臺灣博碩士論文加值系統

腸病毒七十一型屬於微小核糖核酸病毒科(Picornaviridae family)的一員，好感染五歲以下嬰幼兒，是導致手足口病(hand-footand-mouth disease，HFMD)的主要病原之一，亦會造成腦膜炎、癱瘓等神經系統病徵，嚴重者死亡。為了研究腸病毒七十一型的致病機轉，我們實驗室將病毒受器轉殖到小鼠上，建立可被腸病毒七十一型感染的基因轉殖鼠(hSCARB2 Tg mice)，在以此模式進行研究時，我們意外發現一重要現象，若使用橫紋肌肉瘤細胞株(RD cell，Rhabdomyosarcoma)生產病毒，會導致病毒快速發生突變，並使其毒性下降。
我們將由非洲綠猴腎細胞細胞株(Vero cell)生產之病毒命名為EV-V，由RD生產之病毒命名為EV-R，進行一系列實驗比較兩者之差異；在小鼠攻毒實驗中，EV-R不同於EV-V的致死與嚴重神經疾病，只出現輕微的症狀並且全員存活，在組織切片中，也可以見到EV-V導致了更嚴重的肌炎、肌纖維斷裂與淋巴細胞浸潤現象；免疫組織化學染色切片實驗中，亦證實EV-V在組織中的病毒量更高；而定量RT-PCR分析，顯示EV-R病毒複製較少，且誘發之促發炎反應較輕微。
我們將兩種病毒分別進行基因定序，發現EV-R在結構蛋白VP1序列中，有出現三個突變E145G、V146I、S241L，而在5’UTR 494位置由C變成T；病毒-受器親和力試驗結果證實EV-R對於PSGL-1的結合能力大幅上升，然而在SCARB2方面並無顯著差異；我們也透過不同的細胞進行病毒潛入試驗，驗證EV-V與EV-R在感染細胞，侵入階段的差異，以及後續病毒產量上的區別，結果顯示EV-R透過PSGL-1感染細胞的能力上升，且複製更為快速。
這本篇研究中，我們揭示了以往在進行腸病毒七十一型研究被輕忽的細節，以及培養系統對於臨床分離株病毒的影響，和使用合適動物模式的重要性。

Enterovirus 71 (EV71) is a member belonged to Picornaviridae family. The infection of EV71 causes outbreaks of hand-foot-mouth-disease(HFMD) and meningitis, paralysis and even death. To study the pathogenesis of EV71, we had established the transgenic mice expressing EV71 receptor for infection (hSCARB2 Tg mice). In our past study, we had unexpectedly discovered an important phenomenon; using rhabdomyosarcoma cell line (RD) to produce EV71, the progeny EV71 will be rapidly mutated and reduce its toxicity.
In this study, we prepare EV71 in Vero (named as EV-V) or sub-passaged in RD (named as EV-R) cells and then used them as pathogens. Interestingly, EV-R exhibited differential virulence: challenging of hSCARB2 Tg mice with EV71 revealed that EV-V was more virulent than EV-R: 100% of mice that received lethal amounts of EV-V died, while all the mice that received EV-R were survived. Severe pathogenesis correlated with viral burdens and proinflammatory cytokine levels were observed in EV-V-challenged mice, but controversy in EV-R-challenged mice. Histopathological sections of spinal cord and the surrounding muscle show EV-V cause more severe myositis, muscle fiber breakage and lymphocyte infiltration. In immunohistochemical (IHC) staining sections confirmed that EV-V leads a higher amount of virus in the tissue. Quantification reverse transcriptase PCR (Q-RTPCR) results evealed that the EV-R virus replicates less and induces less proinflammatory responses as well.
Consensus sequence analysis revealed EV-R rapidly acquired complete mutations at E145G and S241L and partial mutations at V146I of VP1, and acquired a T to C substitution at nucleotide 494 of the 5’-UTR. EV-R exhibited higher binding affinity for another EV71 receptor, human P-selectin glycoprotein ligand-1(hPSGL-1), than EV-V. Both EV71s exhibited no significant difference in binding to hSCARB2. In virus entry assay and replication rate assays showed that EV-R infect cells through PSGL-1 increased and replication faster.
In summary, we pointed to the neglected details of the past EV71 study that the culture system of virus isolated enterovirus and appropriate animal model are critical key points.

中文摘要 3
Abstract 5
第一章 緒言 7
一、病毒構造 7
二、疫情與防護 8
三、攻毒動物模式 10
四、病毒類種演化 11
第二章 研究目的 13
第三章 實驗材料與方法 14
細胞株及病毒株(Cell and virus strains) 14
動物實驗執行倫理(Ethical regulation of animal experimentation) 15
噬菌斑試驗(plaque formation assay) 15
小鼠攻毒實驗(Virus challenge assay) 15
免疫組織化學染色(Immunohistochemical staining) 16
及時定量反轉錄聚合酶連鎖反應(Real time RT-PCR) 16
病毒基因組定序(Virus genome sequence) 17
腸病毒七十一型生產及濃縮(EV71 production and purification) 17
冷凍電子顯微影像拍攝分析(Cryo electron microscopy, cryoEM) 17
病毒受器親和力試驗(Virus-receptor binding affinity assay) 18
統計分析(Statistical analysis) 19
第四章 實驗結果 20
一、病毒生產與定量 20
二、病毒定序分析與結構模擬 21
三、小鼠攻毒試驗 23
四、攻毒後各組織樣品細胞激素表現分析 24
五、病毒與受器親和力試驗 25
六、病毒侵入與複製速率試驗 26
第五章 結果討論 27
一、腸病毒七十一型點位突變的探討 27
二、培養系統對病毒突變的影響 29
三、此研究的重要性與未來展望 30
第六章 結論 31
第七章 參考文獻 32
附表 45
表一、即時定量反轉錄聚合酶連鎖反應使用之引子列表 45
表二、定序使用之引子列表 46
表三、病毒在不同細胞培養之細胞病變現象與病毒產量表 47
表四、EV-V與EV-R基因序列比較 48
附圖 49
圖一、病毒定序光譜圖 49
圖二、突變點位的結構模擬 50
圖三、EV-V及EV-R電子顯微影像分群 51
圖四、小鼠攻毒實驗存活曲線與神經系統症狀評分 52
圖五、攻毒小鼠脊髓組織切片 53
圖六、蘇木精-伊紅染色及免疫組織化學染色 54
圖七、各組織樣品促發炎激素表現量分析 55
圖八、各組織樣品中病毒量分析 56
圖九、病毒與受器親和力試驗 57
圖十、在單一受器細胞株的病毒侵入與複製試驗 58

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