自身免疫调节因子对巨噬细胞TLRs表达及其活化类型的影响
发布时间：2018-11-26 15:13
【摘要】：自身免疫调节因子(Autoimmune regulator, AIRE)是一种在调控胸腺中枢耐受中发挥着重要作用的转录激活因子。人类Aire基因的突变将导致由自身免疫应答所介导的、以多器官损伤为特征的APECED。其典型临床表现为甲状旁腺功能减退,Addison's病和慢性皮肤粘膜念珠菌感染的三联征。在胸腺中,AIRE主要表达在胸腺髓质上皮细胞(Medullary thymic epithelial cell, mTEC),通过调控该细胞上大量组织限制性抗原(Tissue restricted antigens, TRA)的表达,进而在自身反应性T细胞的阴性选择中起重要作用。AIRE在外周淋巴器官和组织,以及CD14+DC、单核/巨噬细胞、B细胞和粒细胞中也有表达。和胸腺中相比,AIRE在外周中的作用,尤其在血源性细胞中表达的作用,现在还不是十分清楚。而且到目前为止,APECD病人对念珠菌显著易感的原因同样尚不清楚。 为了阐明AIRE在外周血源性细胞中表达的作用和意义,本研究在建立稳定表达AIRE的小鼠巨噬细胞系RAW264.7细胞模型的基础上,探讨AIRE对巨噬细胞上TLRs表达的影响以及可能的调控机制,并检测AIRE调控的TLRs在相应配体刺激后下游靶基因的变化情况；此外,初步分析了AIRE对巨噬细胞活化类型的影响。 1建立AIRE稳定表达的巨噬细胞RAW264.7模型 通过构建含小鼠Aire基因的真核表达质粒pEGFPC1/mAire,经转染、筛选和鉴定,得到了稳定表达GFP-AIRE融合蛋白的RAW264.7细胞(A33-3)以及稳定表达GFP蛋白的RAW264.7细胞(C1-6),为进一步的实验奠定基础。 2 AIRE对巨噬细胞上TLRs表达的影响及其调控机制 为了了解AIRE是否可以调控巨噬细胞上TLRs的表达,我们通过荧光定量PCR和Western blotting检测了A33-3与C1-6细胞之间TLRs表达差异。结果显示,在A33-3细胞中AIRE可以调控TLR1、TLR3和TLR8的表达。通过染色质免疫共沉淀(CHIP)和萤光素酶报告基因活性分析,进一步发现AIRE通过与TLR1、TLR3和TLR8的启动子相互作用,进而调控它们的表达。而且,通过TLR1和TLR3配基的刺激,我们发现AIRE能够增加其相应下游靶基因产物的表达。这些数据提示,在外周抗原提呈细胞(APC)中,AIRE可能通过调控TLRs的表达,进而对病原微生物的识别和外周免疫耐受的调控起着重要作用。 3 AIRE对巨噬细胞活化类型的影响 为了了解AIRE是否对巨噬细胞活化类型产生影响,我们分别用LPS、IL-4以及LPS联合免疫复合物(OVA：抗OVA)刺激A33-3和C1-6细胞,然后分别检测活化后的M1, M2a和M2b型巨噬细胞特异性细胞因子的表达变化情况。结果发现,三种不同的刺激物可以使RAW264.7细胞分别向M1、M2a和M2b三种类型活化,AIRE可能对M1型的活化有促进作用,而对M2型的活化有抑制作用,尤其是M2b型,这提示AIRE可能参与调控巨噬细胞对病原微生物的免疫防御作用。 本研究的意义在于,首次证实在巨噬细胞中AIRE通过与TLR1、TLR3和TLR8的启动子相互作用,能够上调这些基因的表达,提示AIRE可能在APC对病原体的识别和外周免疫耐受调节中发挥重要作用。初步证实在巨噬细胞活化过程中AIRE使其更趋向于M1型活化,有利于机体对病原体(包括真菌)的清除和提高其抗感染能力,这也为了APECED病人对真菌易感的原因提供了一个新的解释。这些研究为我们进一步了解AIRE在外周血源性细胞中表达的作用和意义提供新的实验依据。
[Abstract]:Autoimmune regulator (AIRE) is a kind of transcription activation factor which plays an important role in regulating the tolerance of thymic central nervous system. The mutation of the human Aire gene will result in an APECED mediated by its own immune response, characterized by multiple organ damage. The typical clinical manifestations are hypoparathyroidism, Addison's disease, and a triple sign of chronic cutaneous candidiasis. In the thymus, AIRE is mainly expressed in the thymus and medulla epithelial cell (mTEC), and plays an important role in the negative selection of self-reactive T cells by regulating the expression of a large number of tissue-restricted antigens (TRA) on the cell. AIRE is also expressed in peripheral lymphoid organs and tissues, as well as CD14 + DC, mononuclear/ macrophage, B cells and granulocytes. AIRE's role in the periphery, especially in blood-derived cells, is not very clear, as compared to the thymus. And so far, the reason why the APECD patients have a significant susceptibility to candidiasis is also unknown. In order to clarify the role and significance of the expression of AIRE in peripheral blood-derived cells, the effect of AIRE on the expression of TLRs on macrophages and the possible modulation are discussed on the basis of the establishment of a model of RAW264.7 cells in a mouse macrophage with stable expression of AIRE. The mechanism of control and the detection of the change of the target gene of TLRs regulated by the AIRE after the corresponding ligand were stimulated, and the activation type of the AIRE on the macrophage was preliminarily analyzed. The effect of AIRE on the establishment of an AIRE-stable expression of the macrophage RA W264.7 The RAW264.7 cells (A33-3) stably expressing the GFP-AIRE fusion protein and the RAW26 stably expressing the GFP protein were obtained by constructing a true nuclear expression plasmid pEGFPC1/ mAire containing the mouse Aire gene, transfecting, screening and identifying. 