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系統識別號 U0026-2108201614151700
論文名稱(中文) 腫瘤抑制蛋白質含雙色胺酸功能區氧化還原酶對於腸病毒A71型感染症的調控
論文名稱(英文) Regulation of enterovirus A71 infection by tumor suppressor WW domain-containing oxidoreductase
校院名稱 成功大學
系所名稱(中) 醫學檢驗生物技術學系
系所名稱(英) Department of Medical Laboratory Science and Biotechnology
學年度 104
學期 2
出版年 105
研究生(中文) 陳佩璇
研究生(英文) Pei-Shiuan Chen
學號 T36031060
學位類別 碩士
語文別 英文
論文頁數 84頁
口試委員 指導教授-徐麗君
召集委員-王貞仁
口試委員-陳舜華
口試委員-余佳益
口試委員-王雅芳
中文關鍵字 腸病毒A71型  病毒生活史  含雙色胺酸功能區氧化還原酶  腫瘤抑制蛋白質 
英文關鍵字 Enterovirus A71  virus life cycle  WWOX  tumor suppressor 
學科別分類
中文摘要 人類染色體上容易脆裂的基因WWOX可轉錄產生腫瘤抑制蛋白質-含雙色胺酸功能區氧化還原酶 (WW domain-containing oxidoreductase,也稱作WWOX)。WWOX蛋白質已經被發現可以透過許多訊息傳遞路徑進行調控胚胎發育、骨頭生長代謝、神經損傷。而關於WWOX在感染症方面的研究,先前已經有人發現WWOX會和人類EB病毒(EBV)的病毒蛋白LMP2A以及人類嗜T淋巴球第一型病毒(HTLV-1)的致癌性病毒蛋白Tax進行交互作用,進而誘發腫瘤形成。然而,對於WWOX是否影響病毒的感染過程,目前尚未有這方面的研究。腸病毒A71型是隸屬於小RNA病毒科的腸病毒屬,我們發現到當老鼠有WWOX基因缺陷、或是缺乏WWOX基因的表現,相較於有正常WWOX基因表現的老鼠,腸病毒A71型感染會使老鼠產生比較嚴重的症狀,而且死亡率也較高,且在兩種不同的感染方式,腹腔注射以及口餵都有相似的結果。這些結果顯示,生物體在腸病毒A71型感染的狀況下,WWOX可能扮演調控的角色。為了瞭解宿主蛋白質WWOX是如何影響腸病毒感染的過程,我們也比較人類細胞在培養環境下,腸病毒A71型在缺乏WWOX基因表現的細胞中,相較於控制組細胞的生長狀況。因此,我們的研究發現WWOX在細胞內確實可以阻礙病毒複製的階段。我們將會對於WWOX調控這個現象的機制進行探討。
英文摘要 Human fragile WWOX gene encodes a tumor suppressor WW domain-containing oxidoreductase (designated WWOX). WWOX protein has been shown to regulate embryonic development, bone metabolism, neural injury and apoptosis through various signaling pathways. Previous studies have suggested that WWOX interacts with EB virus latent membrane protein 2A and HTLV-1 oncoprotein Tax, thus leading to tumorigenesis. However, whether WWOX affects viral infection remains unknown. Enterovirus A71 (EV-A71) belongs to the Enterovirus genus within the family of Picornaviridae. In this study, we found that Wwox-/- mice showed higher mortality rates and clinical scores as compared with Wwox+/+ and Wwox+/- littermates after intraperitoneal or oral infection of EV-A71. Higher viral loads were detected in multiple organs of EV-A71-infected Wwox-/- mice. These results suggest that WWOX may downregulate EV-A71 infection in vivo. To investigate how host factor WWOX downregulates EV-A71 infection, we examined EV-A71 life cycle in WWOX-knockdown cell lines. In summary, our results suggest that WWOX negatively regulates EV-A71 infection both in vivo and in vitro. In the future, WWOX-mediated signaling pathways that regulate EV-A71 infection will be further investigated.
論文目次 摘要 I
English Abstract II
Acknowledgements III
Contents V
Figure Index IX
Introduction 1
Enterovirus A71 (EV-A71) infection 1
EV-A71 life cycle 1
Viral proteins participate in EV-A71 RNA replication 2
Host factors in EV-A71 replication 3
WW domain-containing oxidoreductase 3
Functions of WWOX protein 4
The role of WWOX in viral infection 5
Materials and Methods 6
A. Materials 6
A-1 Cell lines 6
A-2 Virus 6
A-3 Reagents and Chemical drugs 7
A-4 Kits 8
A-5 Antibodies 9
A-6 shRNA clones 9
A-7 PCR primers 10
A-8 Consumables 10
A-9 Instruments 11
B. Methods 12
B-1 Cell culture 12
B-2 Generation of Enterovirus A71 and Coxsackievirus A16 13
B-3 Virus infection in mice 14
B-4 Determination of virus dissemination in mouse organs 15
B-5 Plaque assay 15
B-6 Preparation of lentiviral small hairpin RNA (shRNA)- mediated WWOX knockdown cells 16
B-7 Cell viability 17
B-8 Protein extraction 18
B-9 Protein quantification 19
B-10 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting 20
B-11 Neutral red infectious center assay 22
B-12 plasmid DNA purification 23
B-13 In vitro transcription 25
B-14 RNA transfection 27
B-15 RNA extraction 28
B-16 Reverse transcription (RT) 29
B-17 Polymerase chain reaction (PCR) 30
B-18 Real-time PCR 32
B-19 Luciferase reporter assay 33
B-20 Ribosome pull-down assay 34
B-21 Analysis of polysomes and polysome-associated mRNAs 35
Results 39
EV-A71-infected neonatal Wwox+/- mice exhibit higher mortality rates than Wwox+/+ mice. 39
Wwox knockout mice exhibit higher mortality rates, clinical scores and body weight loss as compared with Wwox+/+ and Wwox+/- littermates after i.p. injection of EV-A71. 39
Wwox knockout mice exhibit higher mortality rates, clinical scores and body weight loss as compared with the Wwox+/+ and Wwox+/- littermates after oral infection of EV-A71. 40
EV-A71 infection causes higher viral loads in multiple organs of Wwox-/- mice. 40
WWOX knockdown increases RNA replication and viral production in EV-A71-infected RD cells. 41
Comparable levels of EV-A71 binding to control and WWOX-knockdown RD cells. 43
WWOX may influence EV-A71 entry into host cells. 43
WWOX regulates EV-A71 replication. 44
WWOX negatively regulates EV-A71 translation initiation. 44
WWOX downregulates the association of EV-A71 mRNA with ribosomes. 45
Upregulation of WWOX expression in RD, SK-N-SH and Caco-2 cell lines after EV-A71 or CA16 infection. 46
WWOX protein cleavage after enterovirus infection. 46
Discussion 47
The role of WWOX in physiological functions and cancers 47
The link of WWOX with cancer-associated pathogens 47
WWOX in EV-A71 pathogenesis 48
The role of WWOX during EV-A71 entry into cells 49
WWOX in EV-A71 replication 50
WWOX protein cleavage during EV-A71 infection 50
Conclusion 52
References 53
Figures 58
Figure 1 58
Figure 2 59
Figure 3 61
Figure 4 63
Figure 5 65
Figure 6 66
Figure 7 68
Figure 8 69
Figure 9 71
Figure 10 73
Figure 11 75
Figure 12 77
Figure 13 79
Appendixes 80
Appendix I 80
Appendix II 81
Appendix III 82
Appendix IV 83
Appendix V 84
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