進階搜尋


 
系統識別號 U0026-0812200914095419
論文名稱(中文) 探討自體吞噬泡及單核細胞趨化蛋白-1 之產生在登革病毒感染致病機轉角色
論文名稱(英文) Study on the Role of Autophagosome Formation and Monocyte Chemoattractant Protein-1 (MCP-1) Overexpression in the Pathogenesis of 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 96
學期 2
出版年 97
研究生(中文) 李英瑞
研究生(英文) Ying-Ray Lee
電子信箱 s5891128@mail.ncku.edu.tw
學號 s5891128
學位類別 博士
語文別 英文
論文頁數 125頁
口試委員 指導教授-劉校生
召集委員-黎煥耀
口試委員-陳舜華
口試委員-王貞仁
口試委員-Robert Anderson
口試委員-廖楓
中文關鍵字 單核細胞趨化蛋白-1  自噬泡  登革病毒 
英文關鍵字 MCP-1  Dengue Virus  autophagosome 
學科別分類
中文摘要 登革病毒感染可引發一般性登革熱(Dengue Fever;DF)、登革出血熱(Dengue Hemorrhagic Fever;DHF)及登革休克症候群(Dengue Shock Syndrom;DSS),目前病毒數量的多寡被認為在參與登革病毒感染之致病機制中扮演重要角色。另外,血管滲漏(Vascular Leakage)、血小板減少症(Thrombocytopenia)及血濃縮症
(Hemoconcentration)是登革出血熱(DHF)及登革休克症候群(DSS)之標記,血管通透性之不正常可能造成血液滲漏,而此血管通透性之改變可能受到某些血液內物質之影響;細胞激素被認為在導致血管滲漏上扮演重要角色。細胞自噬現象(Autophagy)是細胞對抗壓力時的反應之一,其目的為回收再利用胞內之不適用蛋白質及胞器。然而其在疾病之致病機轉與微生物感染症之角色仍然不甚清楚,當細胞發生自噬現象時在細胞質中會出現雙層膜結構之自噬泡(Autophagosome)及單層膜結構之自噬溶酶體(Autolysosome);有些病毒感染時會引發細胞自噬現象,並且這些病毒會利用自噬泡進行複製。在本研究中,我們利用GFP-LC3 聚集點及LC3-II 表現增加之方法發確認登革病毒第二型(DV2)之感染會引發細胞自噬現象;另外,我們利用免疫電子顯微鏡方式觀察到DV2 感染後會引發細胞
自噬泡的產生。此外,我們也發現了DV2 感染會抑制mTOR/p70S6K 之訊息傳遞路徑,可能藉此引發細胞自噬現象。另外,由於在DHF 病患的血漿中偵測出極高的單核細胞趨化蛋白-1(Monocyte Chemoattractant Protein-1,MCP-1)之表現。我們發現在DV2 感染下之單核白血球(Monocytes)會表現大量的MCP-1,且此細胞激素之表現量會隨感染之嚴重程度而增加,然而DV2 感染的肝細胞及人類臍靜脈血管內皮細胞(HUVEC)則不會大量表現MCP-1,上述結果顯示登革病毒感染特定細胞會專一性的過量表達MCP-1。利用HUVEC 細胞暴露於基因重組之人類MCP-1 或DV2 感染後之單核白血球的培養液時會造成HUVEC 細胞之滲透性增加,此現象可部分地被MCP-1 之中和型抗體所阻止。此外,HUVEC細胞膜表面之緊密連結蛋白(Tight Junction Protein) ZO-1 之分布會因暴露於MCP-1 而改變分布狀態。綜言之,本論文之主要發現登革病毒感染可引發細胞自噬泡之表現並增加病毒之複製量,而複製後所釋出之大量病毒可進一步增加感染之嚴重程度,並可能導致MCP1-之大量表現,進而增加血管內皮細胞之通透性導致血液滲漏之程度增加。以上發現為出血性登革熱之致病機轉提供一個新的想法。
英文摘要 Dengue virus infection may cause self-limited dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS). The viral load is the key point of the pathogenesis of dengue virus infection. Moreover, vascular leakage, thrombocytopenia and hemoconcentration are the hallmarks of dengue hemorrhage fever (DHF) and dengue shock syndrome (DSS). Permeability aberration may contribute to the plasma leakage, which is most likely affected by certain soluble mediators. Cytokines may play an important role in the vascular leakage. Autophagy, a cellular response against stresses, functions to recycle protein and organelles. However, the roles of autophagy in disease pathogenesis and microbial infection remain unclear. Several viral infections could induce autophagy and replicate using the autophagosome. We found that dengue virus-2 (DV2) infection can induce punctate GFP-microtubule-associated protein light chain (GFP-LC3) and increased expression of the proteolytic derivate LC-II in DV2 infected cells. DV2 infection induced autophagosome formation revealed by immuno-gold labeling of LC3 protein
under the transsimission electron microscopy. The mTOR/p70S6K signaling pathway may be involved in DV2-induced autophagy. Further study reveals that DV2
infection-induced autophagy can enhance the replication of the virus in the infected cells. Furthermore, high levels of monocyte chemoattractant protein-1 (MCP-1) were detected in the plasma of DHF patients. DV2 infection can induce MCP-1 production in monocytes, but not in hepatoma and endothelium cells. The production of MCP-1 is dengue virus-dose dependent. Exposure of human umbilical vein endothelial cells (HUVEC) to recombinant human MCP-1 (rhMCP-1) or the cultured supernatant of DV2-infected human monocytes increased vascular permeability of HUVECs.
