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系統識別號 U0026-2807201003103500
論文名稱(中文) Galectin-1在腸病毒71型感染中所扮演的角色
論文名稱(英文) The role of galectin-1 on EV71 infection
校院名稱 成功大學
系所名稱(中) 微生物及免疫學研究所
系所名稱(英) Department of Microbiology & Immunology
學年度 98
學期 2
出版年 99
研究生(中文) 劉佳明
研究生(英文) Chia-Ming Liu
學號 s4697112
學位類別 碩士
語文別 中文
論文頁數 51頁
口試委員 指導教授-黎煥耀
口試委員-林以行
口試委員-林國儀
中文關鍵字 無菌性腦膜炎  腦幹腦炎 
英文關鍵字 galectin-1 
學科別分類
中文摘要 腸病毒71型屬於Picornaviridae的正單股核醣核酸病毒,其易感染三歲以下孩童並造成ncku-99-s4697112-1.pdf (2065941 bytes)、無菌性腦膜炎及腦幹腦炎等神經性症狀。台灣在1998年爆發腸病毒71型的大流行,共有78名孩童在腸病毒71型感染後併發嚴重的神經性休克和神經性肺水腫而死亡。因此腸病毒71型被認為是繼小兒麻痺病毒後最重要的嗜神經性腸病毒。目前研究指出 scavenger receptor B2 以及human P-selectin glycoprotein ligand-1為腸病毒71型感染所使用的細胞受器。此外也有研究指出DC-SIGN以及sialylated glycans為可與腸病毒71型結合之蛋白質與醣類,但都未能解釋腸病毒71型的組織趨性以及嗜神經性。在我們的研究中,發現腸病毒71型與凝集素以及醣類皆有結合的特性,而腸病毒71型與凝集素的結合需有醣類參與在其中。此外在腸病毒結合細胞的實驗當中,我們發現醣類會影響病毒結合於SK-N-SH細胞表面,但對於RD細胞則無影響,顯示不同的細胞表現不同的腸病毒71型結合蛋白質。我們進一步探討腸病毒71型與哺乳類動物表現的凝集素galectin-1的結合特性,與其是否參與腸病毒71型的致病機轉。利用蛋白質交互作用沉澱試驗(pull down assay)以及酵素免疫分析法(ELISA)分析,發現腸病毒71型與galectin-1具有結合能力,且腸病毒71型與galectin-1的結合會受到醣類影響。在腸病毒71型感染細胞的過程中,galectin-1確實會與病毒蛋白結合。利用流式細胞儀分析細胞表面表現galectin-1的含量,我們發現RD細胞表面未有galectin-1的表現,但SK-N-SH細胞表面則有galecitn-1的表現。利用siRNA抑制galectin-1後發現降低galectin-1的表達會抑制腸病毒71型的細胞感染。更進一步利用galectin-1基因缺失小鼠做 in vivo感染後觀察其變化,發現galectin-1缺失小鼠的死亡率比野生型小鼠低。這些結果指出galectin-1是腸病毒71型的結合蛋白或受體,並在腸病毒71型的感染過程中扮演一個重要的角色。
英文摘要 Enterovirus 71 (EV71) is a small RNA virus that causes aseptic meningitis, brainstem encephalitis as well as hand-foot and mouth disease in young children. In 1998, EV71 infected thousands of children in Taiwan. Four-hundred five (405) children were hospitalized with neurologic disease diagnosed as brainstem encephalitis. Of these, seventy-eight (78) were fatal cases. EV71 appears to be an increasingly important neurotropic enterovirus. Although two proteins (scavenger receptor B2 and human P-selectin glycoprotein ligand-1) were reported to be the putative EV71 receptor, and though DC-SIGN and sialylated glycans can bind EV71 as well, these proteins are not sufficient to explain the neurotropism of EV71. In our study, we found that EV71 can bind to lectins and carbohydrates. The interaction between EV71 and lectins involves carbohydrates. On the EV71 virion binding to cell assay, carbohydrates mediated the interaction between EV71 and SK-N-SH cell, but not RD cells. This suggests that different cell lines might express different EV71 binding proteins. The β-galactoside binding protein, galectin-1, was further investigated for its role on EV71 infection and pathogenesis. By pull down assay and ELISA, we found that EV71 can bind to galectin-1, and carbohydrates contributed to the interaction between EV71 and galectin-1. On EV71 infected cells, galectin-1 interacted with EV71 during infection. SK-N-SH neuron cells express surface galectin-1 whereas RD cells contain only in cytoplasm on flow cytometry analysis. Using galectin-1 siRNA to knockdown the galectin-1 expression resulted in inhibiting EV71 infection. Furthermore, galectin-1 knockout mice were more resistant to EV71 infection-induced death. Our results indicate that galectin-1 is an EV71 binding protein or receptor and plays an important role in EV71 infection.
論文目次 總目錄
中文摘要﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒I
英文摘要﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒II
誌謝﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒III
總目錄﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒IV
圖目錄﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒VI
縮寫索引﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒VII
緒論
一、腸病毒71型病毒學概論﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒1
二、腸病毒71型流行病學﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒2
三、腸病毒71型之臨床症狀﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒2
四、腸病毒71型之致病機轉﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒3
五、凝集素﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒4
六、Galectin-1﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒5
七、研究方向﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒6
材料與方法
實驗材料
一、實驗動物﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒7
二、細胞株﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒7
三、病毒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒7
四、抗體﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒8
五、試劑﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒9
六、塑膠、玻璃製品﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒13
七、儀器﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒14
方法
一、細胞培養﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒15
二、腸病毒71型小鼠適應株製備﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒16
三、病毒培養與定量﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒17
四、病毒結合凝集素與糖類特性分析﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒17
結果
一、確認腸病毒71型﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒22
二、腸病毒71型結合凝集素與單醣的特性﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒22
三、Galectin-1與腸病毒71型的結合能力﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒23
四、Galectin-1與腸病毒71型的結合受到醣類影響﹒﹒﹒﹒﹒﹒﹒﹒﹒24
五、腸病毒71型感染細胞的過程中galectin-1的角色﹒﹒﹒﹒﹒﹒﹒ ﹒24
六、抑制 galectin-1的表現會降低腸病毒71型的感染﹒﹒﹒﹒﹒﹒﹒﹒25
討論﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒27
參考文獻﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒31
圖﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒ 39
作者簡歷﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒﹒51
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