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系統識別號 U0026-0812200915074519
論文名稱(中文) 前基質蛋白抗體對登革病毒感染之影響
論文名稱(英文) Effects of prM antibody on dengue virus infection
校院名稱 成功大學
系所名稱(中) 醫學檢驗生物技術學系碩博士班
系所名稱(英) Department of Medical Laboratory Science and Biotechnology
學年度 97
學期 2
出版年 98
研究生(中文) 簡廷宇
研究生(英文) Ting-yu Chien
電子信箱 t3695108@mail.ncku.edu.tw
學號 t3695108
學位類別 碩士
語文別 中文
論文頁數 74頁
口試委員 指導教授-葉才明
口試委員-林尊湄
口試委員-楊繼江
中文關鍵字 前基質蛋白抗體  登革病毒 
英文關鍵字 dengue virus  prM antibody 
學科別分類
中文摘要 登革病毒感染在全世界熱帶和副熱帶的區域流行威脅人類健康,登革病毒感染可引起一般性的登革熱 (Dengue Fever ; DF) 或較嚴重的登革出血熱 (Dengue Hemorrhagic Fever ; DHF) 及登革休克症候群 (Dengue Shock Syndrome ; DSS) 等病症,而DHF/DSS兩個重要的病徵是血小板減少症 (Thrombocytopenia) 和血漿滲漏(Plasma Leakage);雖然登革病毒在體內的主要標靶細胞是單核球細胞,但是像HMEC-1 (human microvascular endothelial cell)的內皮細胞株也可以在體外的實驗中被登革病毒感染。然而目前造成DHF/DSS的致病機轉還不清楚。在以前的研究中,Dr. Halstead 提出的抗體依靠性增強作用 (antibody-dependent enhancement, ADE)的說法來解釋DHF/DSS產生的原因,然而關於ADE效應其實也還有一些不明瞭的地方。在本研究中我們以登革病毒前基質 (prM) 單株抗體 (70-21) 結合登革病毒的方式來研究ADE效應。我們使用流式細胞儀和螢光顯微鏡等實驗方法觀察到70-21抗體可以結合至一些細胞的表面像是BHK、A549、HMEC-1和Huh7等,雖然70-21抗體可以結合至細胞的表面,但是卻沒有引起細胞凋亡或是增生等現象,然而在抗前基質抗體 (70-21) 的存在下的確可以增加病毒的感染率,並且使得單核球細胞株THP-1分泌比單獨使用登革病毒感染時更多的單核球趨化激素-1 (MCP-1)、巨噬細胞移動抑制因子(MIF)和介白素八(IL-8)。相較之下,在抗膜抗體 (50-2) 的存在下雖然也會增加感染率但是介白素八(IL-8) 的分泌卻變化的不明顯甚至是被抑制的。總括來說在ADE情況之下病毒感染了更多的單核球細胞,使單核球細胞產生較高的細胞激素單核球趨化激素-1、巨噬細胞移動抑制因子、介白素八,這些細胞激素的不正常表現會吸引並活化更多的免疫細胞前來被感染,進而產生更嚴重的免疫反應而使的病情更嚴重,甚至也可能造成血管通透性的改變,最後引起嚴重的出血甚至休克等嚴重症狀。
英文摘要 Dengue virus (DV) is an important re-emerging infectious pathogen in tropical and sub-tropical region worldwide that pose a threat to public health. DV can induce mild dengue fever or severe dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). The major two symptoms of DHF/DSS are thrombocytopenia and plasma leakage. Even though the main target cells of DV in vivo is macrophage or monocyte, many different cell lines such as endothelium (HMEC-1) are susceptible to DV infection in vitro. The antibody dependent enhancement (ADE) has been proposed to explain why DHF/DSS occur mostly in secondary DV infection with different serotypes of DV. However the molecular mechanism of ADE is still unclear. In this study, we used an anti-prM monoclonal antibody (70-21) to study this question. We found anti-prM Ab, but not anti-E Ab (50-2), can cross-react to different cells such as BHK, HMEC-1, and Huh-7 as demonstrated by flow cytometery and immunofluorescent antibody analysis. Although anti-prM Ab could bind to these cells, we didn’t find any damage or other effects to them. However, DV infection rate and the secretion of chemokine MCP-1, MIF and IL-8 of monocytic cell line THP-1 was enhanced by anti-prM antibody. On the contrary, in the presence of anti-E antibody, DV-induced IL-8 production in THP1 cell was inhibited. These results indicated that anti-prM antibody not only can enhance DV infection of monocytes but also increase chemokines secretion, which may contribute to the immunopathogenesis in DHF/DSS by recruiting more immune cells to be infected.
