請用此 Handle URI 來引用此文件：
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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陳培哲(Pei-Jer Chen) | |
| dc.contributor.author | Jo-Hsi Chiang | en |
| dc.contributor.author | 江若熙 | zh_TW |
| dc.date.accessioned | 2021-06-17T02:19:28Z | - |
| dc.date.available | 2019-09-08 | |
| dc.date.copyright | 2017-09-08 | |
| dc.date.issued | 2017 | |
| dc.date.submitted | 2017-08-21 | |
| dc.identifier.citation | 1. Ganem, D. and A.M. Prince, Hepatitis B virus infection--natural history and clinical consequences. N Engl J Med, 2004. 350(11): p. 1118-29.
2. Chou, H.H., et al., Age-related immune clearance of hepatitis B virus infection requires the establishment of gut microbiota. Proc Natl Acad Sci U S A, 2015. 112(7): p. 2175-80. 3. Lee, C.M., et al., Age, gender, and local geographic variations of viral etiology of hepatocellular carcinoma in a hyperendemic area for hepatitis B virus infection. Cancer, 1999. 86(7): p. 1143-50. 4. Sang, S., et al., The Epidemiological Characteristics and Dynamic Transmission of Dengue in China, 2013. PLoS Negl Trop Dis, 2016. 10(11): p. e0005095. 5. Heermann, K.H., et al., Large surface proteins of hepatitis B virus containing the pre-s sequence. Journal of Virology, 1984. 52(2): p. 396-402. 6. Liao, W. and J.H. Ou, Phosphorylation and nuclear localization of the hepatitis B virus core protein: significance of serine in the three repeated SPRRR motifs. J Virol, 1995. 69(2): p. 1025-9. 7. Lan, Y.T., et al., Roles of the three major phosphorylation sites of hepatitis B virus core protein in viral replication. Virology, 1999. 259(2): p. 342-8. 8. Summers, J., A. O'Connell, and I. Millman, Genome of hepatitis B virus: restriction enzyme cleavage and structure of DNA extracted from Dane particles. Proc Natl Acad Sci U S A, 1975. 72(11): p. 4597-601. 9. Wang, N.J., et al., [Liver cancer, liver disease background and virus-like particles in serum among ducks from high incidence area of human hepatocellular carcinoma (author's transl)]. Zhonghua Zhong Liu Za Zhi, 1980. 2(3): p. 174-6. 10. Rabe, B., et al., Nuclear import of hepatitis B virus capsids and release of the viral genome. Proc Natl Acad Sci U S A, 2003. 100(17): p. 9849-54. 11. Sunbul, M., Hepatitis B virus genotypes: global distribution and clinical importance. World J Gastroenterol, 2014. 20(18): p. 5427-34. 12. Guirgis, B.S., R.O. Abbas, and H.M. Azzazy, Hepatitis B virus genotyping: current methods and clinical implications. Int J Infect Dis, 2010. 14(11): p. e941-53. 13. Mayerat, C., A. Mantegani, and P.C. Frei, Does hepatitis B virus (HBV) genotype influence the clinical outcome of HBV infection? J Viral Hepat, 1999. 6(4): p. 299-304. 14. Farza, H., et al., Replication and gene expression of hepatitis B virus in a transgenic mouse that contains the complete viral genome. J Virol, 1988. 62(11): p. 4144-52. 15. Milich, D.R., et al., Is a function of the secreted hepatitis B e antigen to induce immunologic tolerance in utero? Proc Natl Acad Sci U S A, 1990. 87(17): p. 6599-603. 16. Guidotti, L.G., et al., Hepatitis B virus nucleocapsid particles do not cross the hepatocyte nuclear membrane in transgenic mice. J Virol, 1994. 68(9): p. 5469-75. 17. Chisari, F.V., et al., Molecular pathogenesis of hepatocellular carcinoma in hepatitis B virus transgenic mice. Cell, 1989. 59(6): p. 1145-56. 18. Chisari, F.V., et al., A transgenic mouse model of the chronic hepatitis B surface antigen carrier state. Science, 1985. 230(4730): p. 1157-60. 19. Kim, C.M., et al., HBx gene of hepatitis B virus induces liver cancer in transgenic mice. Nature, 1991. 351(6324): p. 317-20. 20. Sun, S. and J. Li, Humanized chimeric mouse models of hepatitis B virus infection. Int J Infect Dis, 2017. 59: p. 131-136. 21. Huang, L.R., et al., An immunocompetent mouse model for the tolerance of human chronic hepatitis B virus infection. Proc Natl Acad Sci U S A, 2006. 103(47): p. 17862-7. 22. Wu, H.L., et al., Temporal aspects of major viral transcript expression in Hep G2 cells transfected with cloned hepatitis B virus DNA: with emphasis on the X transcript. Virology, 1991. 185(2): p. 644-51. 23. Zolotukhin, S., et al., A 'humanized' green fluorescent protein cDNA adapted for high-level expression in mammalian cells. J Virol, 1996. 70(7): p. 4646-54. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/68387 | - |