4.7 cells (C1-6), to lay the foundation for further experiments. The effect of the expression of upper TLRs and its regulatory mechanism in order to understand whether AIRE can regulate the expression of TLRs on macrophages, we detected A33 by fluorescence quantitative PCR and Western blotting. The difference in the expression of TLRs between-3 and C1-6 cells showed that AIRE in A33-3 cells could The expression of TLR1, TLR3 and TLR8 was regulated and the expression of TLR1, TLR3 and TLR8 was analyzed by chromatin immunoprecipitation (CHIP) and luciferase reporter gene activity. The promoter interacts with the promoter to regulate their expression. Furthermore, by the stimulation of the TLR1 and TLR3 ligands, we find that the AI The RE can increase the expression of its corresponding downstream target gene product. These data suggest that, in the peripheral antigen presenting cell (APC), the AIRE may regulate the expression of TLRs and, in turn, the pathogenic microorganism The identification of peripheral immune tolerance plays an important role in the regulation of peripheral immune tolerance Effect. 3 The effect of AIRE on the type of macrophage activation in order to understand whether the AIRE has an effect on the type of macrophage activation, we use LPS, IL-4, and LPS in combination with the immune complex (OVA: anti-OVA), respectively. stimulating A33-3 and C1-6 cells, and then respectively detecting the activated M1 The results showed that three different stimuli could activate RAW264.7 cells to M1, M2a and M2b, respectively. It can promote the activation of M2 type, especially the type of M2b, which suggests AIRE may be involved in regulating the immune defense of macrophages to pathogenic microorganisms. The significance of this study is that the expression of these genes can be up-regulated by interacting with the promoters of TLR1, TLR3 and TLR8 in macrophages for the first time. RE may play an important role in the identification of the pathogen and the regulation of peripheral immune tolerance in the APC. The force, which also provides a new explanation for the reason why the APECED patient is susceptible to the fungus, provides us with a further explanation.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392
本文编号：2358910
[Abstract]:Autoimmune regulator (AIRE) is a kind of transcription activation factor which plays an important role in regulating the tolerance of thymic central nervous system. The mutation of the human Aire gene will result in an APECED mediated by its own immune response, characterized by multiple organ damage. The typical clinical manifestations are hypoparathyroidism, Addison's disease, and a triple sign of chronic cutaneous candidiasis. In the thymus, AIRE is mainly expressed in the thymus and medulla epithelial cell (mTEC), and plays an important role in the negative selection of self-reactive T cells by regulating the expression of a large number of tissue-restricted antigens (TRA) on the cell. AIRE is also expressed in peripheral lymphoid organs and tissues, as well as CD14 + DC, mononuclear/ macrophage, B cells and granulocytes. AIRE's role in the periphery, especially in blood-derived cells, is not very clear, as compared to the thymus. And so far, the reason why the APECD patients have a significant susceptibility to candidiasis is also