蛋白质精氨酸甲基转移酶1在E3大鼠抗原诱导的肺部炎症中的作用机制研究
发布时间：2018-11-27 07:39
【摘要】：研究意义和目的： 支气管哮喘（以下简称哮喘）是一种由多种炎性细胞（如嗜酸性粒细胞、肥大细胞和T淋巴细胞等）浸润所致的反复发作的慢性气道炎症性疾病，临床上以气道高反应为特征，突出表现为可逆性的气道阻塞和发作性呼气性呼吸困难。近年来它的发病率不断上升，而成为严重影响人民健康的多发病。到现在为止，哮喘的发病机制仍未完全阐明，深入研究哮喘发病过程中的生理生化过程，对于我们深入认识哮喘，寻找更有效的诊断与治疗哮喘的手段是必不可少的。 抗原诱导的肺部炎症（antigen induced pulmonary inflammation，AIPI）大鼠模型，是一种经典的哮喘动物模型，它能良好的模拟哮喘的多种发病特点，尤其是哮喘的一个典型特征——肺部炎症。E3大鼠作为一种近交系大鼠，由于其对Th2型炎症的易感性，近年来逐渐开始用于哮喘模型的构建。但是E3大鼠哮喘模型的疾病特征及表型尚没有详尽的研究。因此，我们第一步的研究目的就是构建E3大鼠AIPI模型，观察不同的抗原刺激时长对大鼠哮喘模型各表型的影响。 表观遗传修饰是指通过特定的酶修饰基因组DNA或构成染色质的蛋白，改变染色质的结构，调节靶基因的转录活性。表观遗传修饰参与包括细胞生长发育、分化、信号转导等多种生命过程。表观遗传修饰虽不涉及DNA序列的改变，但可被遗传。蛋白质精氨酸甲基转移酶（PRMTs）可催化组蛋白等多种细胞蛋白发生精氨酸残基甲基化，是一种近年来新发现的表观遗传修饰酶，在多种细胞进程中发挥着重要的调节作用。我们的研究目标是确定参与E3大鼠AIPI模型的重要PRMT，并探寻PRMT1在IL-4诱导的嗜酸性细胞浸润过程中的作用机制。 方法和结果： 1.使用卵清蛋白（OVA）免疫并分别攻击E3大鼠1周、4周和8周，处死后分别观察肺病理变化、肺部炎症细胞浸润情况、血清中NO浓度、血清IgE和OVA特异性抗体及IL-4、TGF-β表达变化。 结果显示无论是抗原刺激1周、4周还是8周，疾病组大鼠肺部均有显著的嗜酸性细胞浸润。抗原刺激1周组主要表现为肺部的炎症反应，如血清中NO水平的升高、迟发型超敏反应、肺IL-4表达水平的升高，炎症细胞的浸润等。而抗原刺激8周组大鼠以气道重塑为主要特征，如肺组织的破坏、胶原生成的增多、粘液分泌、抗体水平的升高、TGF-β的表达上升等。 2. E3大鼠腹腔注射OVA/铝佐剂后鼻腔吸入OVA-PBS攻击构建AIPI模型；使用Real-time RT-PCR检测PRMT1-6、eotaxin-1和CCR3等的表达变化；利用PRMTs的广泛抑制剂AMI-1和小干扰RNA抑制A549细胞中PRMT1的功能；利用基因重组技术特异性的高表达A549肺上皮细胞中的PRMT1；对AIPI模型大鼠使用AMI-1进行干预后观察对哮喘炎症表型的影响。 结果发现：PRMT1在AIPI大鼠的气道和肺泡上皮细胞表达显著升高；使用AMI-1抑制PRMTs的功能或敲低PRMT1的表达后可显著抑制IL-4刺激肺上皮细胞导致的eotaxin-1和CCR3表达改变；A549细胞转染重组质粒后特异性高表达PRMT1且下游基因eotaxin-1和CCR3水平升高；AIPI大鼠给与AMI-1治疗后能够显著降低肺部炎症、下调IL-4的表达和体液免疫反应，，更为显著的是减轻肺部嗜酸性细胞的浸润。 结论： 1. E3大鼠AIPI模型的抗原刺激1周组具有急性炎症的普遍特征，可作为研究急性肺部炎症的良好工具；同时，抗原刺激8周组具有慢性哮喘的显著特征，可用于慢性炎症导致的肺重塑的研究。 2. PRMT1在AIPI大鼠肺上皮细胞中显著上调，IL-4可刺激PRMT1表达水平的升高，干预PRMT1的活性可显著降低IL-4依赖的eotaxin-1的产生，从而影响嗜酸性细胞参与的肺部炎症过程。这些发现将为PRMT1在哮喘疾病中的作用机制研究提供实验基础。
[Abstract]:The significance and purpose of the study: Bronchial asthma (hereinafter referred to as asthma) is a chronic airway inflammatory disease caused by infiltration of various inflammatory cells (such as eosinophils, mast cells and T lymphocytes, etc.), which is clinically indicated as a high airway response. It is characterized by reversible airway obstruction and exhale-expiratory respiratory distress. It is difficult. In recent years, its incidence has been rising, and it has become a multiple of serious impact on the health of the people By now, the pathogenesis of asthma is still not fully set out, the physiological and biochemical process in the course of asthma is studied, and the means of finding more effective diagnosis and treating asthma is essential for us to know the asthma deeply. The model of antigenously induced lung inflammation (AIPI) is a classic model of asthma. It can simulate many of the characteristics of asthma, especially one of the typical characteristics of asthma. Part of the inflammation. E3 rats, as a close-type rat, began to use for asthma models in recent years as a result of their susceptibility to Th2-type inflammation. However, the disease characteristics and phenotype of the model of asthma in the E3 rats were not detailed. Therefore, the first stage of our study was to construct an AIPI model of the E3 rat, to observe the different phenotypes of the model of the asthma model of the rats at different time of stimulation. The epigenetic modification refers to the modification of the structure of the chromatin, the regulation of the target gene, The apparent genetic modification is involved in the development, differentiation and signal transduction of the cells. The epigenetic modification does not involve a change in the DNA sequence, but can be inherited. The methylation of arginine residues in various cell proteins, such as histone, can be catalyzed by a protein arginine methyltransferase (PRMTs), which is a newly discovered epigenetic modification enzyme in recent years, which plays an important role in various cell processes. The objective of our study was to identify the important PRMT involved in the AIPI model of the E3 rat and to explore the process of the infiltration of PRMT1 in the IL-4-induced eosinophil infiltration. The mechanism of action. Methods: 1. The pathological changes of the lung, the infiltration of the inflammatory