| dc.description.abstract | B型肝炎是由B型肝炎病毒感染所引發的肝發炎反應。儘管現行的預防性疫苗有效地降低了B型肝炎的發生率，但在1982年疫苗問世前已感染B型肝炎病毒的人口數量龐大；目前全球約有2.6億慢性B型肝炎帶原者，而每年約90萬人死於慢性B型肝炎病毒感染造成的併發症。B型肝炎病毒目前已知有十種基因型(基因型A至J)，各基因型除了具特定的地域性分佈外，每種基因型對抗病毒療法的免疫反應也都不盡相同。以四種最常見的B型肝炎基因型為例，基因型A分布全球，是最廣泛的一種B型肝炎病毒基因型；基因型B和C是亞洲最常見的B型肝炎病毒基因型；而基因型D則是基因型A之外在歐洲最常見的B型肝炎病毒基因型。由於特殊的地域性分布，這四種基因型的傳染途徑也因此而有些差異。亞洲最廣泛分佈的基因型B和C大多由母子垂直感染途徑傳播，因為大部分感染者年齡偏低，而慢性B型肝炎感染其中的要素即是年齡，故此，大部分亞洲地區的感染者在疫苗問世之前會轉變為慢性帶原者。相較於基因型B和C，基因型A與D的傳播途經大多是在成人間平行傳染，因此較不容易形成慢性帶原；即使如此，基因型A和D仍然存在著一些顯著的差異。在同個地區，同樣群體，藉由相同傳染途徑及感染年齡相仿的這些條件之下，感染基因A的群體卻比感染基因型D的群體容易轉變為慢性帶原者，但是在抗病毒藥物的治療下，基因型D的感染者對干擾素的反應卻遠不及基因型A。有鑑於此，本篇研究希望藉由高壓流體注射小鼠模式去探討基因型A與D這兩者在生物學上的異同以及可能的致病機轉，而本篇研究的假說為。實驗結果顯示，在高壓流體注射之後三天犧牲的C57BL/6小鼠肝臟樣本中，B型肝炎病毒基因型D核心蛋白的表現量顯著地高於基因型A，而這與血清中e抗原的表現量以及HuH-7細胞模式所得出的結果是相應的。相較於核心蛋白的表現量，在HuH-7細胞模式中，基因型A的表面抗原蛋白表現量是遠優於基因型D，這與兩個基因型在細胞培養液和小鼠血清中的表面抗原表現量相呼應。總括而言，B型肝炎病毒基因型A傾向表現較大量的表面抗原蛋白，而基因型D則較傾向表現較大量的核心抗原蛋白。雖然此二種特性與兩種基因型在臨床上的關聯性仍須更進一步的研究，現階段的發現能夠為後續兩基因型的研究提供一個有效的研究平台。 | zh_TW |
| dc.description.abstract | Hepatitis B virus (HBV) has long been a serious global public health threat. With 257 million worldwide chronic carriers, HBV prevails in a region-specific fashion, correlating to its genotypes as well as the means of transmission. Categorized by at least 8% genetic divergence from one another, ten HBV genotypes (A-J) have been identified to date, each with various subgenotypes. Among the four most common HBV genotypes (A-D), while genotype A is pandemic and genotype B and C rampage in Asia, genotype D is somewhat overlooked in spite of its predominance in the Mediterranean countries and the Middle East. Besides the most common transmission route difference (genotype B and C being vertical and genotype A and D horizontal), clinically genotype D displays genotype-specificity in both disease progression and treatment response. When comparing HBV genotype D with genotype A—both found in the same geographic region—patients with genotype D tend to show a weaker response to interferon treatment and a more severe acute liver disease such as fulminant hepatitis than those with genotype A. The well-established hydrodynamic-injection (HDI) technique was utilized here to create a transfection mouse model to investigate HBV genotype D against its genotype A counterpart. Judging by the clinical characteristics and previous scientific literature, I hypothesize that the HBV genotype D would display a lower tendency to persist in the HDI mouse model in comparison to its genotype A equivalent. The results showed that the core protein expression level of HBV genotype D in the HDI C57BL/6 mouse liver samples was about three times that of the genotype A samples. Such finding echoes the mouse serum titers of e-antigen (HBeAg) between the two genotypes as well as the HuH-7 cell model results. For surface protein expression, on the other hand, the HuH-7 cell model evidence suggested that HBV genotype A has about 25 times higher than that of genotype D in western blotting. However, the mouse serum samples and supernatant samples of culture HuH-7 cells only showed a two-to three-fold difference in HBsAg titer level between the two genotype (ELISA). This implies that such stark difference of surface protein expression in western blotting may be attributed to poor HBsAg antibody specificity that favors the recognition of HBV genotype A surface antigen over that of genotype D. Although more intensive studies are required to be carried out to draw meaningful connections linking the findings of this study to clinical significance of the two genotypes, the pAAV-HBV1.3 genotype D clone produced from this study can serve as a powerful tool for all future studies related to HBV genotype D in our laboratory. