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系統識別號 U0026-1302201715591500
論文名稱(中文) 腸病毒A型感染人類核仁蛋白基因轉殖鼠所表現的生物特性
論文名稱(英文) Biological characteristics of human enterovirus species A in human nucleolin transgenic mice
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 105
學期 1
出版年 106
研究生(中文) 林怡珍
研究生(英文) Yi-Chen Lin
學號 t36034131
學位類別 碩士
語文別 中文
論文頁數 50頁
口試委員 指導教授- 張權發.
口試委員-王貞仁
口試委員-王雅芳
口試委員-余佳益
中文關鍵字 腸病毒A型  核仁蛋白  人類核仁蛋白基因轉殖鼠 
英文關鍵字 Enterovirus species A  nucleolin  human nucleolin transgenic (Tg) mice 
學科別分類
中文摘要 腸病毒A型(enterovirus species A, EV-A)是屬於小RNA病毒科 (Picornaviridae) 、腸病毒屬 (Enterovirus;EV) 之病毒。感染腸病毒後,有的會出現手足口病、疱疹性咽峽炎、無菌性腦膜炎、病毒性腦炎、肢體麻痺症候群等。EV-A有25種血清型,例如柯薩奇病毒(CV)A2-A8、A10、A12、A14、A16、腸病毒A 71型(EV-A71)。Receptors在病毒感染的早期是必需的,病毒會辨識細胞表面的receptors而進入宿主細胞。在我們實驗室先前的研究中,我們利用醣蛋白質體的方法,找到會和腸病毒A 71型有交互作用的醣蛋白-核仁蛋白(nucleolin, NCL)。我們發現腸病毒A 71型和柯薩奇病毒 (CV-A2、CV-A4、CV-A6、CV-A10、CV-A16)皆會與核仁蛋白有交互作用,並且抗核蛋白抗體會降低這些病毒附著到宿主細胞。抑制細胞膜上核仁蛋白表現後,腸病毒A71型和柯薩奇病毒結合到細胞能力下降。為了進一步釐清在動物實驗中,核仁蛋白是否在腸病毒A型的感染扮演角色,我們建立了人類核仁蛋白基因轉殖鼠(hNCL-Tg)來觀察腸病毒A型的感染力。利用以下幾株病毒以腹腔注射的方式來感染人類核仁蛋白基因轉殖鼠,病毒株為:腸病毒A71型小鼠適應株MP4、腸病毒A71型臨床分離株94-N2873(C4)、87-N6356(C2)、97-M448(B5)、柯薩奇病毒CV-A6、CV-A10、CV16,觀察並測量其生理變化,包括體重、後肢癱瘓情形及死亡率,發現MP4感染後,人類核仁蛋白基因轉殖鼠臨床症狀較野生型小鼠嚴重,並且死亡率非常高。 柯薩奇病毒A16感染後,人類核仁蛋白基因轉殖鼠臨床症狀亦較野生型小鼠嚴重,並且死亡率也較高。我們進一步分析小鼠腦幹、脊隨、後肢肌肉的病毒量,發現MP4感染後第六天,人類核仁蛋白基因轉殖鼠的脊隨和肌肉裡的病毒量較野生型小鼠高,然而柯薩奇病毒A16感染後,病毒量在人類核仁蛋白基因轉殖鼠與野生型小鼠的組織中沒有顯著差異。根據這些結果,我們認為人類核仁蛋白在腸病毒A型感染中扮演重要的角色,本研究亦提供一個有利於研究腸病毒A型感染的動物模式。
英文摘要 Enterovirus species A (EV-A), one of the four species of EV in the genus Enterovirus of the Picornaviridae family, is known to manifest hand-foot-mouth diseases in young children and may cause severe neurological disorders such as encephalitis and meningitis. This virus EV-A species consists of 25 stereotypes, including coxsackievirus (CV) A2–A8, A10, A12, A14 and A16 and Enterovirus A71 (EV-A71). Receptors are necessary in the early stages of virus infection. The virus recognizes surface receptors and entries into the host cells. In previous study we have identified nucleolin (NCL) as an EV-A71 binding receptor by glycoproteomic approaches. We found that EV-A71 and coxsackieviruses (CV-A2, CV-A4, CV-A6, CV-A10 and CV-A16) interacted directly with nucleolin and an anti-nucleolin antibody reduced the binding of these viruses to host cells. Knockdown of nucleolin decreased the attachment of EV-A71 and coxsackieviruses to RD cells. In order to characterize whether nucleolin involves in EV-As infection in vivo, we established human nucleolin transgenic (hNCL-Tg) mice to evaluate the infectivity of EV-As. The hNCL-Tg mice were intraperitoneally infected with mouse-adapted EV-A71 strain MP4, EV-A71 strains 94-N2873 (C4), 87-N6356 (C2), 97-M448 (B5), and CV-A6 isolated from patients, CV-A10 and CV16. The biological characteristics including body weight, clinical scores of limb paralysis and survival rates were observed. We found that MP4-infected-Tg mice displayed higher clinical scores and progressive limb paralysis prior to death. CV-A16-infected-Tg mice showed more severe limb paralysis and lower survival rates than CV-A16-infected-WT mice. Further, the