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系統識別號 U0026-0104201521104200
論文名稱(中文) 探討含雙色胺酸功能區氧化還原酶與腸病毒71型感染症之交互調控作用
論文名稱(英文) The interplay of WW domain-containing oxidoreductase with enterovirus 71
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 103
學期 2
出版年 104
研究生(中文) 廖紹含
研究生(英文) Shao-Han Liao
學號 T36011044
學位類別 碩士
語文別 英文
論文頁數 66頁
口試委員 指導教授-徐麗君
口試委員-王貞仁
口試委員-詹明修
口試委員-王雅芳
中文關鍵字 含雙色胺酸功能區氧化還原酶  腸病毒71型 
英文關鍵字 WWOX  EV71 
學科別分類
中文摘要 含雙色胺酸功能區氧化還原酶 (WW domain-containing oxidoreductase),又名為WWOX或小鼠WOX1,為一種抑癌蛋白質。 先前研究指出,WWOX蛋白質參與多種訊息傳遞路徑,以發揮抑癌功能、調控細胞凋亡、參與胚胎發育、以及影響神經性疾病的發展。先前亦有研究顯示,WWOX蛋白質與EB病毒 (Epstein-Barr virus) 和人類T細胞白血病毒第1型 (human T cell leukemia virus, HTLV-1) 誘導的癌症調控機制有關。然而,現今尚未有研究證明 WWOX蛋白質是否調控哺乳類細胞受到腸病毒71型 (enterovirus 71; EV71) 感染的作用。為了進一步探討WWOX在EV71感染下可能扮演的角色,我們利用lentiviral shRNA,將人類橫紋肌肉瘤細胞 (human rhabdomyosarcoma, RD) 內WWOX的表現抑制下來。 有趣的是,與控制組細胞相較之下,WWOX蛋白質表現量很低的RD細胞在受到EV71感染後,細胞內EV71的表現量較高。 RD細胞中WWOX蛋白質表現量減少時,並不會改變EV71受器SCARB2與Nucleolin的表現量,病毒結合到細胞表面的情形也沒有受到影響。 Wwox基因剔除小鼠 (knockout, Wwox-/-) 在感染EV71後,比起野生種 (wild-type, Wwox+/+)出現較高死亡率。 綜合以上,我們發現WWOX蛋白質可調控EV71在動物體內及細胞內的感染情形。
英文摘要 WW domain-containing oxidoreductase, designated WWOX or murine WOX1, is a candidate tumor suppressor protein. WWOX has been suggested to be involved in many signaling pathways that regulate tumor suppression, cell death, embryonic development and neuronal diseases. Previous studies have suggested that WWOX is associated with cancer progression induced by Epstein-Barr virus and human T cell leukemia virus. However, whether WWOX controls the infection of mammalian cells by enterovirus 71 (EV71) is still unknown. To investigate the possible role of WWOX in EV71 infection, lentiviral shRNA-mediated knockdown of WWOX expression was used in human rhabdomyosarcoma (RD) cells. Interestingly, higher viral levels were detected in WWOX-knockdown RD cells than the controls after EV71 infection. Comparable expression levels of cellular receptors for EV71, SCARB2 and Nucleolin, were detected in WWOX-knockdown and control RD cells. Knockdown of WWOX expression in RD cells did not affect the binding of EV71 to the cell surface. Wwox gene knockout mice exhibited higher mortality rates than did wild-type littermates after EV71 infection. Taken together, we have demonstrated that WWOX regulates EV71 infection both in vitro and in vivo.
論文目次 中文摘要 I
Abstract II
致謝 III
Contents IV
Figure index VII
Introduction 1
WW domain-containing oxidoreductase 1
Functions of WWOX protein 1
Relationship between WWOX protein and EBV infection 2
WWOX decreases NF-B activation in HTLV-1 infection 2
Enterovirus 71 (EV71) 3
Diseases caused by EV71 infection 3
Transmission and life cycle of EV71 4
EV71 induces autophagy to promote its replication 4
The signaling pathways that regulate EV71 replication in host cells 5
Mouse models for EV71 infection 5
Materials and Methods 7
A. Materials 7
A-1 Cell lines 7
A-2 Chemicals and reagents 7
A-3 Kits 10
A-4 Antibodies 10
A-5 Consumables 11
A-6 Instruments 12
B. Methods 13
B-1 Cell culture 13
B-2 Virus 13
B-3 Plaque assay 14
B-4 WWOX knockdown in cell lines 15
B-5 Virus binding assay: EV71 binding rate at 4℃ 16
B-6 Virus binding assay: ELISA assay 17
B-7 Protein extraction 19
B-8 Protein quantification 20
B-9 Generation and extraction of EV71 21
B-10 RNA extraction 22
B-11 Reverse transcription-polymerase chain reaction (RT-PCR) 23
B-13 Plasmid DNA purification 25
B-14 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting 27
B-15 Hematoxylin and eosin (HE) staining 29
B-16 Immunofluorescence (IF) staining 30
B-17 EV71 infection of Wwox gene knockout mice 31
B-18 Organ dissemination 32
Results 33
EV71 infection upregulates WWOX protein expression in human RD cells 33
WWOX protein impedes in EV71 replication in RD cells 33
EV71 binds equally well to shLuc and shWWOX RD cells 34
Knockdown of WWOX expression enhances endocytosis of EV71 in RD cells 35
Wwox protects mice from lethal infection with EV71 36
Wwox expression decreases the viral loads in multiple organs of EV71-infected mice 37
EV71 infection causes higher mortality rate in T-cell specific Wwox knockout mice 38
Discussion 39
The role of WWOX protein in vitro and in vivo 39
WWOX affects the entry of EV71 into cells 39
Active IRES of WWOX may compete for the initiation factors to inhibit EV71 translation 40
WWOX reduces autophagy to suppress EV71 replication 41
The possible WWOX-mediated signaling pathways for regulating EV71 replication 41
Conclusion 43
References 44
Figures……………………………………………………………………………………..48
Figure 1………………………………………………………………………….………….....48
Figure 2………………………………………………………………………………………..49
Figure 3………………………………………………………………………………………..50
Figure 4………………………………………………………………………………………..52
Figure 5………………………………………………………………………………………..53
Figure 6………………………………………………………………………………………..56
Figure 7………………………………………………………………………………………..57
Figure 8………………………………………………………………………………………..59
Figure 9………………………………………………………………………………………..60
Figure 10………………………………………………………………………………………62
Figure 11………………………………………………………………………………………63
Figure 12………………………………………………………………………………………65
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