900,600,300,150,75,37.5μg/m6个浓度,分别染毒巨噬细胞3、6、12、18、24h,测定巨噬细胞释放IL-1p的水平。结果：(1)三种粒径的标准石英在3、6、12、18、24h这5个时间点均能诱导巨噬细胞释放IL-1p的水平升高,IL-1p的水平变化较明显且较早的时间点为6h,呈现明显的剂量反应关系。 (2)当粉尘浓度在75～300μg/ml之间时,三种粒径标准石英诱导巨噬细胞释放IL-1p的水平有明显的剂量反应关系,且粉尘浓度在300gμg/ml时,释放的IL-1p基本都达到了最高水平。在后续的实验中我们选择了37.5、75、150、300gg/ml作为染毒的浓度组。第二节节不同粒径石英粉尘致巨噬细胞损伤及炎性因子变化目的：探讨不同粒径石英粉尘对巨噬细胞的损伤及炎性反应作用,从而评价粒径在粉尘致炎性及纤维化反应途径中的作用。方法：以PMA诱导THP-1单核细胞株分化的巨噬细胞作为靶细胞,选取两种不同粒径的标准石英(DQ12-PM1,1μm；DQ12-PM3～5,3～5μm),以细胞空白为阴性对照,将粉尘配制成300,150,75,37.5μg/m14个浓度,与巨噬细胞共培养6h,测定细胞活力(MTT)、活性氧(ROS)的释放量及IL-1β、TNF-α、IL-6、IL-β1、IL-18、IL-33的水平。结果：不同粒径的石英粉尘均能损伤巨噬细胞的细胞活力,且随粉尘浓度的升高,巨噬细胞的细胞活力也逐渐下降,均能诱导巨噬细胞释放活性氧(ROS)及炎性细胞因子的表达。在较低浓度组(37.5、75、150μg/ml),粒径为PM1的标准石英诱导巨噬细胞释放IL-1p、TNF-α、IL-6、IL-33的水平略高于粒径为PM3-5的标准石英；在最高浓度组300μg/ml,粒径为PM3-5的标准石英粉尘的作用能力较强。第三节生产性粉尘致巨噬细胞的损伤及炎性因子变化及其与接尘工人健康损害的关联目的：分析生产性粉尘致巨噬细胞炎性因子的反应,评价炎性因子变化与接尘工人健康损害之间的可能关联。方法：上述企业作业点的呼吸性粉尘作为本次试验粉尘,以标准石英为阳性对照组,其他方法同第二部分第一节。结果：瓷厂、钨矿及锡矿粉尘均可导致巨噬细胞的细胞存活率下降、释放炎性细胞因子(IL-1β、TNF-α、IL-6、IL-18、IL-33)及抗炎细胞因子(TGF-β1)水平升高,并随粉尘浓度的升高有一定的剂量反应关系。瓷厂粉尘致巨噬细胞活力下降的程度最显著,锡矿粉尘次之,钨矿粉尘最弱；钨矿和锡矿粉尘诱导巨噬细胞产生前炎性细胞因子(IL-1β、TNF-α(?)(?)IL-6)的作用能力强于瓷厂,在低浓度组(37.5、75μg/ml),钨矿和锡矿粉尘诱导巨噬细胞产生IL-1β的能力显著强于标准石英,而在最高浓度组(300μg/ml),标准石英诱导产生IL-1β的能力显著强于瓷厂、钨矿及锡矿粉尘。钨矿和锡矿粉尘在各浓度组诱导巨噬细胞释放TNF-α的水平显著高于标准石英。但钨矿粉尘几乎未能诱导巨噬细胞产生TGF-β1,,锡矿粉尘几乎未能诱导巨噬细胞产生IL-18及IL-33。钨矿和锡矿粉尘作用于巨噬细胞后诱导产生前炎性细胞因子(IL-1β、TNF-α和IL-6)的能力都较强,而抗炎细胞因子(TGF-β1)的表达却很低；瓷厂粉尘诱导巨噬细胞释放前炎性细胞因子的能力最弱,但抗炎细胞因子的水平却相对较高,与接尘工人患矽肺危险度也是钨矿和锡矿的较高,瓷厂的最低相对应。本部分研究提示IL-1β对评价生产性粉尘致纤维化的有一定的预测作用。第三部分IL-1β在石英导致巨噬细胞炎性反应中的作用目的：探讨IL-1β在石英引发的炎性反应中的作用。方法：以PMA诱导THP-1单核细胞株分化的巨噬细胞作为靶细胞,将重组的IL-1β配制1600,800,400,200,100pg/m15个浓度,染毒巨噬细胞6h或24h；用50ng/ml的LPS预处理巨噬细胞6h,将标准石英粉尘配制成300,150,75,37.5μg/ml4个浓度,与LPS预处理的巨噬细胞共培养6h；用2.5μg/ml IL-1β单克隆抗体预处理细胞,按300,150,75,37.5μg/ml4个浓度的不同粒径的标准石英染毒巨噬细胞6h或24h,均测定细胞培养液上清中IL-1β、TNF-α、IL-6的水平。结果：(1)重组IL-1β作用于巨噬细胞6h和24h后,均可诱导其释放TNF-α、IL-6的水平升高,有明确的剂量反应关系；并随时间的延长,TNF-α、IL-6的水平也逐渐增高；但未能诱导其释放IL-1β,反而重组IL-1β本身的水平呈现下降的趋势,并随时间的延长其下降的趋势越明显。 (2)标准石英作用于LPS预处理的巨噬细胞6h后,能诱导其分泌IL-1β、TNF-α、IL-6的水平均随粉尘浓度的增加而逐渐上升,有明显的剂量反应关系,且三种炎性细胞因子的水平明显高于未用LPS预处理的巨噬细胞。 (3)IL-1β抗体能明显降低高浓度组(150、300μg/m1)标准石英诱导巨噬细胞产生IL-1β的水平,且IL-1β抗体阻断小粒径的标准石英(PM1)诱导释放的IL-1β作用更为显著；IL-1β抗体几乎完全阻断两种粒径的标准石英诱导巨噬细胞释放TNF-α、IL-6。
[Abstract]:In the process of production, the long-term inhalation of 10% of the productive dust of quartz can lead to the occupational disease of silicosis with chronic pulmonary fibrosis as the main pathological change, which is irreversible and can not be cured at present. It is the most serious occupational disease in our country. Development is of great importance for early diagnosis, treatment and prevention of silicosis.