cells in the lungs, the concentration of NO in the serum, the serum IgE and OVA-specific antibodies and the IL-4 were observed after the immunizations of ovalbumin (OVA) and the attack of the E3 rats for 1 week, 4 weeks and 8 weeks, respectively. The expression of TGF-1 was changed. The results showed that both the lung of the disease group and the lung of the rats were either 1 week, 4 weeks or 8 weeks. There was a significant infiltration of eosinophils. The 1-week group showed an inflammatory response to the lungs, such as an increase in the level of NO in the serum, a delayed-type hypersensitivity reaction, and a level of IL-4 expression in the lung. In the group of 8-week-old rats, the airway remodeling was the main feature, such as the destruction of the lung tissue, the increase of collagen production, the secretion of mucus, and the level of the antibody. The expression of TGF-1 was increased, and the expression of OVA-PBS was induced by intraperitoneal injection of OVA/ Al adjuvant in rats. The expression of PRMT1-6, eotaxin-1 and CCR3 was detected by using Real-time RT-PCR. The function of PRMT1 in 49 cells; a high expression of PRMT1 in the lung epithelial cells of A549 lung using a gene recombination technique; and the use of AMI-1 in the AIPI model rats. The results showed that the expression of PRMT1 in the airway and alveolar epithelial cells of AIPI rats increased significantly; the expression of PRMTs or the expression of PRMT1 was inhibited by AMI-1, and the eot caused by IL-4 stimulation of the lung epithelial cells could be significantly inhibited. The expression of axin-1 and CCR3 was changed; after the A549 cells were transfected into the recombinant plasmid, the specific high expression of PRMT1 and the level of the downstream gene eotaxin-1 and CCR3 increased; the AIPI rats were able to significantly reduce the inflammation of the lung after the treatment with the AMI-1, down-regulate the expression of IL-4 and the humoral immune response, By the way, Conclusion: The first week group of AIPI model of the 1. E3 rat has the general characteristics of acute inflammation and can be used as a good tool for the study of acute lung inflammation. It can be used in the study of lung remodeling caused by chronic inflammation. PRMT1 is up-regulated in the lung epithelial cells of AIPI rats, and IL-4 can stimulate the increase of the expression level of PRMT1, and the activity of the intervention of PRMT1 can significantly reduce the eotaxin-1 dependence of IL-4. resulting in a process of pulmonary inflammation that affects the participation of eosinophils. these findings will be prm
【学位授予单位】：西安交通大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R562.25
本文编号：2359898
[Abstract]:The significance and purpose of the study: Bronchial asthma (hereinafter referred to as asthma) is a chronic airway inflammatory disease caused by infiltration of various inflammatory cells (such as eosinophils, mast cells and T lymphocytes, etc.), which is clinically indicated as a high airway response. It is characterized by reversible airway obstruction and exhale-expiratory respiratory distress. It is difficult. In recent years, its incidence has been rising, and it has become a multiple of serious impact on the health of the people By now, the pathogenesis of asthma is still not fully set out, the physiological and biochemical process in the course of asthma is studied, and the means of finding more effective diagnosis and treating asthma is essential for us to know the asthma deeply. The model of antigenously induced lung inflammation (AIPI) is a classic model of asthma. It can simulate many of the characteristics of asthma, especially one of the typical characteristics of asthma. Part of the inflammation. E3 rats, as a close-type rat, began to use for asthma models in recent years as a result of their susceptibility to Th2-type inflammation. However, the disease characteristics and phenotype of the model of asthma in the E3 rats were not detailed. Therefore, the first stage of our study was to construct an AIPI model