【作者】 杨雪松；

【导师】 郝丽； 张立娜；

【作者基本信息】 东北林业大学 ， 生理学， 2020， 硕士

【摘要】 国际癌症研究机构和世界卫生组织将空气颗粒物界定为1级致癌物。石英粉尘同时又是中国最主要的职业危害因素之一,细颗粒物属于吸附性较强的物质,因此其容易变成空气中毒性物质的携带者,通过肺泡毛细血管进入人体的循环系统,从而影响人的身体健康。Th17和Treg作为初始CD4+T细胞的两个新亚型近年以来一直是研究热点之一。ROR γt是人和鼠Th17细胞中非常重要的转录因子,它是表达在免疫细胞上的RORγ的特异性亚型,能够有效促进Th17细胞分化,并可以对Th1细胞中特异性效应因子IL-17的表达和分泌进行调节。这种转录因子分布的组织器官较多,比较主要的包括胸腺、肌肉、肝脏等,另外,它能够参与淋巴结发育和免疫反应,与Th17细胞的形成具有密不可分的关联,并且很大程度上影响人体的自身免疫性、感染性疾病以及肿瘤的生成。本实验以小鼠为研究对象,设定低浓度颗粒物组、中浓度颗粒物组、高浓度颗粒物组,同时以金黄色葡萄球菌感染做对照,设定低浓度金葡菌组、中浓度金葡菌组、高浓度金葡菌组,生理盐水组。通过气管灌注颗粒物的方式感染小鼠,检测其肝脏、脾脏、肺脏组织的RORγ t mRNA表达量变化和蛋白表达量变化;同时采集小鼠肝脏、脾脏、肺脏组织为试验样本,通过HE染色技术,观察组织器官病理学变化;与金黄色葡萄球菌感染的小鼠相比较,推测颗粒物作为刺激源对机体免疫细胞的影响。主要研究结果如下:1.实验组小鼠体重与对照组相比有较明显变化,且颗粒物浓度越高,菌液浓度越高,小鼠体重增长缓慢越明显。其中颗粒物中浓度组和颗粒物高浓度组小鼠食量减少,毛发无光泽且变稀疏,精神沉郁;且颗粒物中浓度组和颗粒物高浓度组小鼠从灌肺第5天开始有咳嗽的症状。2.荧光定量PCR结果显示:在脾脏、肺脏中,ROR-γt因子的表达量与细颗粒物浓度有明显关系,颗粒物浓度越高,ROR-γt因子的表达量越高。其中肺脏作为直接受到刺激的器官,在浓度由10mg/ml提高到20mg/ml时ROR-γ t因子表达量显著增加(p<0.05)。而肝脏中ROR-γt因子的表达量的变化倍数明显没有其他两个器官高;当颗粒浓度为20mg/ml时,肝脏和肺脏中ROR-γt因子的表达量与金黄色葡萄球菌浓度为2*10^10cfu/ml时相近;颗粒浓度为40mg/ml时,脾脏中ROR-γt因子的表达量与金黄色葡萄球菌浓度为1*10^11 cfu/ml时相近。3.蛋白印迹结果显示,在肝脏和肺脏中,ROR-γt蛋白的表达量与细颗粒物浓度有明显关系,颗粒物浓度越高,ROR-γt蛋白的表达量越高;在脾脏中,在颗粒物浓度达到40mg/ml时,ROR-γ t蛋白的表达量才有明显增加(p<0.05)。当颗粒浓度为40mg/ml时,肺脏中ROR-γt蛋白的表达量与金黄色葡萄球菌浓度为2*10^10cfu/ml时相近;颗粒浓度为20mg/ml时,肝脏和脾脏中ROR-γt蛋白的表达量与金黄色葡萄球菌浓度为1*1 0^11cfu/ml时相近。4.切片结果显示,在细颗粒物的刺激下,肺脏受损最为严重,实验组小鼠均有不同程度的支气管扩张和肺泡受损,脾脏在受到刺激后也产生了大量免疫细胞而肝脏并无明显的变化。本实验通过进行外源性刺激,对不同实验组小鼠的细胞免疫因子ROR γt的表达以及小鼠部分器官组织的病理性变化进行分析,对大气细颗粒污染物造成机体的损伤,以及进一步的深入研究提供基础,为大气污染问题以及环境保护问题提供依据,为今后颗粒物污染的治疗提供了新思路和药物干扰的途径。更多还原