More and more studies have shown that the oxidation activity induced by quartz dust and the inflammatory response of the lungs are the key factors that lead to the injury of the body. Recently, the experimental study in vitro and in vivo showed that the quartz dust induced the high level of IL-1p, but the mode of action of IL-1 beta and the health damage caused by it are still unclear.
In order to investigate the degree of inflammatory reaction caused by different dust and the mechanism and influencing factors of induced inflammatory reaction, we collected the productive dust of 3 enterprises (1 ceramic factories in Jiangxi and 1 tungsten mines, 1 tin mines in Guangxi), and standard quartz dust with different particle sizes, and selected PMA to induce macrophage fines in the differentiation of the mononuclear cell lines. As a target cell, cell test in vitro was used to analyze the toxic and inflammatory effects of dust dust in various factories, combined with the health status of the dust exposed workers and comprehensively evaluate the pathogenic ability of dust. This study was divided into three parts: (1) the chemical composition of dust in different factories and the health of dusts, and (2) dust caused to macrophage damage. The relationship between injury and inflammatory reaction and the health damage of workers exposed to dust; (3) the role of IL-1 beta in the inflammatory reaction of macrophages induced by quartz dust.
The first part is about the chemical composition of dust from different factories and the health of workers exposed to dust.
Objective: To explore the chemical composition of dust and its influence on the health of workers exposed to dust.
Methods: the sample of productive dust came from the above 3 enterprises. The content of free silica in dust was determined by pyrophosphoric acid mass spectrometry. The content of chemical elements in dust was determined by atomic absorption spectrometry and atomic fluorescence spectrometry. The 3 enterprises in this section were published and worked for one year from January 1, 1960 to December 31, 1974. All the dust workers above set up a cohort for the study, followed by the end of 2003. The average mortality rate (1973-2003 years) of the national average age of urban residents (1973-2003 years) issued by the Ministry of health was calculated as a comparison of the standardized mortality ratio.
Results: (1) the content of free silica in the dust of porcelain and tin ore is close to 43.39% and 43.60%, respectively, while the free silica content in tungsten dust is higher than that of the porcelain factory and the tin dust, reaching 57.51%..
