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系統識別號 U0026-0812200915060875
論文名稱(中文) 克沙奇B3病毒感染Huh7細胞影響細胞受體的表現
論文名稱(英文) Expression of cellular receptor of coxsackievirus B3 infection in Huh7 cells
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 97
學期 1
出版年 98
研究生(中文) 蔡明勳
研究生(英文) Ming-Hsun Tsai
電子信箱 s4695115@mail.ncku.edu.tw
學號 s4695115
學位類別 碩士
語文別 英文
論文頁數 58頁
口試委員 口試委員-蘇益仁
指導教授-劉清泉
口試委員-黎煥耀
中文關鍵字 克沙奇-腺病毒受體  內化  肝細胞  克沙奇病毒B群  衰變促進因子 
英文關鍵字 Hepatocyte  Internalization  Decay-accelerating factor  Coxsackievirus and adenovirus receptor  Group B Coxsackievirus 
學科別分類
中文摘要 克沙奇病毒B群屬於B型人類腸病毒的成員,包含6種血清型。一些新生兒腸病毒重症與克沙奇病毒B群感染有關,諸如腦膜腦炎、肝炎、心肌炎、肺炎及凝血病變。於1994到2008年,陸續在台灣有克沙奇病毒B群引起新生兒猛爆性肝炎的流行,但克沙奇病毒感染肝細胞則甚少研究報告。細胞受體對於病毒的組織趨向性很重要,已知克沙奇病毒B群利用克沙奇-腺病毒受體(coxsackievirus and adenovirus receptor, CAR)及衰變促進因子(decay- accelerating factor, DAF) 作為主要的受體或副受體來感染細胞,為了研究克沙奇病毒感染時細胞受體的表現,我們以人類肝細胞株Huh7建立體外感染模式。流式細胞儀分析顯示兩受體均高度表現在Huh7細胞,利用抗克沙奇-腺病毒受體的單株抗體可以抑制克沙奇病毒B3型感染,而抗衰變促進因子的單株抗體有協同抑制的效果。感染後8小時細胞表面的克沙奇-腺病毒受體會顯著的減少,而衰變促進因子並不受到影響,這在其他克沙奇病毒株感染也可以觀察到。進一步發現克沙奇病毒感染時會降低克沙奇-腺病毒受體的蛋白質表現,但並不影響轉錄作用也不會轉換成可溶形式釋出。利用共軛焦顯微鏡觀察到感染後6到8小時有明顯的克沙奇-腺病毒受體內化(internalization),並與內吞體/溶體的標記蛋白Lamp1共存,而經由劑量相關pH值改變的內吞作用(endocytosis)可以減緩感染後細胞表面克沙奇-腺病毒受體的減少。綜合上述,克沙奇病毒感染會促進克沙奇-腺病毒受體的內化,進而向下調節細胞受體的表現。
英文摘要 Group B coxsackieviruses (CVB) belong to members of Human Enterovirus B (HEV-B) and contain six serotypes (CVB1-6). They are associated with some severe illness in neonates, including meningoencephalitis, hepatitis, myocarditis, pneumonitis and coagulopathy. There are several epidemics of CVB-associated fulminant hepatitis in Taiwan between 1994 and 2008. Cellular receptors play an important role in the tissue tropism of viral infection. CVB was found to use the coxsackievirus and adenovirus receptor (CAR) and decay-accelerating factor (DAF, CD55) as a primary receptor and coreceptor to infect permissive cells. The previous studies suggested that CVB infection may influence the expressions of CAR and DAF on host cells. To investigate the change of viral receptor expressions of CVB3 infection on Huh7 cells, the in vitro infection model was established. Flow cytometry analysis showed that CAR and DAF are highly expressed on Huh7 cells. By antibody blocking assay, anti-CAR mAb was shown to block CVB3 infection on Huh7 cells, while anti-DAF mAb has synergistic inhibitory effect. At 8 hours of CVB3 infection, cellular expression of CAR was markedly reduced on Huh7 cells. Reduction of CAR expression was also observed among different CVB serotypes. CVB3 infection reduced CAR expression in protein level, but not in transcription level and not associated with converting to soluble form on Huh7 cells. Confocal microscopy analysis illustrated that internalization of CAR is associated with endosomal/lysosomal marker, Lamp1 at 8 hours postinfection. Moreover, low-pH mediated endocytosis may be involved in the mechanism of CAR internalization and digestion in dose-dependent manner. In conclusion, CVB infection could enhance CAR internalization and downregulate cellular CAR expression.
論文目次 Abstract in Chinese ii
Abstract iii
Acknowledgment iv
Contents v
Figure list vii
Abbreviations viii
Chapter 1 - Introduction 1
1.1 Classification of group B coxsackievirus 1
1.2 Epidemiology of group B coxsackievirus 1
1.3 Group B coxsackievirus and human diseases 2
1.4 Coxsackievirus structure 4
1.5 Life cycle of group B coxsackievirus 5
1.6 Cellular Receptors for group B coxsackievirus 6
1.6.1 Coxsackievirus and Adenovirus Receptor 7
1.6.2 Decay-Accelerating Factor 9
1.7 Expression and modulation of cellular receptor 11
1.8 Research motivation 13
1.9 Aims of study 13
Chapter 2 - Materials and Methods 14
2.1 Cells and viruses 14
2.2 Antibodies 14
2.3 Flow cytometry 15
2.4 Plaque assay 16
2.5 Antibody blocking assay 16
2.6 Enzyme-Linked ImmunoSorbent Assay 16
2.7 Reverse transcription polymerase chain reaction 17
2.7.1 RNA extraction 17
2.7.2 Reverse transcription 17
2.7.3 Polymerase chain reaction 18
2.8 Western blot analysis 18
2.9 Immunofluorescence assay 19
Chapter 3 - Results 21
3.1 Identification of receptor usage 21
3.2 Expression of cellular receptor on infected cells 22
3.3 Detection of soluble coxsackievirus and adenovirus receptor 23
3.4 Expressions of coxsackievirus and adenovirus receptor in RNA level and protein level 24
3.5 The intracellular localization of coxsackievirus and adenovirus receptor 25
3.6 Disruption of low-pH mediated internalization 26
Chapter 4 - Discussion 28
Chapter 5 -Conclusion 34
Chapter 6 –References 35
Figures 45
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