【Abstract】 The IARC and the WHO have classified air particles as grade 1 carcinogens.Quartz dust is also one of the major occupational hazards in China.As fine particles are highly adsorbent substances,they are easy to become carriers of toxic substances in the air and enter the circulatory system through alveolar capillaries,thus causing harm to multiple systems of the whole body.Two new subtypes of CD4+T cells,Th17 and Treg,have been the focus of recent research.RORyt,a specifical subtype of RORy expressed in immune cells,is the main transcription factor of human and mouse in Thl7 cells.It can not only promote the differentiation of Th17 cells,but also regulate the expression and secretion of IL-17,a specific effector of Th1 cells.RORγ is expressed in thymus,muscle,brain,heart,kidney,liver and lung,and is involved in lymph node development,immune response and production of Th17 cell.RORyt is closely related to the occurrence and development of various immune diseases,infectious diseases and tumors.In this experiment,mice were taken as the research objects,and the low-concentration particulate matter group,the medium-concentration particulate matter group,the high-concentration particulate matter group,the low-concentration staphylococcus aureus group,the medium-concentration staphylococcus aureus group,the high-concentration staphylococcus aureus group and the normal saline group were set.The changes in the mRNA expression level and protein expression level of ROR exposing t in liver,spleen and lung tissues of the mice were detected by injecting particulate matter into the trachea;At the same time,the liver,spleen and lung tissues of mice were collected as test samples,and the pathological changes of tissues and organs in each group were observed by HE staining technique;And the effect of particulate matter on immune cells of the body was speculated by comparing mice infected with staphylococcus aureus.The main findings are as follows:1.The weight of mice in the experimental group was significantly different from that in the control group,and the higher the concentration of particulate matter was,the higher the concentration of bacterial solution was,and the slower the growth of body weight was.Mice in the medium-concentration particulate matter group and the high-concentration particulate matter group ate less food,their hair was dull and sparse,and their spirits were depressed.Mice in these groups began to have cough symptoms on day 5 after lung irrigation.2.Real-time quantitative PCR results showed:In spleen and lung,the expression level of RORyt factor was significantly related to the concentration of fine particles,and the higher the concentration of particles was,the higher the expression level of RORyt factor was.Among them,the lung,as the organ directly stimulated,significantly increased the expression level of RORγt factor when the concentration increased from 10mg/ml to 20mg/ml(p<0.05).However.the expression of RORγt factor in the liver was not significantly higher than that in the other two organs.When the particle concentration was 20mg/ml.the expression level of RORγt factor in liver and lung was similar to the staphylococcus aureus group when the concentration was 2*10^1 0cfu/ml.At the particle concentration of 40mg/ml.the expression level of RORγt factor in the spleen was similar to that at the concentration of staphylococcus aureus of 1*10^11cfu/ml.3.Western blot results showed that there was a significant relationship between the expression of RORγt protein and the concentration of fine particles in liver and lung,and the higher the concentration of particles was.the higher the expression of RORγt protein was.In the spleen,the expression level of RORγt protein increased significantly when the particle concentration reached 40mg/ml(p<0.05).At the particle concentration of 40mg/ml.the expression of RORγt protein in the lungs was similar to the staphylococcus aureus group at the concentration of 2*10^0cfu/ml.At the particle concentration of 20mg/ml.the expression level of RORγt protein in the liver and spleen was similar to the staphylococcus aureus group at the concentration of 1*10^11 cfu/ml.4.Section results showed that lung damage was the most serious under the stimulation of fine particulate matter,and the experimental group of mice all had different degrees of bronchiectasis and alveolar damage.The spleen also produced a large number of immune cells after stimulation,while the liver showed no significant changes.In this experiment.the expression of RORyt.a cellular immune factor,and pathological changes in some organs and tissues of mice in different experimental groups were analyzed by exogenous stimulation.Provide the basis for further in-depth research for damaged to the body caused by fine particulate pollutants in the atmosphere,provide evidence for air pollution and environmental protection,provide a new way for the treatment of particulate matter pollution and a new way for drug interference.更多还原