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研究生:徐偉展
研究生(外文):Wei-Chan Hsu
論文名稱:補體分子C5a受體交互作用之研究
論文名稱(外文):The interaction between C5a receptors
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:60
中文關鍵詞:補體
外文關鍵詞:complementC5a
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補體在先天性免疫系統中,對於抵禦細菌感染扮演了重要的角色。在細菌感染發生時,補體可以經由三種活化途徑,進而組裝出C5 轉化酵素,將C5切割成C5a與C5b : C5b會在細菌表面形成MAC (membrane attack complex)將細菌溶解;C5a則具有強化免疫反應的功能,能刺激免疫細胞的趨化反應,也能活化嗜中性白血球與巨噬細胞,增強發炎反應。C5a有兩種已知受體(C5aR與C5L2),C5aR是典型的G蛋白耦合受體,C5L2則屬於同類受體家族,但是缺少了重要的DRY訊息傳遞片段,原被推論為是沒有訊息傳遞功能的誘騙受體。但根據近年來在C5L2基因剔除小鼠上的研究指出,C5L2在C5a引發之發炎相關之免疫反應中扮演重要的調控角色。目前的研究指出C5L2應是ㄧ種具備功能性的受體,但是它是如何發揮作用,其完整機制尚待進一步的研究。已知許多G蛋白耦合受體能夠藉由形成多分子聚合受體的方式以調控其下游訊息傳遞。C5aR也曾被報導能自行形成雙分子聚合受體。在之前的研究曾發現,缺少C5L2的骨髓嗜中性白血球細胞膜上表現了較低量的C5aR。由此,我們推論C5L2可能藉由與C5aR形成複合體的方式,以調控C5aR的膜表現量及調控C5a所引發的訊息。本研究目的為探討C5L2能否與C5aR交互作用,進而產生多分子聚合受體結構。在本研究中,我們首先驗證了C5L2基因缺失的骨髓嗜中性白血球僅能表現低量的膜型C5aR。接著也利用穩定表現高量膜型C5aR的293T轉殖株細胞,驗證了提升C5L2的表現量確實能同步提升C5aR的膜表現量。我們更進一步利用免疫沉澱的方法,我們也驗證了在蛋白質層次上,C5aR確實能與C5L2形成受體蛋白複合體。在共軛焦螢光顯微鏡下,我們也能清楚觀察到C5aR與C5L2的表現位置高度重疊。另外,我們也以螢光共振能量轉移的方式進一步確認了C5aR與C5L2分子層次上的交互作用關係。總歸而言,本研究首次證明了C5L2與C5aR能夠交互作用,產生多分子聚合受體結構。
The complement system plays important roles in innate immune responses against infectious microbes. It is under cascade activation immediately upon infection. The complement component C5 was cleaved into C5a and C5b after activation. The small fragment C5a activates immune cells, such as neutrophils and macrophages, and enhances inflammatory responses. Two types of C5a receptors, C5aR and C5L2, have been reported. Both of them belong to the GPCR family. Under C5a stimulation, C5aR transduces signals through G-protein and activates immune cells. C5L2, lacking the G-protein coupling DRY domain, was originally thought as a non-functional decoy receptor. However, recent studies showed that C5L2 plays important roles in C5a-mediated immune responses. The mechanism about how C5L2 participates C5a signaling still remains unclear. Many GPCRs mediate their signals by forming multi-molecular receptor complex. We speculate that C5L2 and C5aR may mediate C5a-induced signals by forming multi-recptor complex. In this study, we first confirmed that C5aR expression on C5L2 deficient bone marrow neutrophil surface is dramaticly diminished. Surface C5aR level is altered by increasing C5L2 expression in C5aR-overexpressed stable 293T transfectants. C5L2-C5aR interaction was determined by biochemical co-immunopreciptation experiment. The colocalization of C5aR and C5L2 was then monitored by con-focal microscopy observation. The molecular interaction between C5aR and C5L2 was further confirmed by the fluorescence resonance energy transfer (FRET) analysis. Whether this interaction dedicates the C5a-mediated signaling still remains an interesting question and will be further determined comprehensively. Taken together, we reveal a novel mechanism that C5L2 could modulate C5a-mediated signaling through the molecular interaction with C5aR.
目錄
摘要 1
英文摘要 2
前言 3
1.先天性免疫系統 3
2.補體系統 4
3.C5a 受體: C5aR與C5L2 6
實驗目的 9
材料方法 11
材料 11
方法 17
實驗結果 23
1.嗜中性白血球細胞膜上之C5aR表現量會受到C5L2影響 23
1.1以流式細胞儀偵測嗜中性白血球膜上C5aR表現 23
1.2在C5aR穩定表現轉殖株中增加C5L2的表現,能夠增強細胞膜上C5aR表現 23
2. C5L2能與C5aR交互作用而形成蛋白複合體結構 25
2.1利用免疫沉澱法證明C5aR-C5L2蛋白分子彼此能交互作用 25
2.2利用共軛焦螢光顯微鏡證明C5aR與C5L2分子形成複合受體 27
2.3利用螢光共振能量轉移證明C5aR-C5L2形成複合體 28
3. C5L2影響C5a傳遞訊息 29
3.1將C5L2表現載體轉殖入C5aR穩定表現細胞株後,對於C5a訊息的影響 30
3.2在 C5L2基因剔除肺部纖維組織母細胞重新送入C5L2對於C5a傳遞訊息之幫助 30
3.3以攜帶C5L2YFP表現載體感染C5L2基因剔除嗜中性白血球 31
討論 32
1.C5aR細胞膜表現情形受到C5L2調控 32
2.C5L2能與C5aR交互作用形成複合受體 33
3.C5L2造成細胞凋亡之可能性 34
4.C5L2對於C5a下游訊息傳遞能力 35
5. C5L2與C5aR交互作用對於C5a訊息傳遞的影響 36
參考文獻 38
實驗圖表 42
附錄 59
參考文獻
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