論文目次 總目錄
中文摘要……………………………………………Ⅰ
英文摘要………………………………………Ⅱ
誌謝………………...………………………………III
總目錄…………………………………………………IV
圖目錄…………………………………VIII
緒論…………………………………….1
登革病毒的發現和歷史及流行病學…………………1
登革病毒的起源……………………………2
病毒的宿主和媒介……………………………2
登革病毒的特性………………………………2
登革病毒感染之臨床表現…………………..4
登革熱…………………………..……..5
登革出血熱及登革休克症候群………………5
治療………………………………… ……………6
引發登革出血熱及登革休克症候群之機制的理論……6
登革病毒感染細胞之機制…………….9
登革病毒感染之目標細胞及其影響………….10
登革病毒引起的發炎反應和相關的細胞激素…………13
單核白血球系學趨化蛋白-1 (Monocyte chemoattractant protein-1; MCP-1)之特性………14
單核白血球化學趨化蛋白-1 與疾病之關係………15
第八介白素(Interleukin-8; IL-8)之特性………16
第八介白素與疾病之關係………..…16
巨噬細胞移動抑制因子(Macrophage migration inhibitory factor; MIF)之特性...............17
巨噬細胞移動抑制因子與疾病之關係……18
研究動機與實驗設計………………….20
材料與方法……………………………21
材料..…………............…...21
1.儀器..…………………………......…...21
2.試劑..…………...………………..…...22
3.抗體..……………………. .…..…....…...23
4.病毒..………………………......…...24
5.細胞株..…………….…...24
方法..………………............….....24
1.病毒接種與分離..……… ……………………...24
2.病毒斑分析(Plaque assay)..…………….24
3.細胞培養..….…………………….....25
4.單株抗體的製備..……………………25
5.單株抗體特性試驗..………….…..26
6.間接免疫螢光染色法(IFA)實驗..………….26
7.流式細胞偵測儀(Flow cytometry)偵測抗前基質(Anti-prM)抗體(70-21)結合至人類細胞或老鼠細胞和細胞內病毒抗原..……..........26
8.酵素免疫分析法..………………………27
9.西方墨點法..………………..…………27
10. MTT試驗………………………………...28
11. 細胞凋亡試驗…….……………28
結果………………..29
ㄧ、登革抗套模(anti-E;50-2)抗體和抗前基質(anti-prM;70-21)抗體的特性.......29
1.登革anti-E抗體(50-2)和anti-prM抗體(70-21)可以辨識到登革病毒(圖1) …………………....…..29
2.登革anti-E抗體(50-2)和anti-prM抗體(70-21)可以辨識到在
BHK細胞的細胞質內的登革病毒(圖2) …….……..29
3.登革anti-E抗體(50-2)和anti-prM抗體(70-21)分別可以特異的辨識到登革病毒蛋白(圖三) ………….29
二、登革抗前基質抗體(anti-prM antibody)可以結合到缺乏FcγR的細胞和其影響……………….30
1.登革抗前基質抗體可以結合到缺乏FcγR的人類和老鼠細胞(圖四、五)…………………………30
2.登革抗前基質抗體可以結合到缺乏FcγR的人類內皮細胞並且可以被專一性的M3胜抑制(圖六)………30
3.登革抗前基質抗體對細胞形態的影響(圖七) ……31
4.登革抗前基質抗體對細胞增殖的影響(圖八) …..31
5.登革抗前基質抗體對細胞產生細胞凋亡的影響(圖九) ………31
6.登革抗前基質抗體對細胞產生細胞激素的影響(圖十) ………31
三、登革抗套膜抗體和抗前基質抗體可以增強登革病毒感染具有FcγR的細胞和其影響………………………..32
1.登革抗前基質抗體和抗套膜抗體可以增強登革病毒感染具有FcγR的細胞THP1(圖十一)………….....……32
2.在ADE情況下對THP1 分泌MCP-1的影響(圖十二) ….32
3.在ADE情況下對THP1分泌 IL-8的影響(圖十三) …….33
4.在ADE情況下對THP1分泌 MIF的影響(圖十四) ……………. 33
四、登革抗前基質抗體可以增強登革病毒感染不具有FcγR的細胞和其影響……………………..……….…....33
1.登革抗前基質抗體可以增強登革病毒感染不具有FcγR的細胞 HMEC-1、A549(圖十五) ……………....…33
2.在ADE情況下對不具有FcγR的細胞分泌IL-8的影響(圖十六) ...............34
討論…………………....……....35
抗前基質抗體能夠結合至不帶有Fc受器的細胞及對其生理功能的影響……………………………………35
抗前基質抗體(70-21)和抗套膜抗體(50-2)可以增加登革病毒感染具有FcγR的細胞…..36
ADE效應下造成MCP-1的過量表現………………....36
ADE效應下造成IL-8的過量表現……………38
ADE效應下造成MIF的過量表現…………….38
抗前基質抗體(70-21)可以增加登革病毒感染不具有FcγR的細胞..39
結論………………....…………...40
參考文獻……………...…41
圖附錄……………58
作者簡歷……………....…..………....74
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