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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陳培哲 | |
| dc.contributor.author | Hsin-Wei Wang | en |
| dc.contributor.author | 王新惟 | zh_TW |
| dc.date.accessioned | 2021-06-15T11:50:01Z | - |
| dc.date.available | 2016-08-26 | |
| dc.date.copyright | 2016-08-26 | |
| dc.date.issued | 2016 | |
| dc.date.submitted | 2016-08-12 | |
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Elena, S.F., et al., Phylogeny of viroids, viroidlike satellite RNAs, and the viroidlike domain of hepatitis delta virus RNA. Proc Natl Acad Sci U S A, 1991. 88(13): p. 5631-4. 35. Jenkins, G.M., et al., Testing the extent of sequence similarity among viroids, satellite RNAs, and hepatitis delta virus. J Mol Evol, 2000. 50(1): p. 98-102. 36. Salehi-Ashtiani, K., et al., A genomewide search for ribozymes reveals an HDV-like sequence in the human CPEB3 gene. Science, 2006. 13(5794): p. 1788-92. 37. Gripon, P., et al., Hepatitis B virus infection of adult human hepatocytes cultured in the presence of dimethyl sulfoxide. J Virol, 1988. 62(11): p. 4136-43. 38. Gudima, S., et al., Primary human hepatocytes are susceptible to infection by hepatitis delta virus assembled with envelope proteins of woodchuck hepatitis virus. J Virol, 2008. 82(15): p. 7276-83. 39. Gudima, S., et al., Assembly of hepatitis delta virus: particle characterization, including the ability to infect primary human hepatocytes. J Virol, 2007. 81(7): p. 3608-17. 40. Gripon, P., et al., Infection of a human hepatoma cell line by hepatitis B virus. Proc Natl Acad Sci U S A, 2002. 99(24): p. 15655-60. 41. Sureau, C., The use of hepatocytes to investigate HDV infection: the HDV/HepaRG model. Methods Mol Biol, 2010. 640: p. 463-73. 42. Mu, J.J., D.S. Chen, and P.J. Chen, The conserved serine 177 in the delta antigen of hepatitis delta virus is one putative phosphorylation site and is required for efficient viral RNA replication. J Virol, 2001. 75(19): p. 9087-95. 43. Wang, C.J., et al., Small-form hepatitis B surface antigen is sufficient to help in the assembly of hepatitis delta virus-like particles. J Virol, 1991. 65(12): p. 6630-6. 44. Iwamoto, M., et al., Evaluation and identification of hepatitis B virus entry inhibitors using HepG2 cells overexpressing a membrane transporter NTCP. Biochem Biophys Res Commun, 2014. 443(3): p. 808-13. 45. Huang, C.R., et al., Lysine-71 in the large delta antigen of hepatitis delta virus clade 3 modulates its localization and secretion. Virus Res, 2012. 170(1-2): p. 75-84. 46. Mu, J.J., et al., Characterization of the phosphorylated forms and the phosphorylated residues of hepatitis delta virus delta antigens. J Virol, 1999. 73(12): p. 10540-5. 47. Lewis, G.D. and T.G. Metcalf, Polyethylene glycol precipitation for recovery of pathogenic viruses, including hepatitis A virus and human rotavirus, from oyster, water, and sediment samples. Appl Environ Microbiol, 1988. 54(8): p.1983-8. 48. Nowak, S.A. and T. Chou, Mechanisms of receptor/ coreceptor-mediated entry of enveloped viruses. Biophys J, 2009. 96(7): p. 2624-36. 49. Schulze, A., et al., Hepatocyte polarization is essential for the productive entry of the hepatitis B virus. Hepatology, 2012. 55(2): p. 373-83. 50. Ni, Y., et al., Hepatitis B and D viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes. Gastroenterology, 2014. 146(4): p. 1070-83. 51. Lentz, B.R., Polymer-induced membrane fusion: potential mechanism and relation to cell fusion events. Chem Phys Lipids, 1994. 73(1-2): p. 91-106. 52. Malinin, V.S., P. Frederik, and B.R. Lentz, Osmotic and curvature stress affect PEG- induced fusion of lipid vesicles but not mixing of their lipids. Biophys J, 2002. 82(4): p. 2090-100. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/49812 | - |
