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系統識別號 U0026-0812200910194191
論文名稱(中文) 登革病毒感染產生自體抗體所引發的病理效應
論文名稱(英文) Pathological effects of autoantibodies produced during dengue virus infection
校院名稱 成功大學
系所名稱(中) 基礎醫學研究所
系所名稱(英) Institute of Basic Medical Sciences
學年度 90
學期 2
出版年 91
研究生(中文) 林秋烽
研究生(英文) Chiou-Feng Lin
電子信箱 chioufenglin@yahoo.com.tw
學號 s5887108
學位類別 博士
語文別 中文
論文頁數 147頁
口試委員 口試委員-劉校生
指導教授-林以行
口試委員-黎煥耀
召集委員-葉才明
口試委員-陳舜華
口試委員-林宜玲
口試委員-廖經倫
口試委員-黃奇英
中文關鍵字 抗登革病毒非結構性蛋白1抗體  登革出血熱  登革病毒  自體免疫 
英文關鍵字 dengue hemorrhagic fever  dengue virus  autoimmunity  anti-DV NS1 antibody 
學科別分類
中文摘要 登革病毒 (dengue virus; DV) 的感染是以埃及斑蚊所媒介的病毒性傳染病,對於人類健康有嚴重的威脅性。臨床上,感染DV的病患會出現輕微的登革熱 (dengue fever; DF) 症狀甚或演變至嚴重的登革出血熱和登革休克症候群 (dengue hemorrhagic fever and dengue shock syndrome; DHF/DSS)。目前所知,嚴重性血小板減少症、血管病變及出血性症狀是DHF/DSS的主要病徵,即使有許多致病假說來解釋病理的發生,然而DV感染最後導致DHF/DSS的真正機制仍未清楚。一般認為除了來自病毒的直接參與外,宿主因子包括免疫細胞、補體的活化以及細胞激素的異常表現亦可能參與DHF/DSS的病程發展。在本論文裡,我們探討宿主對抗DV感染產生的免疫反應是否可能參與登革病症的發生。實驗結果顯示在登革病患血清中測得抗血小板及抗內皮細胞的自體抗體存在,並且,在DHF/DSS病患血清中自體抗體的含量比典型DF的病患較高,而這樣的現象在測試其它病毒如日本腦炎、C型肝炎或腸病毒71型感染的病患或正常人血清中並沒有發現。我們認為DV感染除了會直接傷害宿主細胞外,病毒引發宿主產生自體免疫反應也可能參與登革病症的發生。我們進一步研究登革病患血清中自體抗體的特性,且對此不正當的自體免疫反應其所可能引起之病理效應做一探討。利用登革第二型病毒非結構性蛋白1 (NS1) 之cDNA,將之構築在E. coli的表現系統進而表達重組DV NS1蛋白。實驗證明登革病患血清中抗體與細胞的結合作用會被經由DV NS1的競爭結合處理所抑制,而這樣的結果顯示了抗DV NS1抗體 (anti-DV NS1) 至少可能是血清中與血小板或內皮細胞發生結合作用的抗體之一。為了直接證明anti-DV NS1會產生自體免疫反應,我們以DV NS1蛋白免疫小鼠進而純化得到anti-DV NS1抗體,實驗證明這些抗體可以與血小板及內皮細胞發生結合作用。有趣的是,登革病患血清抗體或小鼠的anti-DV NS1抗體在補體的存在下會引起血小板及內皮細胞的溶裂作用,此外,anti-DV NS1抗體亦會導致內皮細胞經由caspase的活化而進行細胞凋亡。針對anti-DV NS1抗體刺激內皮細胞所造成訊息傳遞之研究,結果顯示抗體可引起內皮細胞內蛋白質的磷酸化現象以及NF-kB蛋白的活化作用。進一步的實驗發現anti-DV NS1抗體會誘發iNOS與NO的表現量增加,且抗體所引發之內皮細胞凋亡則會被NOS抑制劑L-NAME所抑制。根究其作用機制顯示anti-DV NS1抗體刺激內皮細胞會使得Bcl-2和Bcl-xL mRNA及蛋白質表現下降,Bax及p53表現則增高,而這樣的情形均可被L-NAME所抑制。綜而言之,anti-DV NS1抗體會引發內皮細胞內訊息傳遞作用並誘導細胞走向死亡,這過程與機轉可能是由於一連串活化細胞之訊息傳遞途徑促進NO的產生而提高了Bax和p53並降低Bcl-2及Bcl-xL表現所導致的結果。根據本論文的研究結果,我們認為血小板及內皮細胞自體抗體的產生會造成細胞失去正常機能,這樣的效應可能在DV感染所引起的致病機制上扮演了一個重要的角色。本研究的發現可以做為研發登革疫苗時的重要考量,可考慮的方向包括以基因修飾的方法將DV NS1蛋白具有引發自體免疫病理效應的片段或抗原決定位加以突變或去除,藉此排除DV NS1疫苗其可能引發自體抗體並與細胞發生結合作用的影響。
英文摘要 Dengue virus (DV) infection, an important infectious mosquito-borne disease transmitted by Aedes aegypti, represents a serious public health threat to people throughout the world. Clinical manifestations of patients with DV infection range in severity from dengue fever (DF) to life-threatening dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS). Severe thrombocytopenia, vascular leakage, and hemorrhagic syndrome are the major hallmarks associated with DHF/DSS; the mechanisms are not well understood, although several hypotheses have been suggested. In addition to a direct viral damage to the host, the host immune responses, including immune cell activation, complement activation, and cytokine production, may also play some roles. In the present study, the possible role of host immunity in the pathogenesis of DV infection was investigated. Results show that anti-platelet and anti-endothelial cell autoantibodies are present in DV-infected patient sera, but not in JEV-, HCV-, or Enterovirus 71-infected and normal control sera. The levels of autoantibodies in DHF/DSS patient sera are higher than those in patients with DF. We propose that in addition to the direct effect of DV, the production of autoantibodies in DV infection may also be involved in the pathogenesis and progression of dengue disease. The cDNA that encodes DV nonstructural protein 1 (NS1) protein is constructed in the E. coli expression system. Results show that the cell binding activity of patient sera can be inhibited by pretreatment