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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陳培哲 | |
| dc.contributor.author | Po-Wen Wang | en |
| dc.contributor.author | 王柏文 | zh_TW |
| dc.date.accessioned | 2021-06-17T05:00:04Z | - |
| dc.date.available | 2020-08-01 | |
| dc.date.copyright | 2018-08-01 | |
| dc.date.issued | 2018 | |
| dc.date.submitted | 2018-07-25 | |
| dc.identifier.citation | References
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/71234 | - |
| dc.description.abstract | 慢性B型肝炎在全世界是一個重大的公共衛生議題，目前的治療未能有效地使慢性B型肝炎患者痊癒。在臨床研究上，HBV剪接與慢性B型肝炎具有高度的正相關性。探討HBV剪接在B型肝炎病毒感染中所扮演的角色，或許能提供HBV抗病毒藥物一個嶄新的靶點。先前的研究在肝癌細胞和小鼠中發現，轉染帶有剪接功能缺失的HBV質體，並不影響HBV病毒的複製。然而，利用人肝嵌合小鼠模型，感染由免疫缺陷小鼠所製造的HBV病毒，證實了HBV剪接對於建立有效的HBV感染是必要的。因為轉染模型只能模擬部分HBV的生命週期，在本篇研究中，肝癌細胞感染模型將被用來探討HBV剪接在HBV感染中所扮演的角色。研究結果指出，HBV基因組的複製、RNA轉錄與蛋白質轉譯並不仰賴HBV剪接。本篇研究與人肝嵌合小鼠模型的結果相異，可能源自於兩者研究模式上的差異，包括HBV質體、病毒來源與感染細胞株等因子。本篇論文將針對這些可能的因子做進一步的討論。 | zh_TW |
| dc.description.abstract | Chronic Hepatitis B infection remains a public health issue worldwide. Currently, there is still no effective therapy for chronic HBV infection. Clinical studies have found that chronic infection was associated with HBV spliced variants. Understanding the role of HBV splicing in viral life cycle should provide a new strategy for HBV treatment. Previously, it had been demonstrated that HBV splicing variants were not essential for viral replication in transfection model in vivo and in vitro. However, another study had revealed that splicing-deficient HBV, generated from SCID mice by in vivo transfection, failed to establish efficient HBV infection in humanized liver chimeric mice, suggesting the important role of HBV splicing. Because of the transfection model may not completely recapitulate the viral life cycle, in this study, we exploited an in vitro infection model to examine the role of HBV splicing. Splicing-deficient HBV virions, produced from hepatoma cell line Huh7 by in vitro transfection, were used to infect NTCP-overexpressing hepatoma cell line HepG2-NTCP-C4. Our data indicated that viral replication, transcription and translation have no difference between cells infected with WT or splicing-deficient HBV. In contrast to the restricted HBV infection in vivo, the successful HBV infection in vitro might due to several factors, such as replicons, virus-producing cells and virus-infected cells, which will be discussed in the thesis. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T05:00:04Z (GMT). No. of bitstreams: 1 ntu-107-R05445119-1.pdf: 8799624 bytes, checksum: ceed36b529c5c404318378bc8fa54e57 (MD5) Previous issue date: 2018 | en |
| dc.description.tableofcontents | 口試委員審定書 i
謝辭 ii 中文摘要 iii Abstract iv Table of contents vi 1. Introduction 1 1.1. Background of HBV 1 1.1.1. General information 1 1.1.2. Structure of HBV particles 1 1.1.3. Genome organization 2 1.1.4. Life cycle of HBV infection 4 1.2. HBV RNA splicing 8 1.2.1. Mechanisms and regulations 8 1.2.2. Species of spliced RNAs 10 1.2.3. Novel proteins from spliced mRNAs 12 1.3. HBV in vitro infection models 14 1.3.1. Primary hepatocytes 14 1.3.2. HepaRG cells 17 1.3.3. Hepatoma cell lines 17 1.3.4. Pluripotent stem cells 19 1.4. Hypothesis 20 2. Materials and Methods 22 2.1. Plasmid 22 2.2. Site-directed mutagenesis 23 2.3. Cell culture 24 2.4. In vitro HBV virion production and preparation 24 2.5. In vivo HBV virion production and preparation 25 2.6. HBV virion quantification 26 2.7. In vitro HBV infection 27 2.8. ELISA 28 2.9. Southern blot 28 2.10. Northern blot 30 2.11. Western blot 31 2.12. Native agarose gel 32 2.13. Immunofluorescence assay 33 2.14. Reverse transcription 33 3. Results 35 3.1. Establishment of an efficient HBV infection model in vitro. 