出版年:

2002

研究生:

邱淑貞

研究生(英文姓名):

Shu-Chen Chiu

論文名稱:

抗登革病毒非結構性蛋白1抗體在體外及體內模式上病理角色之研究

英文論文名稱:

Studies on the Pathological Role of Antibodies Against Dengue Virus Nonstructural Protein 1 Both in Vitro and in Vivo

指導教授:

林以行 教授

指導教授(英文姓名):

Dr. Yee-Shin Lin

學位類別:

碩士

校院名稱:

國立成功大學　

系所名稱:

微生物暨免疫學研究所

學號:

s46891128

學年度:

90

語文別:

中文

論文頁數:

89

關鍵詞:

登革病毒 ; 非結構性蛋白1

英文關鍵詞:

dengue virus ; Nonstructural Protein 1

被引用次數:

0

[ 摘要 ]

中文摘要
登革病毒 (dengue virus, DV) 屬於黃熱病毒科的一員，其傳染方式是藉由埃及斑蚊或白線斑蚊所媒介。臨床上，感染登革病毒的病人會有不同嚴重程度的症狀產生，輕則典型的發燒 (dengue fever)，重則會有出血熱 (dengue hemorrhagic fever; DHF)，甚至休克 (dengue shock syndrome; DSS) 的現象產生。血小板減少症、血管病變及出血性症狀是DHF/DSS主要的病徵，雖然如此，DV造成DHF/DSS的致病機轉仍不清楚。本實驗室先前的研究顯示，登革第二型病毒非結構性蛋白1抗體 (anti-DV NS1) 可以交互結合至內皮細胞，並且會造成細胞功能喪失。進一步證實登革病人血清中anti-DV NS1抗體的確會和內皮細胞結合並造成細胞凋亡的結果。在本論文中，進一步探討anti-DV NS1在體內及體外病理的角色。我們純化構築於E. coli的DV2 NS1蛋白，並免疫小鼠進一步取得anti-DV NS1抗體。由免疫染色法證實anti-DV NS1的確會與人類微血管內皮細胞 (HMEC-1) 進行交叉結合反應，不僅如此，抗體亦可以結合至小鼠的血管內皮細胞。萃取內皮細胞的膜蛋白經由西方墨點法觀察其與anti-NS1抗體結合，結果顯示分子量大約在95、130及150 kDa的位置分別有結合的反應。此外，將HMEC-1給予anti-DV NS1處理1小時後，再繼續培養12或24小時，也可觀察到明顯的細胞凋亡現象產生。進一步探討細胞活化訊息傳遞的機轉，結果顯示內皮細胞在anti-DV NS1刺激之後會有IL-6、IL-8及MCP-1的表現，而RANTES並沒有產生。利用流式細胞儀的方式證實，HMEC-1在給予anti-DV NS1處理後，ICAM-1會被誘導出，而且其黏著性有增高結合PBMC的趨勢。除此之外，為了要瞭解anti-DV NS1在體內的致病機轉，我們被動給予BALB/c小鼠anti-DV NS1，發現小鼠血清中AST、ALT值會明顯上升，而由病理切片觀察可知，在肝細胞中除了有一些吞噬細胞的浸潤外，還會有細胞凋亡現象的產生。綜而言之，anti-DV NS1在DV感染的過程中的確參與了重要的致病機轉，而這種機轉似乎與自體免疫的產生有著密切的關係。

[ 英文摘要 ]

英文摘要
Dengue virus (DV), the member of Flaviviridae, are transmitted by Aedes aegypti or Aedes albopictus. In clinical, patients with DV infection show a wide range of disease severity from mild febrile illness as dengue fever to life-threatening dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS). Thrombocytopenia, vascular leakage, and hemorrhagic syndrome are the hallmarks of the DHF/DSS. However, the pathogenesis of DHF/DSS is poorly understood. Previous studies in our laboratory showed the endothelial cell dysfunction caused by antibodies (Abs) against DV nonstructural protein 1 (NS1). Anti-NS1 Abs from dengue patients cross-reacted with endothelial cells and caused endothelial cell apoptosis. In this study, anti-NS1 Abs were generated in mice and their effects on endothelial cells (HMEC-1) were examined. The cross-reactivity of anti-NS1 Abs with HMEC-1 cells was detected by immunostaining. The binding activity of anti-NS1 Abs was also observed in vascular endothelium of mouse vessel ex vivo. Western blot analysis demonstrated that proteins with molecular weights of approximately 95-, 130- and 150-kDa could be detected following the hybridization of anti-NS1 Abs with endothelial cell extract membrane fractions. Anti-NS1-induced HMEC-1 apoptosis could be observed when Abs were incubated with cells for 1 h, followed by 12 or 24 h further cultivation. The cytokine and chemokine production after anti-NS1 stimulation was also examined. Results showed that the expression of IL-6、IL-8 and MCP-1, but not RANTES, in endothelial cells increased after treatment with anti-NS1 Abs. Furthermore, we found that expression of ICAM-1 was induced and the adhesion ability of PBMC was upregulated after anti-NS1 stimulation. In the animal studies, the levels of AST and ALT in mouse sera increased after passive administration of anti-NS1 Abs. The pathological effects of anti-NS1 Abs in mouse liver revealed macrophages infiltratration and some cells undergoing apoptosis. Taken together, these findings may provide the evidence on the pathological role of anti-NS1 Abs in DV infection.