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系統識別號 U0026-1608201419392600
論文名稱(中文) 以奈米探針協助探討腸病毒七十一型入侵宿主的超微結構分析
論文名稱(英文) Nanoprobe assisted ultrastructural analysis of EV71 entry.
校院名稱 成功大學
系所名稱(中) 口腔醫學研究所
系所名稱(英) Institute of Oral Medicine
學年度 102
學期 2
出版年 103
研究生(中文) 王雅慧
研究生(英文) Ya-Hui Wang
學號 T46011020
學位類別 碩士
語文別 英文
論文頁數 67頁
口試委員 指導教授-謝達斌
共同指導教授-吳尚蓉
口試委員-陳玉玲
口試委員-張權發
中文關鍵字 腸病毒七十一型  免疫探針  超薄切片  免疫金包埋後標定  免疫螢光  穿透式電子顯微鏡  共軛焦電子顯微鏡 
英文關鍵字 EV71  SCARB2  NCL  nanoprobe  ultra-thin sectioning  post-embedding nanoprobe labeling  immunofluorescence  TEM 
學科別分類
中文摘要 腸病毒71型 (enterovirus 71, EV71) 為手足口疾病的致病原,屬於Picornaviridae的正股核糖核酸病毒。1998年台灣爆發腸病毒71型的大流行,多名孩童在腸病毒71型感染後併發嚴重的神經性症狀像是神經性休克和神經性肺水腫。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。而腸病毒感染症目前並沒有特效藥,只能採取支持療法(如退燒、止咳、打點滴等)。因此目前研究都著手於研發疫苗或抗病毒藥物的研發。

了解腸病毒71型使用何種細胞受器感染細胞可以幫助我們進一步了解病毒的致病機制。Yamayoshi S.et al., 在2009年提出了Scavenger receptor B2, member 2 (SCARB2) 為腸病毒71型的細胞受器。然而最近研究發現,在病毒感染宿主的階段中可能有另一因子幫助腸病毒71型感染。由成大醫學檢驗生物技術學系張教授的先前實驗結果發現 Nucleolin (NCL) 在病毒71型的感染過程中扮演著重要角色。為了更進一步的證實此NCL 在病毒感染的重要性,我們從結構觀點來著手,設計了兩種奈米探針,分別為直徑大小3 nm及13 nm的奈米探針來專一性的標定SCARB2及NCL,藉由奈米探針在穿透式電子顯微鏡下的高對比度,定位並定量地分析病毒感染細胞時細胞膜上受體(SCARB2及NCL)的分布,探討其交互作用。我們實驗結果發現,病毒感染細胞時,細胞膜上的SCARB2及NCL似乎較靠近病毒,協助病毒入侵細胞。此外,若想更深入研究病毒進入細胞後,病毒與受體的交互作用是否繼續存在,奈米探針亦可應用在超薄切片中標定細胞內的病毒及受體。

此研究證實奈米探針技術可成功標定目標蛋白,定位及定量EV71入侵細胞時的決定分子。未來此技術可應用在低溫電子顯微鏡下標定出病毒感染過程的關鍵決定分子及利於病毒入侵細胞時動態超微結構分析。
英文摘要 Enterovirus 71 (EV71) is one of the major causative agents of hand, foot, and mouth disease (HFMD). EV71 is a genus of positive-sense single-stranded RNA virus and is the member of Picornaviridae family. In 1998, Taiwan faced the outbreak of EV71, there were many children complicated by severe neurological symptoms such as acute encephalitis, acute flaccid paralysis, and cardiopulmonary failure. Therefore, EV71 is considered to be the most important neurotropic enterovirus after the eradication of the polio virus. To date, there is still no efficient vaccine or drug treatment found to against the enterovirus, only the supportive therapy has been applied.

Identification of receptor and explore the virus-receptor interaction can facilitate research in pathogenesis mechanisms of viral entry. In 2009, Yamayoshi S. found that Scavenger receptor B2, member 2 (SCARB2) is a cellular receptor for EV71. In addition, recent study suggested that there might be other factors aiding in virus infection during early stage of infection. It was found that NCL on the cell surface may play important roles on mechanisms of virus I nfection. (Unpublished data, Prof. Chuan-Fa, Chang). To prove this hypothesis, two kinds of nanoprobes with diameter of 3 nm and 13 nm were prepared to label anti-SCARB2 and anti-NCL, respectively. The transmission electron microscope (TEM) was utilized to observe the virus and cellular receptor interaction. The naoprobes provide high contrast so that the receptors can be localized on the cell. Our results shows that receptors SCARB2 and NCL seem close to EV71 upon EV71 infection implying these two receptors play important roles during EV71 infection. For further study, the nanoprobes can be utilized to label the receptors in the ultra-thin section to figure out the relationship between EV71 and receptor in the cell. We anticipate that our nanoprobe labeling and developed imaging strategy would significantly improve Cryo-EM observation of virion infection kinetic study to reveal the key structural determinants governing the pathogenic process.
論文目次 中文摘要 2
Abstract 4
Acknowledgement 6
Figure contents 10
Abbreviations 11
1. Introduction 13
1.1 Enterovirus 71 is a common virus that caused foot and mouth disease 13
1.1.1 EV71 cause serious neurological complications 13
1.1.2 EV71 life cycle. 14
1.1.3 The EV71 infection stage 15
1.1.4 Recent studies show that there are other factors helping EV71 infection 16
1.2 TEM images and nanotechnology: An emerging trend 17
1.2.1 Advanced TEM images : Cryo-electron microscopy (Cryo-EM) 18
1.2.2 From traditional ultra-thin section images to advanced images of whole mount samples 18
1.2.3 Nanotechnology derived advanced imaging probes 19
1.2.4 Gold nanoparticle for bio-labeling 20
1.3 Rationale 21
2. Materials and Method 22
2.1 NG particles preparation 22
2.1.1 NG13 particles preparation 22
2.1.2 NG3 particles preparation 22
2.2 Detected nanoparticles quality and morphology 23
2.2.1 UV-vis spectroscopy checked nanoparticle quality 23
2.2.2 Transmission electron microscopy inspection nanoparticles morphology 23
2.2.3 Inductively Coupled Plasma (ICP) learned concentration standard curve of nanoparticle 24
2.3 Modifying amino group on particles surface 24
2.3.1 Modifying amino group on 13nm nano-god particles surface 25
2.3.2 Modified amino group on 3nm nano-god particles surface 25
2.3.3 How many antibodies can be conjugated to nanogold 26
2.3.4 Conjugated NG13 nanoparticle to anti-NCL 27
2.3.5 Conjugated NG3 nanoparticle to anti-SCARB2 28
2.4 ELISA for check nanoprobe efficacy 28
2.5 RD cell culture and EV71 infection 29
2.6 Immunoflurescence assay 29
2.7 Confocal image for EV71 infection 30
2.8 Whole-mounted cells method and nanoprobe labeling 31
2.10 Post embedding nanoprobe labeling 32
3. Results 33
3.1 Quality and morphology of nanogold particles 33
3.2 Prepared nanoprobes 33
3.4 Immunofluorescence assay 34
3.5 Molecular localization under confocal microscopy 35
3.6 Whole-mounted cells and nanoprobe labeling 35
3.7 Post-embedding nanoprobe labeling 35
4. Discussion 37
5. Conclusion 41
6. References 42
7. Figure and Legend 54
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