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研究生:陳冠安
研究生(外文):Guann-An Chen
論文名稱:TRADD蛋白在TLR4訊息傳遞中扮演角色之研究
論文名稱(外文):Study of the Role of TRADD in TLR4 Signaling
指導教授:陳念榮
指導教授(外文):Nien-Jung Chen
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:微生物及免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:64
中文關鍵詞:類鐸受體腫瘤壞死因子
外文關鍵詞:TRADDTNFTLRdynasore
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類鐸受體(TLRs)在發炎反應中扮演了非常重要的角色,它能夠辨識外來抗原引起免疫反應,產生與發炎反應相關的細胞激素。在受到LPS刺激時,TLR4能同時活化MyD88主導(MyD88-dependent pathway)以及TRIF主導(TRIF-dependent pathway)的兩種訊息傳遞途徑,而活化發炎反應前期及晚期的基因表現。目前對於當TLR4被刺激活化後,這兩種訊息傳遞途徑之間的作用調控機制了解並不清楚。近年來的研究發現,越來越多的細胞內訊息傳遞途徑之間有cross-talk的現象,下游的訊息傳遞分子往往不只單純的在一個傳遞途徑扮演調控者的角色,而是能在不同的訊息傳導途徑中進行交互調控,進而組成一個更複雜的訊息傳遞網絡。已知在腫瘤壞死因子受體(TNFR)的訊息傳遞下游一個重要的中介體蛋白TRADD (TNF receptor-associated death domain)也參與了TLR訊息傳遞的調控。目前了解較清楚的是TRADD在TLR3-TRIF所引發的訊息中所扮演的角色。然而在同樣也能利用TRIF傳遞訊息的TLR4下游,TRADD的角色還沒有清楚的定論。在本研究中,我們藉由生物化學的角度證實了TRADD可以與TLR4下游的訊息傳遞分子(MyD88、Mal、受體的TIR domain以及RIP)產生交互作用,並且我們利用藥物Dynasore (一種endocytosis的抑制劑,可以阻斷TRIF主導的訊息)處理TRADD缺失的細胞可以觀察到在LPS刺激後能引發比野生型細胞更強的NF-�羠訊息以及細胞激素產生,間接證明了TRADD除了能正向調控TLR4下游TRIF主導的訊息傳遞途徑之外,TRADD也在MyD88所主導的訊息傳遞途徑當中也扮演了令人意外的負向調控角色。在此,我們提出了TRADD在參與TLR4訊息傳遞途徑時所扮演新的調控角色,我們也推測TRADD可能在TLR4訊息前、晚期訊號的切換上發揮功能。
Toll-like receptors (TLRs) play very important roles in inflammatory response. They recognize pathogen-associated molecular patterns (PAMPs) and induce cells to produce inflammatory cytokines, chemokines and interferons. Upon LPS stimulation, TLR4 activates both MyD88-dependent (early stage) and TRIF-dependent pathway (late stage), but the mechanism involved in regulation of these two pathways remains unclear. Recently, the key adaptor protein of TNFR1 signaling, TRADD (TNF receptor-associated death domain), had been reported involved not only in TNFR1 signaling but also in TLR3 signaling through the TRIF-dependent pathway. Whether TRADD plays roles in TLR4-mediated signaling remains an unsolved question. In this study, by using biochemical approach, we demonstrated the interaction between TRADD and the signaling components of TLR4 (such as MyD88, Mal, TIR domain of receptor and RIP). It was well demonstrated that TRADD plays a positive role in TRIF-dependent signaling. Here we focus on dissecting the role of TRADD in the early stage MyD88-dependent signals. With the treatment of Dynasore (an inhibitor of endocytosis which blocks TRIF-dependent signals), LPS induce an increased NF-�羠-mediated signal and enhanced cytokines production in TRADD deficient cells than in wild-type cells. Taken together, our results reveal a novel negative regulatory role of TRADD in Mal-MyD88 related signaling-mediated by TLR4 and it is presumed that TRADD may play role as a regulator in the interchange of TLR4 signaling.
中文摘要 1
英文摘要 2

前言 3
1.先天免疫反應與發炎反應 3
1.1 Toll-like receptor 3
1.2 TLR的訊息傳遞 5
2. 腫瘤壞死因子 5
2.1 腫瘤壞死因子超家族 6
2.2 腫瘤壞死因子受體(TNFR)超家族 6
2.3 TNF在發炎反應的訊息傳遞 7
2.4 TRADD的功能與角色 8
3. TRADD調控TLR的訊息傳遞路徑 9

實驗目的 10

材料 11
1. 試劑與溶液 11
2. 培養基與培養液 17

實驗方法 19
1. 細菌培養 19
2. DNA製備 19
3. 質體選殖 20
4. 細菌轉形 21
5. 細胞培養與轉染 21
6. 小鼠基因型鑑定 22
7. QRT-PCR 23
8. NF-�羠冷光酶報導基因試驗 24
9. 西方點墨法 24

實驗結果 27
I. TRADD與TLR4下游mediator之間的交互作用 27
II. TRADD與不同TIR domain之間的交互作用 28
II-1 TRADD全長與TIR domain之間的交互作用 28
II-2 TRADD N端與TIR domain之間的交互作用 29
III. 探討TRADD對TLR4下游mediator所主導的訊息傳遞之調控 29
III-1. 在過量表現的模式上觀察TRADD在MyD88途徑扮演的角色 29
III-2. TRADD對於受體TIR domain所主導的訊息傳遞之調控 30
IV. 探討TRADD在MyD88-dependent pathway的功能 32
IV-1. 分隔MyD88途徑以及TRIF途徑的訊息傳遞 32
IV-2. 去除TNF所造成的第二重效果(secondary effect) 33
IV-3. 在小鼠腹腔巨噬細胞上觀察TRADD在MyD88途徑扮演的角色 33
IV-4. 在小鼠肺部纖維母細胞觀察TRADD在MyD88途徑扮演的角色 34
IV-5. 在小鼠骨髓衍生巨噬細胞上觀察TRADD在MyD88途徑扮演的
角色 35
V. TRADD在TLR4訊息傳遞調控的機制 35

討論 36
TRADD與TLR4下游mediator之間的交互作用 37
TRADD與TIR domain之間的交互作用 38
TRADD在MyD88的訊息傳遞途徑扮演的角色 38
TRADD在TLR4訊息傳遞扮演的角色 41
結語 42

參考文獻 43

圖表 47

附錄 61
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