(2) the content of other elements is very low in the ceramic factory except for the highest content of aluminum (10.64%), the content of iron in tungsten dust is the highest, the content of the zinc element is the lowest, and the lowest is 0.09%. The content of calcium, zinc and arsenic in tin dust is the highest, which is 9.62%, 0.33%, 0.34%, and the content of aluminum is the lowest, 2.80%.
(3) the number of workers in the ceramic factory was 907, the number of pneumoconiosis was 148, the incidence was 2.82 per thousand. The standardized death ratio (SMR) was 1.15, the mortality ratio of pneumoconiosis, malignant tumor and lung cancer was 85.26,0.58,0.77, the number of wolfram coal mines was 2350, the number of pneumoconiosis was 788, the incidence was 8.44 per thousand. The ratio of death to death was 1.24, and the ratio of death to the standardized death ratio (SMR) was 1.24, The mortality ratio of pneumoconiosis, malignant tumor and lung cancer was 101.80,0.67,0.73, the number of tin mines was 3108, and the number of pneumoconiosis was 466, the incidence of the incidence was 1.07 of the 2.42%o. total death ratio (SMR), and the mortality ratio of pneumoconiosis, malignant tumor and lung cancer was 48.67,1.44,2.43., respectively.
The content of free silica in dust is the key factor affecting the incidence of pneumoconiosis. The content of aluminum in the dust of the ceramic factory is higher than that of the other 2 types of metal mines, which may be the main cause of the high accumulation of silica dust in the ceramic factory workers and the relatively low incidence of pneumoconiosis.
The second part is the relationship between dust induced macrophage injury and inflammatory reaction and the health damage of workers exposed to dust.
Model selection of silica induced macrophage producing IL-1 beta
Objective: To explore the effects, time effects and dose effects of silica dust on macrophages producing IL-1p.
Methods: the macrophages differentiated from THP-1 mononuclear cells were used as target cells by PMA. The standard quartz DQ12 (DQ12-PM1,1 mu m, DQ12-PM3-5,3 ~ 5 m, DQ12-PM5,5 u m) was selected as 900600300150,75,37.5 mu g/m6 concentration, and macrophage 3,6,12,18,24h was poisoned, and the level of macrophage release was measured.
Results: (1) the standard quartz of three particle sizes could induce the increase of the level of macrophage releasing IL-1p at the 5 time points of 3,6,12,18,24h. The level of IL-1p was significantly changed and the earlier time point was 6h, showing a significant dose response relationship.
(2) when the dust concentration is 75~300 g/ml, there is a significant dose response relationship between three kinds of standard quartz induced macrophages to release IL-1p. And when the dust concentration is 300g mu g/ml, the release of IL-1p basically reaches the highest level. In the follow-up experiment, we selected 37.5,75150300gg/ml as the concentration group.
Macrophage injury and inflammatory factor changes induced by second different size quartz dust
Objective: To investigate the effect of different particle size quartz dust on the damage and inflammatory reaction of macrophages, and to evaluate the effect of particle size on the inflammatory and fibrotic reaction pathways of dust.
Methods: the macrophages differentiated by PMA THP-1 mononuclear cells were used as target cells, and two different particle sizes of standard quartz (DQ12-PM1,1 mu m; DQ12-PM3 to 5,3 ~ 5 m) were selected as negative control, and the dust was prepared into 300150,75,37.5 u g/m14 concentration and co cultured with macrophage cells for 6h, and the activity oxygen (MTT) and reactive oxygen species were measured. ROS release and IL-1 beta, TNF- alpha, IL-6, IL- beta 1, IL-18, IL-33 levels.
Results: the quartz dust with different particle sizes could damage the cell viability of macrophages, and the cell viability of macrophages decreased with the increase of dust concentration, which could induce the release of reactive oxygen species (ROS) and the expression of inflammatory cytokines in macrophages. In a lower concentration group (37.5,75150 mu g/ml), a standard quartz with a particle size of PM1 was induced to induce macrophages. The level of IL-1p, TNF- alpha, IL-6 and IL-33 was slightly higher than the standard quartz with the particle size of PM3-5, and the standard quartz dust with a particle size of PM3-5 was stronger than the standard quartz with the highest concentration of PM3-5 in the highest concentration group.