of the E3 rat, to observe the different phenotypes of the model of the asthma model of the rats at different time of stimulation. The epigenetic modification refers to the modification of the structure of the chromatin, the regulation of the target gene, The apparent genetic modification is involved in the development, differentiation and signal transduction of the cells. The epigenetic modification does not involve a change in the DNA sequence, but can be inherited. The methylation of arginine residues in various cell proteins, such as histone, can be catalyzed by a protein arginine methyltransferase (PRMTs), which is a newly discovered epigenetic modification enzyme in recent years, which plays an important role in various cell processes. The objective of our study was to identify the important PRMT involved in the AIPI model of the E3 rat and to explore the process of the infiltration of PRMT1 in the IL-4-induced eosinophil infiltration. The mechanism of action. Methods: 1. The pathological changes of the lung, the infiltration of the inflammatory cells in the lungs, the concentration of NO in the serum, the serum IgE and OVA-specific antibodies and the IL-4 were observed after the immunizations of ovalbumin (OVA) and the attack of the E3 rats for 1 week, 4 weeks and 8 weeks, respectively. The expression of TGF-1 was changed. The results showed that both the lung of the disease group and the lung of the rats were either 1 week, 4 weeks or 8 weeks. There was a significant infiltration of eosinophils. The 1-week group showed an inflammatory response to the lungs, such as an increase in the level of NO in the serum, a delayed-type hypersensitivity reaction, and a level of IL-4 expression in the lung. In the group of 8-week-old rats, the airway remodeling was the main feature, such as the destruction of the lung tissue, the increase of collagen production, the secretion of mucus, and the level of the antibody. The expression of TGF-1 was increased, and the expression of OVA-PBS was induced by intraperitoneal injection of OVA/ Al adjuvant in rats. The expression of PRMT1-6, eotaxin-1 and CCR3 was detected by using Real-time RT-PCR. The function of PRMT1 in 49 cells; a high expression of PRMT1 in the lung epithelial cells of A549 lung using a gene recombination technique; and the use of AMI-1 in the AIPI model rats. The results showed that the expression of PRMT1 in the airway and alveolar epithelial cells of AIPI rats increased significantly; the expression of PRMTs or the expression of PRMT1 was inhibited by AMI-1, and the eot caused by IL-4 stimulation of the lung epithelial cells could be significantly inhibited. The expression of axin-1 and CCR3 was changed; after the A549 cells were transfected into the recombinant plasmid, the specific high expression of PRMT1 and the level of the downstream gene eotaxin-1 and CCR3 increased; the AIPI rats were able to significantly reduce the inflammation of the lung after the treatment with the AMI-1, down-regulate the expression of IL-4 and the humoral immune response, By the way, Conclusion: The first week group of AIPI model of the 1. E3 rat has the general characteristics of acute inflammation and can be used as a good tool for the study of acute lung inflammation. It can be used in the study of lung remodeling caused by chronic inflammation. PRMT1 is up-regulated in the lung epithelial cells of AIPI rats, and IL-4 can stimulate the increase of the expression level of PRMT1, and the activity of the intervention of PRMT1 can significantly reduce the eotaxin-1 dependence of IL-4. resulting in a process of pulmonary inflammation that affects the participation of eosinophils. these findings will be prm
【学位授予单位】：西安交通大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R562.25
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相关期刊论文 前2条
1 孙青竹;焦芳芳;杨旭东;钟波;蒋梅花;李国良;吕彬;韩燕;宁启兰;张富军;孙健;吕社民;;蛋白精氨酸甲基转移酶在E3大鼠哮喘模型中表达变化的研究[J];南方医科大学学报;2010年04期
2 欧阳若芸;胡成平;陈平;朱锦琪;黄信刚;;哮喘中Th_1/Th_2类细胞因子免疫失衡对神经生长因子表达的影响[J];中南大学学报(医学版);2007年01期
本文编号：2359898
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