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系統識別號 U0026-2707201000180100
論文名稱(中文) 抗登革病毒套膜蛋白及前趨膜蛋白抗體在登革出血上所扮演的角色
論文名稱(英文) The role of anti-dengue E/prM antibodies on dengue hemorrhage
校院名稱 成功大學
系所名稱(中) 微生物及免疫學研究所
系所名稱(英) Department of Microbiology & Immunology
學年度 98
學期 2
出版年 99
研究生(中文) 王政育
研究生(英文) Cheng-Yu Wang
學號 s4697106
學位類別 碩士
語文別 中文
論文頁數 55頁
口試委員 指導教授-黎煥耀
口試委員-林以行
口試委員-林國儀
中文關鍵字 登革熱  登革病毒  血小板  血管內皮細胞 
英文關鍵字 Dengue  platelet  endothelial cell 
學科別分類
中文摘要 登革熱在疾病的表現可以從發燒到有生命危險的登革出血熱/登革熱休克症候群,血小板低下與血漿滲漏是登革出血熱/登革熱休克症候群的關鍵臨床表現,目前登革出血熱/登革熱休克症候群的致病機轉認為是免疫致病機轉。因為抗原分子構型相似,一些抗登革病毒抗體可以交叉反應結合到血小板與血管內皮細胞,並且在登革熱出血熱/登革熱休克症候群的病人身上發現有大量的抗自體血小板與血管內皮細胞抗體。但是這些自體抗體如何造成登革出血熱/登革熱休克症候群的詳細機轉仍然不清楚。為了在大鼠建立登革病毒感染引起疾病的動物模式,因此我們分析來自小鼠的抗登革病毒單株抗體,是否具有交叉反應,能結合到大鼠血小板。我們發現一些單株抗體具有交叉反應,可以結合到大鼠血小板,例如抗登革前趨膜蛋白抗體 (anti-prM mAb 188-38) 與抗登革套膜蛋白抗體 (anti-E mAb 50-2)。並且登革病毒可以增加抗登革套膜蛋白抗體與抗登革前趨膜蛋白抗體結合到血小板。根據ELISA與流式細胞儀的結果,我們推論登革病毒可以直接結合到血小板,並且隨著病毒濃度升高,登革病毒結合到血小板的量也升高。登革病毒結合到血小板後可以直接毒殺血小板或透過活化補體導致血小板被破壞。同時抗登革抗體結合到血小板上的登革病毒後會抑制血小板受凝集素刺激後的凝集。另外登革病毒結合到血管內皮細胞後,也增加抗登革病毒抗體結合到血管內皮細胞的量,也增加了抗登革病毒抗體對血管內皮細胞的破壞。我們實驗上所觀察到的登革病毒可以直接結合到血小板與血管內皮細胞,抗登革病毒抗體和登革病毒可以造成血小板或血管內皮細胞的毒殺或影響其功能。這發現提供了一個新的機制探討登革出血熱,值得更進一步研究。
英文摘要 Dengue disease has a wide range of clinical manifes-tations from a mild febrile illness to a life-threatening dengue hemorrhage fever (DHF)/dengue shock syndrome (DSS). Plasma leakage and thrombocytopenia are the cardinal features of DHF/DSS, and an immune- pathogenesis theory for development of DHF/DSS is suggested. A molecular mimicry exists between dengue antigen and cellular protein, some anti-dengue antibodies cross- react to platelets and endothelial cells. There are the anti-platelet and anti-endothelial cell autoantibodies in dengue disease, and the autoantibody levels in DHF/DSS patient sera are higher than those in dengue fever patient sera. However, the mechanism on how these autoantibodies cause DHF/DSS is not clearly understood. By using S.D. rat as the animal to investigate the dengue disease, we found that mouse anti-dengue prM monoclone antibody (188-38) can cross-react to rat platelets. Not all of the mouse anti-dengue E/prM monoclonal antibody has the cross-reactivity to human or rat platelets. The dengue virus can enhance the mouse anti-dengue E/prM monoclonal antibody binding to platelets in vitro. This phenomenon is enhanced when the amount of virus increased. In addition, ELISA data shows that dengue virus could bind to human and rat platelets. Dengue virus has direct cytotoxic effect on platelets after binding to them. Dengue virus could also initiate complement mediated-lysis of platelets. Furthermore, anti-dengue virus antibody binding to platelets inhibit thrombin-induced platelet aggregation. In addition to platelets, we also exam human microvascular endothelial cell in vitro. We found that dengue virus could increase the anti-dengue prM (155-49)/E (50-2) monoclonal antibody binding to human endothelial cell in vitro. Dengue virus has direct cytotoxic effect on endothelial cells after binding to them. And dengue virus as like anti-dengue antibody binding to endothelial cells could directly initiate complement mediated-lysis of endothelial cells. In conclusion, anti-dengue virus antibody in combination of dengue virus can cause platelets or endothelial cells cytotoxicity or dysfunction. These findings provide a new mechanism for dengue hemorrhage and need further investigation.
論文目次 考試合格證明.............................................I
誌謝....................................................II
中文摘要................................................IV
英文摘要.................................................V
總目錄.................................................VII
圖目錄..................................................IX
表目錄..................................................XI
縮寫索引...............................................XII

序論.....................................................1
材料與方法..............................................12

A. 材料
1. 實驗動物.............................................12
2. 細胞株...............................................12
3. 抗體.................................................12
4. 試劑.................................................13
5. 塑膠玻璃製品.........................................15
6. 儀器.................................................16

B. 方法
1. 從小鼠腹水純化抗體...................................17
2. 登革病毒的培養.......................................18
3. 登革病毒力價 (Titer) 濃度的測定......................18
4. 血小板的製備.........................................19
5. 以流式細胞儀分析抗登革病毒單株抗體結合到血小板.......19
6. 以ELISA分析抗登革病毒單株抗體結合到血小板............19
7. 補體媒介的血小板破壞 (Complement-mediated lysis of
platelets) 檢測......................................20
8. 血小板凝集測試.......................................21
9. 以流式細胞儀分析抗登革病毒單株抗體結合到HMEC-1.......21
10.補體媒介的HMEC-1破壞 (Complement-mediated lysis of
HMEC-1) 檢測.........................................21
11.統計.................................................22

結果....................................................23
一. 抗登革套膜蛋白抗體與抗登革前趨膜蛋白抗體可以結合到血
小板................................................23
二. 登革病毒可以增加抗登革套膜蛋白抗體與抗登革前趨膜蛋白
抗體結合到血小板....................................23
三. 登革病毒可以結合到血小板............................24
四. 登革病毒結合到血小板後,可以活化補體造成細胞溶解....24
五. 抗登革病毒抗體結合到血小板的登革病毒後,會抑制血小板
受刺激後的凝集......................................25
六. 登革病毒可以增加抗登革套膜蛋白抗體與抗登革前趨膜蛋白
抗體結合到微血管內皮細胞............................25
七. 登革病毒結合到微血管內皮細胞後,可以活化補體造成細胞溶
解..................................................26

討論....................................................27
參考文獻................................................33
圖......................................................42
表......................................................54
作者簡歷................................................55
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