自身免疫調(diào)節(jié)因子對(duì)巨噬細(xì)胞TLRs表達(dá)及其活化類型的影響
發(fā)布時(shí)間：2018-11-26 15:13
【摘要】：自身免疫調(diào)節(jié)因子(Autoimmune regulator, AIRE)是一種在調(diào)控胸腺中樞耐受中發(fā)揮著重要作用的轉(zhuǎn)錄激活因子。人類Aire基因的突變將導(dǎo)致由自身免疫應(yīng)答所介導(dǎo)的、以多器官損傷為特征的APECED。其典型臨床表現(xiàn)為甲狀旁腺功能減退,Addison's病和慢性皮膚粘膜念珠菌感染的三聯(lián)征。在胸腺中,AIRE主要表達(dá)在胸腺髓質(zhì)上皮細(xì)胞(Medullary thymic epithelial cell, mTEC),通過調(diào)控該細(xì)胞上大量組織限制性抗原(Tissue restricted antigens, TRA)的表達(dá),進(jìn)而在自身反應(yīng)性T細(xì)胞的陰性選擇中起重要作用。AIRE在外周淋巴器官和組織,以及CD14+DC、單核/巨噬細(xì)胞、B細(xì)胞和粒細(xì)胞中也有表達(dá)。和胸腺中相比,AIRE在外周中的作用,尤其在血源性細(xì)胞中表達(dá)的作用,現(xiàn)在還不是十分清楚。而且到目前為止,APECD病人對(duì)念珠菌顯著易感的原因同樣尚不清楚。 為了闡明AIRE在外周血源性細(xì)胞中表達(dá)的作用和意義,本研究在建立穩(wěn)定表達(dá)AIRE的小鼠巨噬細(xì)胞系RAW264.7細(xì)胞模型的基礎(chǔ)上,探討AIRE對(duì)巨噬細(xì)胞上TLRs表達(dá)的影響以及可能的調(diào)控機(jī)制,并檢測(cè)AIRE調(diào)控的TLRs在相應(yīng)配體刺激后下游靶基因的變化情況；此外,初步分析了AIRE對(duì)巨噬細(xì)胞活化類型的影響。 1建立AIRE穩(wěn)定表達(dá)的巨噬細(xì)胞RAW264.7模型 通過構(gòu)建含小鼠Aire基因的真核表達(dá)質(zhì)粒pEGFPC1/mAire,經(jīng)轉(zhuǎn)染、篩選和鑒定,得到了穩(wěn)定表達(dá)GFP-AIRE融合蛋白的RAW264.7細(xì)胞(A33-3)以及穩(wěn)定表達(dá)GFP蛋白的RAW264.7細(xì)胞(C1-6),為進(jìn)一步的實(shí)驗(yàn)奠定基礎(chǔ)。 2 AIRE對(duì)巨噬細(xì)胞上TLRs表達(dá)的影響及其調(diào)控機(jī)制 為了了解AIRE是否可以調(diào)控巨噬細(xì)胞上TLRs的表達(dá),我們通過熒光定量PCR和Western blotting檢測(cè)了A33-3與C1-6細(xì)胞之間TLRs表達(dá)差異。結(jié)果顯示,在A33-3細(xì)胞中AIRE可以調(diào)控TLR1、TLR3和TLR8的表達(dá)。通過染色質(zhì)免疫共沉淀(CHIP)和螢光素酶報(bào)告基因活性分析,進(jìn)一步發(fā)現(xiàn)AIRE通過與TLR1、TLR3和TLR8的啟動(dòng)子相互作用,進(jìn)而調(diào)控它們的表達(dá)。而且,通過TLR1和TLR3配基的刺激,我們發(fā)現(xiàn)AIRE能夠增加其相應(yīng)下游靶基因產(chǎn)物的表達(dá)。這些數(shù)據(jù)提示,在外周抗原提呈細(xì)胞(APC)中,AIRE可能通過調(diào)控TLRs的表達(dá),進(jìn)而對(duì)病原微生物的識(shí)別和外周免疫耐受的調(diào)控起著重要作用。 3 AIRE對(duì)巨噬細(xì)胞活化類型的影響 為了了解AIRE是否對(duì)巨噬細(xì)胞活化類型產(chǎn)生影響,我們分別用LPS、IL-4以及LPS聯(lián)合免疫復(fù)合物(OVA：抗OVA)刺激A33-3和C1-6細(xì)胞,然后分別檢測(cè)活化后的M1, M2a和M2b型巨噬細(xì)胞特異性細(xì)胞因子的表達(dá)變化情況。結(jié)果發(fā)現(xiàn),三種不同的刺激物可以使RAW264.7細(xì)胞分別向M1、M2a和M2b三種類型活化,AIRE可能對(duì)M1型的活化有促進(jìn)作用,而對(duì)M2型的活化有抑制作用,尤其是M2b型,這提示AIRE可能參與調(diào)控巨噬細(xì)胞對(duì)病原微生物的免疫防御作用。 本研究的意義在于,首次證實(shí)在巨噬細(xì)胞中AIRE通過與TLR1、TLR3和TLR8的啟動(dòng)子相互作用,能夠上調(diào)這些基因的表達(dá),提示AIRE可能在APC對(duì)病原體的識(shí)別和外周免疫耐受調(diào)節(jié)中發(fā)揮重要作用。初步證實(shí)在巨噬細(xì)胞活化過程中AIRE使其更趨向于M1型活化,有利于機(jī)體對(duì)病原體(包括真菌)的清除和提高其抗感染能力,這也為了APECED病人對(duì)真菌易感的原因提供了一個(gè)新的解釋。這些研究為我們進(jìn)一步了解AIRE在外周血源性細(xì)胞中表達(dá)的作用和意義提供新的實(shí)驗(yàn)依據(jù)。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392
本文編號(hào)：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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392
【共引文獻(xiàn)】
相關(guān)博士學(xué)位論文 前2條
1 石亮;自身免疫調(diào)節(jié)因子對(duì)大分子自噬及細(xì)胞吞噬功能的影響[D];華中科技大學(xué);2010年
2 吳靜;巨噬細(xì)胞內(nèi)DNA-PKcs協(xié)同Aire作用調(diào)節(jié)TLRs的表達(dá)[D];吉林大學(xué);2012年
相關(guān)碩士學(xué)位論文 前1條
1 和知非;自身免疫調(diào)節(jié)因子對(duì)RAW264.7細(xì)胞中CCL22表達(dá)影響的研究[D];吉林大學(xué);2010年
，本文編號(hào)：2358910
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