viral load in brainstem, spinal cord and limb muscles were also investigated. We found that MP4-infected-Tg mice showed higher viral load than MP4-infected-WT mice in spinal cord and muscle at day 6 postinfection. However, viral load in these tissues of CV-A16-infected-Tg mice had no difference between CV-A16-infected-WT mice. Based on the above, it is suggested that human nucleolin should play a key role in EV-As infection, and hNCL-Tg mice should be a useful animal model for the investigation of EV-As infection.
論文目次 Abstract I
摘要 III
Table of Contents IV
Abbreviations VII
Chapter 1. Introduction 1
1. Enteroviruses (EVs) 1
1.1 Classification 1
1.2 Clinical symptoms and epidemiology 1
2. Receptors and attachment factors of EV-A71 2
3. Nucleolin 3
3.1 Function and position 3
3.2 Roles of NCL in virus infection 4
4. Animal models 4
Chapter 2. Objective 6
Chapter 3. Materials and Methods 7
1. Virus amplification 7
2. Plaque assay 7
3. Animal model 8
3.1 Establishment 8
3.2 Genotyping 8
3.3 Western blot analysis 8
3.4 Infection 9
3.5 Viral load in tissue 9
4. Statistical analysis 10
Chapter 4. Results 11
Previous studies 11
Confirmation and verification of hNCL-Tg mice. 11
hNCL increases EV-As infection 11
hNCL expresses in brain stem, spinal cord and muscle of Tg mice 14
Virus titer in organ of virus infected mice 14
Different disease manifestations were observed in MP4 and CV-A16 infected hNCL-Tg mice 15
Skin rashes were observed in MP4 infected hNCL-Tg mice 15
Chapter 5. Discussion 16
Chapter 6. Conclusion 21
References 22
Figure 26
Table 41
Appendix 43
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Figure 1. Verification of human nucleolin transgenic mice (hNCL-Tg mice). 26
Figure 2. Body weight of wild type and hNCL-Tg mice 27
Figure3.The clinical scores and survival rates of wild-type and hNCL-Tg mice challenged with MP4. 28
Figure 4. The clinical scores and body weight of wild-type and hNCL-Tg mice challenged with EV-A71 94-N2873(C4). 29
Figure 5. The clinical scores and body weight of wild-type and hNCL-Tg mice challenged with 87-N6356(C2). 30
Figure 6. The clinical scores and body weight of wild-type and hNCL-Tg mice challenged with 97-M448(B5). 31
Figure 7. The clinical scores of wild-type mice challenged with CV-A16. 32
Figure 8. The clinical scores and survival rates of wild-type and hNCL-Tg mice challenged with CV-A16. 33
Figure 9. The clinical scores and survival rates of wild-type and hNCL-Tg mice challenged with CV-A6. 34
Figure 10. The clinical scores of wild-type and hNCL-Tg mice challenged with CV-A10. 35
Figure 11. The expression of fusion red in tissue of wild-type and hNCL-Tg mice 36
Figure 12. The viral load in central nervous and muscle of wild type and hNCL-Tg mice infected with MP4 strain 37
Figure 13. The viral load in central nervous and muscle of wild type and hNCL-Tg mice infected with CV-A16 38
Figure 14. Disease manifestations (limb paralysis) of hNCL-Tg mice infected with MP4 and CV-A16. 39
Figure 15. Disease manifestations (skin rashes) of hNCL-Tg mice infected with MP4. 40
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