35 3.2. Selecting HBV replicon for production of infectious virions 36 3.3. Splicing-deficient HBV is not essential for HBV replication in Huh7 transfection model in vitro. 37 3.4. Splicing-deficient HBV is not essential for HBV replication in HepG2-NTCP-C4 infection model in vitro. 39 3.5. Possible factors that affect the infectivity of splicing-deficient HBV in HepG2-NTCP-C4 infection model in vitro. 40 4. Discussion 42 5. References 46 6. Figures 54 6.1. HBV infection in adherent and non-adherent HepG2-NTCP-C4 cell line. 56 6.2. HBV virions produced from Huh7 transfected with pCMV-HBV (D) support productive HBV infection in HepG2-NTCP-C4 cell line. 60 6.3. HBV splicing is not essential for viral replication in Huh7 in vitro transfection model. 66 6.4. HBV splicing is not essential for viral replication in HepG2-NTCP-C4 in vitro infection model. 72 6.5. Balb/c-derived HBV was unable to infect HepG2-NTCP-C4 cell line. 77 6.6. Polyethylene glycol (PEG) 8000 does not significantly affect the infectivity of splicing-deficient HBV 81 6.7. Second-round infection is limited in HepG2-NTCP-C4 cell line. 83 7. Appendix 84 7.1. General life cycle of HBV 84 7.2. HBV splicing (I) 85 7.3. HBV splicing (II) 86 7.4. Plasmid map – pAAV-HBV (genotype A) 1.2 87 7.5. Plasmid map – pAAV-HBV (genotype D) 1.3 88 7.6. Plasmid map – pHBV (genotype D) 1.3 89 7.7. Plasmid map – pCMV-HBV (genotype D) 1.05 90 | |
| dc.language.iso | en | |
| dc.subject | HBV剪接 | zh_TW |
| dc.subject | 肝癌細胞感染模型 | zh_TW |
| dc.subject | 肝癌細胞轉染模型 | zh_TW |
| dc.subject | 慢性B型肝炎感染 | zh_TW |
| dc.subject | 人肝嵌合小鼠 | zh_TW |
| dc.subject | in vitro Huh7 transfection model | en |
| dc.subject | in vitro HepG2-NTCP-C4 infection model | en |
| dc.subject | humanized liver chimeric mice | en |
| dc.subject | HBV splicing | en |
| dc.subject | Chronic Hepatitis B infection | en |
| dc.title | 探討B型肝炎病毒剪接在HepG2-NTCP-C4感染模型中所扮演的角色 | zh_TW |
| dc.title | The role of HBV spliced variants in HBV in vitro infection in HepG2-NTCP-C4 cell line | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 106-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 陶秘華,葉秀慧 | |
| dc.subject.keyword | 慢性B型肝炎感染,HBV剪接,人肝嵌合小鼠,肝癌細胞感染模型,肝癌細胞轉染模型, | zh_TW |
| dc.subject.keyword | Chronic Hepatitis B infection,HBV splicing,humanized liver chimeric mice,in vitro HepG2-NTCP-C4 infection model,in vitro Huh7 transfection model, | en |
| dc.relation.page | 90 | |
| dc.identifier.doi | 10.6342/NTU201801930 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2018-07-26 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 微生物學研究所 | zh_TW |
| 顯示於系所單位： | 微生物學科所 | |
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| ntu-107-1.pdf 未授權公開取用 | 8.59 MB | Adobe PDF |
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