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標題: | 一個新穎的鋅指蛋白於發炎反應中所扮演的角色 The functional role of a novel zinc finger protein in inflammation |
作者: | Yu-Ching Chen 陳俞靜 |
指導教授: | 徐立中(Li-Chung Hsu) |
關鍵字: | 發炎反應,鋅指蛋白, Toll like receptor,inflammation,zinc finger protein, |
出版年 : | 2012 |
學位: | 碩士 |
摘要: | 先天性免疫系統是宿主抵抗病原菌感染的第一道防線,先天性免疫反應可經由各種不同的 pattern-recognition receptors (PRRs) 所啟動。這些PRRs 包括Toll-like receptors (TLRs)、RIG-I-like receptors、 NOD-like receptors以及 C-type lectin receptors. PRRs 可透過辨識細菌、病毒和真菌表現的特殊分子結構 (稱之為pathogen-associated molecular patterns (PAMPs)) 來進一步活化先天免疫反應,例如:誘發發炎細胞激素、趨化素、第一型干擾素等。除了PAMPs之外,近年來,PRRs也被報導能夠偵測細胞受到傷害時所釋出之胞內物質 (統稱為damage-associated molecular patterns或DAMPs) 進而修復細胞。然而,過量的發炎細胞激素會造成過度的發炎反應,因而導致許多疾病的發生,例如:敗血症,癌症,甚至一些慢性發炎的疾病。因此,先天性免疫反應是需要被嚴密調控的。儘管在過去已有非常多關於Toll-like receptors 訊息傳遞的研究,但是我們對於Toll-like receptors 如何調控免疫反應的了解依舊是不完整的。我們實驗室之前發現一個包含鋅指(zinc finger)的蛋白質— ZFAND5 在巨噬細胞受到lipopolysaccharide (LPS)的刺激之下表現量會提升。ZFAND5 在N端的位置包含一個類似A20的zinc finger domain,以及在C端包含一個類似於AN1的zinc finger domain。ZFAND5先前曾被報導過與TLR訊息傳遞中的IKKγ、RIP、 TRAF6和polyubiquitin有交互作用,然而ZFAND5在TLR中所扮演的角色仍是不清楚的。在這篇研究中,我們希望找出ZFAND5在TLR中的功能以及如何調控TLR的分子機制。為了解決這個問題,我們首先降低ZFAND5在Raw 264.7類巨噬細胞株及骨髓衍生巨噬細胞中的表現量,接著觀察免疫反應是否因此受到影響。我們發現到當ZFAND5的表現量下降時,受到LPS刺激後巨噬細胞中的前發炎性細胞激素以及干擾素反應基因亦會受到抑制。除此之外,ZFAND5缺乏的細胞中,轉錄因子p65和IRF3在LPS刺激後,入核的量也會減少。我們還發現使用其他的TLR ligands包含Pam3CSK4和Poly I:C刺激,前發炎性細胞激素以及干擾素反應基因亦會在ZFAND5的表現量下降時受到抑制。我們更進一步的探討發現,ZFAND5 可能透過調控IKK複合體的活性以及加強TRAF3的K63-linked ubiquitination來分別調節在LPS刺激後MyD88和TRIF的傳遞路徑。 綜合以上的結論,我們的數據暗示著ZFAND5在TLR的訊息傳遞路徑中可能扮演正調控的角色,當然,我們還需要更多的研究來找出ZFAND5在TLR4訊息傳遞中的分子機制。 Innate immunity is the first line of host defense against pathogen infection. The innate immune response is initiated by various pattern recognition receptors (PRRs), which include Toll-like receptors, RIG-I-like receptors, NOD-like receptors, and C-type lectin receptors. PRRs can sense bacteria, viruses and fungi via so called pathogen-associated molecular patterns (PAMPs) and activate the innate immune response, including the induction of inflammatory cytokines, chemokines, and type I interferons. It has recently been shown that PRRs also recognize DAMPs (Damage-associated molecular patterns) released upon tissue injury and modulate inflammation and tissue repair. However, overexpression of inflammatory cytokines causes excessive inflammatory response, which contributes to many human diseases, such as sepsis, cancer, and chronic inflammatory diseases. Thus, it is important to tightly control the innate immune response. Extensive efforts have been made to elucidate TLRs signal pathways, but our understanding of regulation of TLR-mediated immune response remains fragmentary. We previously identified a zinc finger protein, ZFAND5, which is up-regulated in macrophage after LPS stimulation. ZFAND5 contains an A20-like zinc finger domain (ZnF-A20) at its N terminus and an AN1-like domain (ZnF-AN1) at its C terminus. It has been reported that ZFAND5 interacts with IKKγ, RIP, TRAF6 and polyubiquitin which all are involved in TLR signaling pathway. However, the role of ZFAND5 in TLR function is still unknown. Here, we aim to study the function of ZFAND5 in regulation of TLR signaling and its molecular mechanism. To address this question, we first knockdowned ZFAND5 in Raw264.7 macrophage-like cells and bone marrow derived macrophages (BMDMs) and then examined whether the immune response is affected. We found that the expression of proinflammatory cytokines and interferon-responsive genes were down-regulated in ZFAND5-silenced macrophages after LPS stimulation. The nuclear translocation of transcription factors, p65 and IRF3, were also decreased in LPS-induced ZFAND5-deficient macrophages. In addition, induction of cytokines expression was suppressed in ZFAND5 knockdown cells in response to other TLR ligands including Pam3CSK4 and Poly I:C. Furthermore, we found that ZFAND5 regulated the MyD88- and TRIF-dependent pathways in response to LPS possible through modulating the activation of IKK complex and enhancing K63-linked ubiquitination of TRAF3, respectively. Taken together, our data strongly suggest that ZFAND5 function as a positive regulator in the TLRs signaling pathway. More studies are required to elucidate the molecular mechanism of ZFAND5 in regulating TLR4 signaling. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/64369 |
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顯示於系所單位: | 分子醫學研究所 |
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