臺灣博碩士論文加值系統

登革病毒感染往往導致輕微的登革熱或者威脅生命的登革出血熱/登革休克症狀。然而，對於登革感染的致病機轉至今仍無完全的瞭解。在本實驗室先前的研究中發現，無論是在主動模式下給予小鼠登革病毒非結構性蛋白1 (DV NS1) 或是被動給予小鼠抗DV NS1抗體，都發現小鼠肝臟中的AST/ALT濃度有增高的情形，但其腎臟之BUN值並無顯著的變化。另外也發現抗DV NS1抗體可與小鼠的下腔靜脈結合。在本論文中，我們進一步探究抗DV NS1抗體在受損的器官中可能扮演的角色。結果發現在主動給予DV NS1小鼠的肝臟血管內皮層有抗體的堆積且伴隨著有細胞凋亡的情形，但在腎臟並無觀察到有特異性抗體的堆積以及細胞凋亡的現象。另一方面，我們也發現抗DV NS1抗體可直接與正常小鼠肝臟血管內皮層結合，但在腎臟並沒有發現特異性的結合情形。除此之外，主動給予DV NS1的小鼠肝臟中有巨噬細胞浸潤的情形。我們推測因DV NS1抗體刺激而造成的內皮細胞功能喪失可能與肝臟中浸潤的細胞一同造成肝臟的損害。先前本實驗室在體外實驗中觀察到在抗DV NS1抗體刺激內皮細胞後會造成免疫活化作用，包括細胞激素、化學趨化激素以及細胞黏著分子的表現量上升。在本論文中，我們發現這些物質的活化與NF-kB相關，藉由抑制其活性可降低IL-6、IL-8、MCP-1及部分ICAM-1的表現。綜合以上體內及體外的研究結果，對於抗DV NS1抗體刺激所造成的自體免疫作用，並導致免疫活化現象，可能顯示抗DV NS1抗體在登革感染的致病機轉中扮演了一個重要的角色。

Dengue virus (DV) infection causes dengue fever or severe life-threatening dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). However, the pathogenesis of DHF/DSS is still not well understood. Previous studies in our laboratory showed that the AST and ALT, but not BUN, in mouse sera increased after either active immunization with DV nonstructural protein 1 (NS1) or passive administration with anti-DV NS1 antibodies (Abs). Furthermore, anti-DV NS1 Abs could bind to mouse vessel endothelium in inferior vena. In this study, we investigate the potential pathogenic role of anti-DV NS1 Abs in the damaged organs. Mice actively immunized with DV NS1 protein revealed the presence of anti-DV NS1 Abs on vessel endothelium in the liver. However, in the kidney, mice actively immunized with DV NS1 protein, JEV NS1 protein or PBS show similar binding patterns. Studies using organ sections from normal mice showed that anti-DV NS1 Abs, but not anti-JEV NS1 Abs, could directly bind to vessel endothelium in the liver sections. However, no specific cross-reaction of anti-DV NS1 Abs with mouse kidney vessel endothelium. Interestingly, gross and histological examinations revealed tissue damage with the presence of macrophage infiltration in the liver of NS1-immunized mice. We speculate that anti-DV NS1-induced endothelial cell dysfunction might result in liver tissue damage, at least in part, by infiltrated cells. Previous in vitro studies in our laboratory showed immune activation including cytokine and chemokine production in endothelial cells after treatment with anti-DV NS1 Abs. In this study, we demonstrated the involvement of upstream regulator NF-kB in immune activation by EMSA. After treatment with NF-kB inhibitor pyrrolidine dithiocarbamate, the expressions of IL-6、IL-8 and MCP-1 which could be stimulated by anti-DV NS1 Abs were inhibited. Taken together, endothelial cell damage and abnormal activation after stimulation by anti-DV NS1 Abs have been examined both in vivo and in vitro. These results imply the involvement of anti-DV NS1 Abs in the pathogenesis of DV infection.

中文摘要
英文摘要
致謝
目錄
圖目錄
符號及縮寫索引表
緒論
材料與方法
A. 材料
A-1 實驗動物
A-2 細菌株
A-3 細胞株
A-4 試劑藥品
A-5 抗體
A-6 耗材
A-7 儀器
B. 方法
B-1 NS1重組蛋白之製備
B-1.1 NS1重組蛋白之誘導表現
B-1.2 NS1重組蛋白的純化
B-2 SDS-PAGE 膠體電泳
B-2.1 SDS-PAGE之蛋白質電泳步驟
B-2.2以細菌為樣本時的處理
B-3 蛋白質定量
B-4 NS1重組蛋白免疫小鼠及血清的製備
B-5 抗體的製備與特性分析
B-5.1 抗體的製備
B-5.2 抗體特性的分析－以酵素結合免疫吸附法 (Enzyme-linked immunosorbent assay, ELISA) 測定抗體的效價
B-6 rNS1免疫小鼠病理現象之觀察
B-6.1 石蠟組織切片的製備與HE stain
B-6.2 冷凍切片的製備
B-6.3 以螢光染色觀察器官的病理變化
B-7 以免疫組織化學染色法(Immunohistochemistry, IHC) 觀察細胞死亡的情形
B-7.1 玻片的前處理與組織切片的製備請參考B-6.1及B-6.2
B-7.2 組織切片的TUNEL反應
B-8 以電泳移動偏移分析( Electrophoretic Mobility Shift Assays，EMSA ) 探討核蛋白之活化
B-8.1細胞核內蛋白質萃取
B-8.2 EMSA 的操作
B-9以免疫細胞染色 (immunocytochemistrys taining) 法觀察細胞激素、化學趨化激素以及細胞黏著分子的表現
B-9.1 Cytokine、chemokine以及adhesion molecule之細胞染色
結果
I. 探討登革病毒非結構性蛋白1抗體與血管內皮層發生交叉結合作用
1. 抗登革病毒非結構性蛋白1 (DV NS1) 抗體致病性角色的研究
2.以DV NS1重組蛋白免疫小鼠建立主動模式研究抗體致病性
3.主動模式中觀察抗DV NS1抗體造成肝臟及腎臟之病理現象
II. 探討抗登革病毒非結構性蛋白1抗體刺激內皮細胞發生訊息傳遞作用
1. 抗DV NS1抗體刺激內皮細胞造成NF-kB的活化
2.探討NF-kB在抗登革病毒非結構性蛋白1抗體活化內皮細胞過程中所扮演的角色
III. 抗登革病毒非結構性蛋白1抗體造成內皮系統異常活化與肝臟病變的關係
討論
參考文獻
圖
附錄
作者簡歷

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