第九型伊科病毒(Echo 9)在分類上屬於小RNA病毒科(Picornaviridae)的腸病毒屬(Enterovirus)，會引發嚴重的神經系統病變如無菌性腦膜炎及類似小兒痲痺病毒感染後的肢體麻痺。1998至今，台灣爆發有關伊科病毒病例中第九型伊柯病毒佔重要部分，然而目前臨床上仍然沒有抗第九型伊科病毒的專一性抗病毒藥物。
腸病毒的正股RNA利用其5'端未轉譯區(5’-UTR)的內部核醣體進入區（IRES）進行病毒的轉譯，部分腸病毒IRES依賴型轉譯起始(IRES-dependent translation initiation)受特定的細胞因子稱作IRES trans-acting factors (ITAFs)所調控。這些 ITAFs 包括 La autoantigen, polypyrimidine tract binding protein (PTB), upstream of N-ras (Unr) 及 poly(rC) binding protein 2 (PCBP2)等。然而目前有關腸病毒ITAFs的研究多侷限於小兒痲痺病毒及鼻病毒，而這些細胞因子在第九型伊科病毒IRES轉譯中所扮演的角色仍不清楚。
本篇利用RNA 干擾策略及雙冷光報導基因法，我們發現在RD細胞（Rhabdomyosarcoma cells）中抑制PCBP2的表現會造成第九型伊科病毒IRES的活性下降，且會造成病毒蛋白表現顯著下降及病毒顆粒生成顯著減少。因此我們發現了PCBP2蛋白為第九型伊科病毒重要的ITAF之一。

Echovirus Type 9 (Echo 9), a member of the genus Enterovirus of the family Picornaviridae, is a common human pathogen associated with severe neurological manifestations including aseptic meningitis and poliomyelitis-like paralysis. Echo 9 has been implicated in a number of enterovirus-associated outbreaks in Taiwan since 1998. However, there has not been any specific anti-Echo 9 agent available for clinical use.
Translation of the enterovirus plus-strand RNA genome occurs via internal ribosomal entry site (IRES) at the 5’ untranslated region (5’-UTR). The initiation of IRES-dependent translation in some enteroviruses is modulated by certain cellular factors termed the IRES trans-acting factors (ITAFs); these ITAFs include La autoantigen, polypyrimidine tract binding protein (PTB), upstream of N-ras (Unr) and poly(rC) binding protein 2 (PCBP2). Studies on the enteroviral ITAF have mostly been limited to polioviruses and rhinoviruses, and the roles that these cellular factors play in Echo 9 IRES-driven translation remains elusive.
Utilizing the RNA interference strategy and dual-luciferase assay, we showed that knockdown of PCBP2, but not other ITAFs, could significantly decrease the Echo 9 IRES activity in Rhabdomyosarcoma cells. The decreased level of PCBP2 also led to reduced viral protein expression and viral particle production. We reasoned that PCBP2, among the ITAFs investigated, played the most significant role in controlling Echo 9 IRES.

中文摘要 4
Abstract 5
第一章 緒論 6
第一節 腸病毒概述 6
第二節 第九型伊科病毒 9
第三節 內部核醣體進入區 10
(Internal Ribosomal Entry Site, IRES) 10
第四節、核醣核酸干擾方法 15
第五節、研究動機 17
第二章 實驗材料與方法 18
第一節 細胞株的培養與操作 18
第二節 病毒的培養與定量 21
第三節 質體建構 24
第四節 利用免疫螢光染色法觀察第九型伊科病毒感染RD細胞的情形 36
第五節 利用siRNA抑制細胞內ITAFs表現 38
第六節 抑制ITAFs對第九型伊科病毒的影響 45
第三章 實驗結果 51
第一節 第九型伊科病毒感染RD細胞所產生的細胞病變及細胞受病毒感染的情形 51
第二節 抑制細胞內ITAFs對第九型伊科病毒之影響 52
第四章 討論 55
第五章 圖表 60
圖一、RD 細胞以1M.O.I.第九型伊科病毒感染後第6、8、10小時後的細胞病變及免疫螢光染色分析。 60
圖二、抑制PCBP2對第九型伊科病毒之影響 61
圖三、抑制PTB對第九型伊科病毒之影響 62
圖四、抑制Unr對第九型伊科病毒之影響 63
圖五、抑制La對第九型伊科病毒之影響 64
圖六、抑制ITAFs對第九型伊科病毒IRES activity之影響 65
圖七a、以免疫螢光染色分析抑制PCBP2、PTB、Unr、La對第九型伊科病毒之影響（6小時） 66
圖七b、以免疫螢光染色分析抑制PCBP2、PTB、Unr、La對第九型伊科病毒之影響（12小時） 67
表一、抑制ITAFs時第九型伊科病毒雙冷光報導基因質體之F/R 68
第六章 附錄 69
附錄一、 小RNA病毒科分類 69
附錄二、腸病毒屬分類 70
附錄三、腸病毒的基因體結構 70
附錄四、腸病毒的複製週期 71
附錄五、小RNA病毒IRES的分類 72
附錄六、siRNA的結構 73
附錄七、核醣核酸干擾機制 73
附錄八、1970年至2005年美國疾病管制局統計出前十五名出現的腸病毒血清型，第九型伊科病毒為第一名佔11.8% 74
附錄九、第九型伊科病毒於1970-2005年間呈現每三到五年爆發流行之週期性 74
附錄十、1998～2008年腸病毒感染併發重症疫情 75
附錄十一、2000~2005年台灣分離出Echovirus型別 75
附錄十二、2008~2009年疾管局合約實驗室統計資料中分離的伊科病毒型別 76
附錄十三、1998～2008年腸病毒感染併發重症病例檢出病原之病毒型別分佈圖 76
附錄十四、1998～2008年腸病毒感染併發重症死亡病例中可分離病原之病毒型別分佈圖 77
附錄十五、質體pCREL-EMCV 77
附錄十六、不同病毒轉譯起始其IRES所作用的細胞ITAF 78
第七章 參考文獻 79

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