【摘要】：研究意義和目的： 支氣管哮喘（以下簡(jiǎn)稱哮喘）是一種由多種炎性細(xì)胞（如嗜酸性粒細(xì)胞、肥大細(xì)胞和T淋巴細(xì)胞等）浸潤(rùn)所致的反復(fù)發(fā)作的慢性氣道炎癥性疾病，臨床上以氣道高反應(yīng)為特征，突出表現(xiàn)為可逆性的氣道阻塞和發(fā)作性呼氣性呼吸困難。近年來(lái)它的發(fā)病率不斷上升，而成為嚴(yán)重影響人民健康的多發(fā)病。到現(xiàn)在為止，哮喘的發(fā)病機(jī)制仍未完全闡明，深入研究哮喘發(fā)病過(guò)程中的生理生化過(guò)程，對(duì)于我們深入認(rèn)識(shí)哮喘，尋找更有效的診斷與治療哮喘的手段是必不可少的。 抗原誘導(dǎo)的肺部炎癥（antigen induced pulmonary inflammation，AIPI）大鼠模型，是一種經(jīng)典的哮喘動(dòng)物模型，它能良好的模擬哮喘的多種發(fā)病特點(diǎn)，尤其是哮喘的一個(gè)典型特征——肺部炎癥。E3大鼠作為一種近交系大鼠，由于其對(duì)Th2型炎癥的易感性，近年來(lái)逐漸開(kāi)始用于哮喘模型的構(gòu)建。但是E3大鼠哮喘模型的疾病特征及表型尚沒(méi)有詳盡的研究。因此，我們第一步的研究目的就是構(gòu)建E3大鼠AIPI模型，觀察不同的抗原刺激時(shí)長(zhǎng)對(duì)大鼠哮喘模型各表型的影響。 表觀遺傳修飾是指通過(guò)特定的酶修飾基因組DNA或構(gòu)成染色質(zhì)的蛋白，改變?nèi)旧|(zhì)的結(jié)構(gòu)，調(diào)節(jié)靶基因的轉(zhuǎn)錄活性。表觀遺傳修飾參與包括細(xì)胞生長(zhǎng)發(fā)育、分化、信號(hào)轉(zhuǎn)導(dǎo)等多種生命過(guò)程。表觀遺傳修飾雖不涉及DNA序列的改變，但可被遺傳。蛋白質(zhì)精氨酸甲基轉(zhuǎn)移酶（PRMTs）可催化組蛋白等多種細(xì)胞蛋白發(fā)生精氨酸殘基甲基化，是一種近年來(lái)新發(fā)現(xiàn)的表觀遺傳修飾酶，在多種細(xì)胞進(jìn)程中發(fā)揮著重要的調(diào)節(jié)作用。我們的研究目標(biāo)是確定參與E3大鼠AIPI模型的重要PRMT，并探尋PRMT1在IL-4誘導(dǎo)的嗜酸性細(xì)胞浸潤(rùn)過(guò)程中的作用機(jī)制。 方法和結(jié)果： 1.使用卵清蛋白（OVA）免疫并分別攻擊E3大鼠1周、4周和8周，處死后分別觀察肺病理變化、肺部炎癥細(xì)胞浸潤(rùn)情況、血清中NO濃度、血清IgE和OVA特異性抗體及IL-4、TGF-β表達(dá)變化。 結(jié)果顯示無(wú)論是抗原刺激1周、4周還是8周，疾病組大鼠肺部均有顯著的嗜酸性細(xì)胞浸潤(rùn)�？乖碳�1周組主要表現(xiàn)為肺部的炎癥反應(yīng)，如血清中NO水平的升高、遲發(fā)型超敏反應(yīng)、肺IL-4表達(dá)水平的升高，炎癥細(xì)胞的浸潤(rùn)等。而抗原刺激8周組大鼠以氣道重塑為主要特征，如肺組織的破壞、膠原生成的增多、粘液分泌、抗體水平的升高、TGF-β的表達(dá)上升等。 2. E3大鼠腹腔注射OVA/鋁佐劑后鼻腔吸入OVA-PBS攻擊構(gòu)建AIPI模型；使用Real-time RT-PCR檢測(cè)PRMT1-6、eotaxin-1和CCR3等的表達(dá)變化；利用PRMTs的廣泛抑制劑AMI-1和小干擾RNA抑制A549細(xì)胞中PRMT1的功能；利用基因重組技術(shù)特異性的高表達(dá)A549肺上皮細(xì)胞中的PRMT1；對(duì)AIPI模型大鼠使用AMI-1進(jìn)行干預(yù)后觀察對(duì)哮喘炎癥表型的影響。 結(jié)果發(fā)現(xiàn)：PRMT1在AIPI大鼠的氣道和肺泡上皮細(xì)胞表達(dá)顯著升高；使用AMI-1抑制PRMTs的功能或敲低PRMT1的表達(dá)后可顯著抑制IL-4刺激肺上皮細(xì)胞導(dǎo)致的eotaxin-1和CCR3表達(dá)改變；A549細(xì)胞轉(zhuǎn)染重組質(zhì)粒后特異性高表達(dá)PRMT1且下游基因eotaxin-1和CCR3水平升高；AIPI大鼠給與AMI-1治療后能夠顯著降低肺部炎癥、下調(diào)IL-4的表達(dá)和體液免疫反應(yīng)，，更為顯著的是減輕肺部嗜酸性細(xì)胞的浸潤(rùn)。 結(jié)論： 1. E3大鼠AIPI模型的抗原刺激1周組具有急性炎癥的普遍特征，可作為研究急性肺部炎癥的良好工具；同時(shí)，抗原刺激8周組具有慢性哮喘的顯著特征，可用于慢性炎癥導(dǎo)致的肺重塑的研究。 2. PRMT1在AIPI大鼠肺上皮細(xì)胞中顯著上調(diào)，IL-4可刺激PRMT1表達(dá)水平的升高，干預(yù)PRMT1的活性可顯著降低IL-4依賴的eotaxin-1的產(chǎn)生，從而影響嗜酸性細(xì)胞參與的肺部炎癥過(guò)程。這些發(fā)現(xiàn)將為PRMT1在哮喘疾病中的作用機(jī)制研究提供實(shí)驗(yàn)基礎(chǔ)。
[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
【學(xué)位授予單位】：西安交通大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R562.25

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