unknown. In order to clarify the role and significance of the expression of AIRE in peripheral blood-derived cells, the effect of AIRE on the expression of TLRs on macrophages and the possible modulation are discussed on the basis of the establishment of a model of RAW264.7 cells in a mouse macrophage with stable expression of AIRE. The mechanism of control and the detection of the change of the target gene of TLRs regulated by the AIRE after the corresponding ligand were stimulated, and the activation type of the AIRE on the macrophage was preliminarily analyzed. The effect of AIRE on the establishment of an AIRE-stable expression of the macrophage RA W264.7 The RAW264.7 cells (A33-3) stably expressing the GFP-AIRE fusion protein and the RAW26 stably expressing the GFP protein were obtained by constructing a true nuclear expression plasmid pEGFPC1/ mAire containing the mouse Aire gene, transfecting, screening and identifying. 4.7 cells (C1-6), to lay the foundation for further experiments. The effect of the expression of upper TLRs and its regulatory mechanism in order to understand whether AIRE can regulate the expression of TLRs on macrophages, we detected A33 by fluorescence quantitative PCR and Western blotting. The difference in the expression of TLRs between-3 and C1-6 cells showed that AIRE in A33-3 cells could The expression of TLR1, TLR3 and TLR8 was regulated and the expression of TLR1, TLR3 and TLR8 was analyzed by chromatin immunoprecipitation (CHIP) and luciferase reporter gene activity. The promoter interacts with the promoter to regulate their expression. Furthermore, by the stimulation of the TLR1 and TLR3 ligands, we find that the AI The RE can increase the expression of its corresponding downstream target gene product. These data suggest that, in the peripheral antigen presenting cell (APC), the AIRE may regulate the expression of TLRs and, in turn, the pathogenic microorganism The identification of peripheral immune tolerance plays an important role in the regulation of peripheral immune tolerance Effect. 3 The effect of AIRE on the type of macrophage activation in order to understand whether the AIRE has an effect on the type of macrophage activation, we use LPS, IL-4, and LPS in combination with the immune complex (OVA: anti-OVA), respectively. stimulating A33-3 and C1-6 cells, and then respectively detecting the activated M1 The results showed that three different stimuli could activate RAW264.7 cells to M1, M2a and M2b, respectively. It can promote the activation of M2 type, especially the type of M2b, which suggests AIRE may be involved in regulating the immune defense of macrophages to pathogenic microorganisms. The significance of this study is that the expression of these genes can be up-regulated by interacting with the promoters of TLR1, TLR3 and TLR8 in macrophages for the first time. RE may play an important role in the identification of the pathogen and the regulation of peripheral immune tolerance in the APC. The force, which also provides a new explanation for the reason why the APECED patient is susceptible to the fungus, provides us with a further explanation.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392
【共引文献】
相关博士学位论文 前2条
1 石亮;自身免疫调节因子对大分子自噬及细胞吞噬功能的影响[D];华中科技大学;2010年
2 吴静;巨噬细胞内DNA-PKcs协同Aire作用调节TLRs的表达[D];吉林大学;2012年
相关硕士学位论文 前1条
1 和知非;自身免疫调节因子对RAW264.7细胞中CCL22表达影响的研究[D];吉林大学;2010年
，本文编号：2358910
本文链接：https://www.wllwen.com/xiyixuelunwen/2358910.html
最近更新
教材专著