| dc.description.abstract | D 型肝炎病毒(Hepatitis Delta Virus, HDV)是已知能夠感染人類最小的 RNA 病毒，具有獨特的環狀 RNA 結構。D 型肝炎病毒需要 B 型肝炎病毒的外套膜幫助其包裹出完整的病毒粒子並成功進行感染，它也被稱作為是 B 型肝炎病毒的衛星病毒。全世界約有 3.5 億人口為慢性 B 型肝炎患者，其中百分之五的患者併有 D 型肝炎，雖然感染比率不高，但風險在於，併有 D 型肝炎感染的患者其症狀比起單獨感染 B 型肝炎者往往更加猛烈，發展為猛爆性肝炎的比例大幅提升，且使用抗B 型肝炎病毒的藥物並沒辦法緩解病症。一直以來，對於 D 型肝炎病毒的致病機制，以及進入宿主體內之後所導致的免疫反應並不清楚。近期的文獻，利用基因轉殖鼠 (hNTCP- Transgenic mice)研究先天與後天免疫對於 D 型肝炎病毒感染的影響，結果顯示，先天免疫在 D 型肝炎病毒清除上扮演著不可或缺的角色。先天免疫很重要的一環在於，利用「模式識別受體 (PRR, pattern recognition receptor) 」辨認病原體的保守結構—「病原體相關分子模式 (PAMP，pathogen-associated molecular pattern)，以啟動下游抗菌反應，我們感興趣的是，D 型肝炎病毒其獨特的環狀 RNA 結構，進入細胞後是否有專門的受體去辨認它。在另一篇利用人肝嵌合鼠模型 (chimeric mice with humanized liver)觀察 D 型肝炎病毒感染後宿主體內基因調控情形的研究，其結果指出 RIG-I 及 TLR3 等專門辨認病毒核酸的模式識別受體，基因表現量的確會因為 D 型肝炎病毒感染而上升。基於這些初步的觀察，我們希望能夠在細胞模型上進一步探討這些相關的免疫反應，因此，需要一個能夠穩定感染 D 型肝炎病毒且感染率達一定程度的系統。在本篇研究中，我們使用兩株穩定過表現 B 型肝炎病毒進入細胞所需之接受器—鈉離子牛磺膽酸共轉運蛋白的肝癌細胞株(HepG2-hNTCP-C4, HepG2-hNTCP-SW1)進行 D 型肝炎病毒感染實驗。結果顯示，只有 hNTCP-C4 此肝癌細胞株能夠被 D 型肝炎病毒所感染。在病毒感染當量(Genome equivalent/ cell) 為 1000 時，感染後第 6 天細胞感染率僅 0.46%；當病毒感染當量提高到 5000，感染率達 4.3%。另外，我們嘗試了不同感染條件，調整 PEG 以及 DMSO 的濃度，期望能改善感染率，但發現在 NTCP-C4 這株細胞，不論提高 PEG 濃度或是加入 DMSO，皆無法使感染率提升。我們之後的目標，是希望能夠利用此 D 型肝炎病毒感染模型找出可能辨認 D 型肝炎病毒的模式識別受體，並作為分析細胞如何產生抗病毒機制的工具，不過如果要在病毒感染後進行相關的免疫反應研究，細胞感染率預期至少要達到五成。 | zh_TW |
| dc.description.abstract | Hepatitis D Virus (HDV) is the simplest RNA virus with a unique circular genome. It is a defective RNA virus because it has to rely on Hepatitis B Virus (HBV) to assemble new virions and propagate infection. Without HBV, HDV would be unable to finish its life cycle; therefore, it is called the satellite virus of HBV. Approximately 350 million people worldwide are chronically infected with HBV. Although there is only about 5% of HBV carriers infected with HDV, the risk is that it could lead to the most severe forms of hepatitis— fulminant hepatitis. Also, antivirals against HBV do not ameliorate hepatitis D. It has long been unclear to what extent cellular immune responses attribute to liver disease and why immune responses fail to control viral replication in persistent HDV infection. Recently, two studies in mouse model have given some hints on the relation between innate immunity and HDV infection. One of the two studies, using hNTCP-transgenic mice to investigate how innate and adaptive immunity affect the clearance of HDV. Their data indicated that it is innate immunity but not adaptive immunity plays a role in HDV clearance. Pattern-recognition receptors (PRRs), which detect pathogens and initiate host antimicrobial responses such as producing type I interferons and proinflammatory cytokines, are important parts of innate immunity. We are interested in whether such unique circular RNA structure of HDV can be recognized by specific host PRRs. The other studies, using chimeric mice with humanized liver to analyze regulation of genes in host liver after HDV infection. Genes of PRRs in sensing viral nucleic acid are indeed up-regulated after HDV infection. According to those preliminary observation, we would like to further explore such matter in cell models; hence, a stable HDV infection system is needed. In this study, we used two specific HepG2 cell lines (HepG2-hNTCP-C4 and HepG2-hNTCP-SW1) which both stably overexpress an HBV entry receptor, human Sodium taurocholate cotransporting polypeptide (NTCP). Our results showed only NTCP-C4 cells are susceptible to HDV infection, and at 1000 genome equivalent/ cell, only 0.46% of the cells were infected at Day 6 post infection. To see a 4.3% infection rate, the genome equivalent/ cell would need to increase to 5000. In addition, with the aim of enhancing infection efficiency, we tried different conditions during HDV infection by adjusting concentration of PEG and DMSO. However, it seems that increase of PEG concentration or addition of DMSO both not improve the infection process. For our final goal is to find out possible PRRs detecting hepatitis delta virus, and evaluate how anti-viral response be initiated via this cell system in the future. HDV infection rate has to reach a certain level to induced sequential host immune responses. To see immune signaling mechanisms, a 50% infection rate might be necessary. We hope this infection model could shed light on the interactions between host immunity and HDV infected cells, and provide help in future study. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T11:50:01Z (GMT). No. of bitstreams: 1 ntu-105-R03448008-1.pdf: 1951334 bytes, checksum: 951b048ec12a7279a31f282890064f28 (MD5) Previous issue date: 2016 | en |