Third sections of dust induced macrophage injury and inflammatory factors and their relationship with health damage of exposed workers
Objective: to analyze the reaction of inflammatory cytokines in macrophages induced by productive dust, and to evaluate the possible correlation between the changes of inflammatory factors and the health damage of workers exposed to dust.
Methods: respirable dust at the above operation points was taken as the dust in this experiment. The standard quartz was used as the positive control group, and the other methods were the first part of the second part.
Results: porcelain factory, tungsten ore and tin dust can lead to the decrease of cell survival rate of macrophage, the release of inflammatory cytokines (IL-1 beta, TNF- a, IL-6, IL-18, IL-33) and anti-inflammatory cytokine (TGF- beta 1) level, and the increase of the concentration of dust has a certain anti response relationship. The degree of the decrease of macrophage vitality caused by the dust of porcelain factory is the most obvious At the same time, tungsten dust is the weakest, tungsten ore dust is the weakest; IL-1 beta, TNF- alpha (?) IL-6) induced by tungsten and tin dust is stronger than porcelain factory. In low concentration group (37.5,75 mu g/ml), the ability of tungsten ore and tin dust to induce the production of IL-1 beta in megagocytic cells is stronger than that of standard quartz, but at the highest concentration Group (300 g/ml), the ability of standard quartz to induce the production of IL-1 beta was significantly stronger than that of porcelain factory, tungsten ore and tin dust. The level of TNF- alpha released by macrophages in each concentration group was significantly higher than that of standard quartz. But tungsten dust could hardly induce macrophage to produce TGF- beta 1, and tin dust almost failed to induce macrophage. Producing IL-18 and IL-33.
The ability of tungsten ore and tin dust to induce proinflammatory cytokines (IL-1, TNF- A and IL-6) after the action of macrophages is strong, but the expression of anti-inflammatory cytokine (TGF- beta 1) is very low, and the ability of porcelain factory dust to induce macrophage to release inflammatory cytokines is the weakest, but the level of anti-inflammatory cytokines is relatively high, and the dust is connected to the dust. The risk of silicosis in workers is also higher than that of tungsten ores and tin mines, and the lowest in porcelain factories. This part of this study suggests that IL-1 beta has a certain predictive effect on evaluating the fibrosis of productive dust.
The third part is the role of IL-1 beta in the inflammatory response of macrophages induced by quartz.
Objective: To investigate the role of IL-1 beta in the inflammatory reaction induced by quartz.
Methods: the macrophages differentiated from THP-1 mononuclear cells were used as target cells by PMA, and the recombinant IL-1 beta was prepared for 1600800400200100pg/m15 concentration, 6h or 24h of macrophages, and 6h of macrophages was pretreated with 50ng/ml LPS, and the standard quartz dust was prepared into 300150,75,37.5 um g/ml4 concentration, and the macrophage pretreated with LPS. 6h was co cultured. The cells were pretreated with 2.5 g/ml IL-1 beta monoclonal antibody, and 6h or 24h of macrophages were infected by standard quartz with different particle sizes of 300150,75,37.5 mu g/ml4. The level of IL-1 beta, TNF- alpha and IL-6 in the supernatant of cell culture liquid was measured.
Results: (1) the effect of recombinant IL-1 beta on macrophage 6h and 24h could induce the release of TNF- alpha, the level of IL-6 increased, and there was a definite dose response relationship; and the level of TNF- alpha and IL-6 increased gradually with the prolongation of time, but failed to induce the release of IL-1 beta, instead, the level of IL-1 beta itself showed a downward trend, and with time. The more obvious the trend of prolonging its decline.
(2) standard quartz can induce the secretion of IL-1 beta, TNF- alpha and IL-6 after the effect of LPS pretreated macrophage 6h. The level of the standard quartz increases with the increase of dust concentration, and there is a significant dose response relationship, and the level of the three inflammatory cytokines is significantly higher than that of the macrophages without the LPS pretreated.
(3) IL-1 beta antibody could significantly reduce the level of IL-1 beta in the standard quartz induced macrophages in the high concentration group (150300 g/m1), and the IL-1 beta antibody blocked the IL-1 beta induced by the standard quartz (PM1) induced by the small size of the quartz (PM1), and the IL-1 beta antibody almost completely blocked the release of TNF- a by two kinds of standard quartz induced macrophages. IL-6.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R131

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