with DV NS1. The antibodies (Abs) against DV NS1 (anti-DV NS1) produced after DV infection may, at least in part, account for the cross-reactivity of patient sera with platelets and endothelial cells. To provide direct evidence that anti-DV NS1 Abs play a role in the autoimmune response, anti-DV NS1 Abs have been generated from DV NS1-immunized mice. Results show that anti-DV NS1 Abs cross-react with platelets and endothelial cells. Interestingly, both dengue patient sera and mouse anti-DV NS1 Abs cause platelet and endothelial cell lysis in the presence of complement. Furthermore, anti-DV NS1 Abs induce endothelial cell apoptosis in a caspase-dependent manner. The signal transduction triggered by anti-DV NS1 Abs shows an increase in protein tyrosine phosphorylation and NF-kB activation. Further studies show an induction of iNOS expression and NO production, and that treatment with the NOS inhibitor L-NAME blocks cell apoptosis. The mRNA and protein expressions of Bcl-2 and Bcl-xL decrease while Bax and p53 increase after anti-DV NS1 Abs treatment, an effect that could also be inhibited by L-NAME. Taken together, anti-DV NS1 Abs trigger the intracellular signaling that leads to apoptotic cell death in endothelial cells via production of NO, upregulation of Bax and p53, and downregulation of Bcl-2 and Bcl-xL. Based on the results of this study, we suggest that the generation of autoantibodies against platelets and endothelial cells resulting in their dysfunction may play a role in the pathogenesis of dengue virus infection. The findings in the present study may provide valuable information for dengue vaccine development. The possible approaches include gene modifications of DV NS1 to truncate or mutate the epitopes that may cause the pathogenic effects.
論文目次 總目錄………………………………………………………………………………I
授權書………………………………………………………………………………III
考試合格證明………………………………………………………………………IV
中文摘要……………………………………………………………………………V
英文摘要……………………………………………………………………………VII
誌謝…………………………………………………………………………………IX
表目錄………………………………………………………………………………X
圖目錄………………………………………………………………………………XI
縮寫索引……………………………………………………………………………XIV
緒論…………………………………………………………………………………1
研究動機與實驗設計………………………………………………………………15
一、 探討登革病患血清中抗體與血小板及內皮細胞的關係……………15
二、 探討抗登革病毒非結構性蛋白1抗體引發血小板及內皮細胞的效應16
材料與方法…………………………………………………………………………17
A 材料………………………………………………………………………………17
A-1 實驗動物………………………………………………………………………17
A-2 細菌、病毒及細胞株…………………………………………………………17
A-3 病患血清………………………………………………………………………17
A-4 重組蛋白與peptide合成……………………………………………………18
A-5 試劑藥品………………………………………………………………………18
A-6 抗體……………………………………………………………………………20
A-7 耗材……………………………………………………………………………21
A-8 儀器……………………………………………………………………………22
B 方法………………………………………………………………………………23
B-1 病患血清的分離與保存………………………………………………………23
B-2 小鼠模式的研究………………………………………………………………23
B-3 細胞的分離與培養……………………………………………………………24
B-4 抗體的製備與特性分析………………………………………………………27
B-5 細胞與抗體的結合分析………………………………………………………28
B-6 細胞與抗體的作用……………………………………………………………30
B-7 細胞凋亡的分析………………………………………………………………33
B-8 蛋白質與核酸物質的分析……………………………………………………34
B-9 統計方法………………………………………………………………………42
結果…………………………………………………………………………………43
一、 登革病毒感染產生抗血小板自體抗體………………………………43
二、 登革病毒感染產生抗內皮細胞自體抗體……………………………51
三、 抗登革病毒非結構性蛋白1抗體引發內皮細胞凋亡…………………55
討論…………………………………………………………………………………60
一、 登革病毒感染產生抗血小板自體抗體的研究………………………60
二、 登革病毒感染產生抗內皮細胞自體抗體的研究……………………66
三、 抗登革病毒非結構性蛋白1抗體引發內皮細胞凋亡的機制…………72
四、 登革病毒感染引發之免疫反應參與登革致病機制…………………76
結論…………………………………………………………………………………82
參考文獻……………………………………………………………………………85
表附錄……………………………………………………………………………103
圖附錄……………………………………………………………………………109
論文發表…………………………………………………………………………143
作者簡歷…………………………………………………………………………144
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