| dc.description.tableofcontents | 口試委員會審定書 .................................................................... i
誌謝 ........................................................................................ ii 中文摘要 .............................................................................. V ABSTRACT ...................................................................... VII LIST OF ABBREVIATIONS ............................................. IX CHAPTER 1: INTRODUCTION ......................................... 1 1.1 History and epidemiology of HDV ............................................................. 1 1.2 Characterization of HDV..................................................... 2 1.3 HDV entry and replication .................................................. 2 1.4 Delta antigen(s) ................................................................... 3 1.5 Viroid analogy ..................................................................... 4 1.6 HBV and HDV infection models ........................................ 5 1.7 The aim of this study ........................................................... 6 CHAPTER 2: MATERIAL AND METHODS ............................................. 7 2.1 Plasmid construct ................................................................ 7 2.2 Cell culture and DNA transfection .............................................................. 8 2.3 Virus preparation and quantification ........................................................... 9 2.4 HDV infection ................................................................... 10 2.5 Biological analysis ............................................................ 11 CHAPTER 3: RESULTS .................................................... 14 3.1 Check the integrity of type l IFN pathway in HepG2 and hNTCP-stable-expressed cell lines ................................................................. 14 3.2 Kinetics of viral RNA synthesis and virus production ...................................................... 15 3.3 Quantification of HDV viral particles ....................................................... 16 3.4 HDV infection of cell expressing human sodium taurocholate co-transporting polypeptide ……………………………………………………………..17 3.5 Concentration titration of PEG and DMSO during HDV infection .................................. 18 CHAPTER 4: DISCUSSIONS ............................................ 19 CHAPTER 5: FIGURES ..................................................... 22 Figure 1. Examine the integrity of type l IFN pathway in HepG2 and hNTCP-stable-expressed cell .......................................................................... 23 Figure 2. Kinetics of HDV genomic RNA synthesis and viral particle production in Huh7 cells. ................................................................ 25 Figure 3. Quantification of HDV viral particles for later infection experiments .................... 27 Figure 4. HDV infection of cell expressing human sodium taurocholate co-transporting polypeptide ...................................................... 30 Figure 5. Concentration titration of PEG and DMSO during HDV infection ......................... 33 REFERENCE .............................................. 35 | |
| dc.language.iso | en | |
| dc.title | 建立 D 型肝炎病毒感染之細胞模式以探討相關先天免疫反應研究 | zh_TW |
| dc.title | Hepatitis D Virus Infection in Cell Culture for Innate Immune Response Study | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 104-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 徐立中,趙玫 | |
| dc.subject.keyword | D 型肝炎病毒,B 型肝炎病毒,病毒感染,宿主免疫反應, | zh_TW |
| dc.subject.keyword | Hepatitis D virus,Hepatitis B Virus,HDV infection,host immune response, | en |
| dc.relation.page | 38 | |
| dc.identifier.doi | 10.6342/NTU201602133 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2016-08-12 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 分子醫學研究所 | zh_TW |
| 顯示於系所單位： | 分子醫學研究所 | |
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