Neutralizing monoclonal antibody of MCP-1 partially reduced vascular permeability. The distribution of the tight junction protein ZO-1 on the cellular membrane of
HUVEC was disrupted either by pretreatment of recombinant MCP-1 or the conditioned medium from DV2-infected monocytes. All together, we unravel that dengue virus infection can induce autophagosome formation and increase viral replication. Moreover, increased viral load sustains the overexpression of MCP-1, which further increases the degree of the vascular permeability. Our findings open a
new direction toward understanding the pathogenesis of DHF.
論文目次 Contents ..................................I
Abstract in Chinese .......................IV
Abstract ..................................VI
Table list ................................VII
Figure list ...............................VIII
Abbreviations .............................X
Introduction ..............................1
Specific aims .............................24
Materials and methods .....................26
I. Materials
1. Cell lines .............................26
2. Viruses ................................26
3. Patient sera ...........................26
4. Antibodies .............................27
5. Instruments ............................28
6. Reagents ...............................29
7. Buffers ................................33
II. Methods
1. Virus culture ..........................35
2. Plaque assay ...........................36
3. Cell culture ...........................36
4. Plasmid preparation ....................37
5. Plasmid transfection ...................38
6. Immunofluorescent staining .............38
7. Transmission electron microscope .......38
8. Western blot analysis ..................39
9. Flow cytometry analysis ................39
10. ELISA assay ...........................40
11. RNA extraction ........................40
12. RT-PCR analysis .......................40
13. Vascular endothelial permeability assay ..41
14. Statistical analysis ..................42
Results ...................................43
I. Dengue virus infection induces autophagosome formation .................................43
II. The mechanisms involved in dengue virus induced activation of autophagic machinery.........47
III. The relationship between activation of autophagic machinery and viral replication ...........49
IV. Dengue virus infection induces MCP-1 overexpression ............................52
V. The relationship between MCP-1 and the permeability change of vascular endothelial cells ......55
Discussion ................................59
I. Dengue virus infection induces autophagosome formation ..................................59
II. The mechanisms involved in dengue virus induced autophagy machinery ........................61
III. The relationship between the activation of autophagic machinery and viral replication ............63
IV. Dengue virus infection induces MCP-1 overexpression .............................67
V. The relationship between MCP-1 and the permeability of vascular endothelial cells .................69
Conclusion .................................72
References .................................73
Tables .....................................95
Figures ....................................96
Schematic outline of this study ............122
Author’s curriculum vitae .................123
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lipopolysaccharide. J Virol 76, 9877-87.
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Azizan, A., Sweat, J., Espino, C., Gemmer, J., Stark, L. & Kazanis, D. (2006). Differential proinflammatory and angiogenesis-specific cytokine production in human pulmonary endothelial cells, HPMEC-ST1.6R infected with dengue-2 and dengue-3 virus. J Virol Methods 138, 211-7.
Baehrecke, E. H. (2005). Autophagy: dual roles in life and death? Nat Rev Mol Cell Biol 6, 505-10.
Benarroch, D., Egloff, M. P., Mulard, L., Guerreiro, C., Romette, J. L. & Canard, B. (2004a). A structural basis for the inhibition of the NS5 dengue virus mRNA 2'-O-methyltransferase domain by ribavirin 5'-triphosphate. J Biol Chem 279, 35638-43.
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