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T02:19:28Z (GMT). No. of bitstreams: 1 ntu-106-R04445133-1.pdf: 1876142 bytes, checksum: 487acabeaa0d3b048c0cf6c7d27b19a7 (MD5) Previous issue date: 2017 | en |
| dc.description.tableofcontents | Table of Contents
國立臺灣大學碩士學位論文口試委員會審定書 i 誌 謝 ii 中文摘要 III Abstract V CHAPTER 1: INTRODUCTION 1 1.1 Hepatitis B Virus (HBV) Overview 1 1.1.1 HBV Virology 4 1.1.2 Genotypes 7 1.2 Models Used for the Study of HBV 9 1.3 The Hypothesis of this Study 15 CHAPTER 2: MATERIAL AND METHODS 15 2.1 HBV plasmid construct and AAV vector construct 15 2.1.1 pAAV-HBV1.2 genotype A plasmid 16 2.1.2 pAAV-HBV1.3 genotype D plasmid 17 2.2 Hydrodynamically Injected (HDI) HBV Transfection Mouse Model 18 2.3 Western Blotting 18 2.3.1 Protein Sample Preparation 19 2.3.2 Western Blotting Procedures 22 2.4 Northern Blotting 25 2.4.1 RNA Sample Preparation 26 2.4.2 Northern Blotting Procedures 27 2.5 Southern Blotting 31 2.5.1 DNA Sample Preparation 32 2.5.2 Southern Blotting Procedures 33 CHAPTER 3: RESULTS 34 3.1 Cloning a pAAV-HBV1.3 Genotype D Construct 34 3.2 Comparing HBV Replication between Genotype A and Genotype D with the pAAV Vector background in the Transfected HuH-7 Cultured Cell Model 41 3.2.1 Western Blotting: HBV Replication at the Protein Level 41 3.2.2 Northern Blotting: HBV Replication at the RNA Level 45 3.2.3 Southern Blotting: HBV Replication at the DNA Level 46 3.3 Comparing HBV Replication between Genotype A and Genotype D with the pAAV Vector background in Hydrodynamically Injected Mouse Model 47 3.3.1 Western Blotting: HBV Replication at the Protein Level 48 3.3.2 Northern Blotting: HBV Replication at the RNA Level 49 3.3.3 Persistent HBV Mouse Model: HDI CBA mouse model 50 CHAPTER 4: DISCUSSIONS 51 4.1 Poor HBV Surface Protein Detection among HBV Genotype D Clones 51 4.2 Southern Blotting Troubleshooting 52 4.3 Factors influencing HBV persistency in the HDI persistence CBA mouse model 53 CHAPTER 5: SUMMARY 56 REFERENCE 59 FIGURES 61 Figure 1. Cloning a pAAV-HBV1.3 Genotype D Construct. 61 Figure 2. HBV Replication between Genotype A and Genotype D in the Transfected HuH-7 Cultured Cell Model 63 Figure 3. HBV Replication between Genotype A and Genotype D in HDI BALB/c and CBA Mouse Model 64 APPENDIX 65 Appendix 1. pAAV-HBV1.2 Genotype A Plasmid Construct 65 Appendix 2. pAAV-HBV1.3 Genotype D Construct 66 Appendix 3. HBV1.3 Genotype D (with endogenous promotor) Construct 67 Appendix 4. The Amino Acid Sequence Comparison between HBV Genotype A and Genotype D at the S Region. 68 Figure Index Figure 1. Cloning a pAAV-HBV1.3 Genotype D Construct. 61 Figure 2. HBV Replication between Genotype A and Genotype D in the Transfected HuH-7 Cultured Cell Model 63 Figure 3. HBV Replication between Genotype A and Genotype D in HDI BALB/c and CBA Mouse Model 64 | |
| dc.language.iso | en | |
| dc.subject | 高壓流體注射小鼠模式 | zh_TW |
| dc.subject | B型肝炎病毒A基因型 | zh_TW |
| dc.subject | B型肝炎病毒D基因型 | zh_TW |
| dc.subject | 表面抗原 | zh_TW |
| dc.subject | B型肝炎病毒基因型 | zh_TW |
| dc.subject | 核心抗原 | zh_TW |
| dc.subject | HBV genotype A | en |
| dc.subject | surface protein | en |
| dc.subject | HBV genotypes | en |
| dc.subject | core protein | en |
| dc.subject | hydrodynamic injection mouse model | en |
| dc.subject | HBV genotype D | en |
| dc.title | 以高壓流體注射之小鼠模式探討B型肝炎病毒基因型A與D之生物學差異 | zh_TW |
| dc.title | The Biology between Hepatitis B Virus Genotype A and D in the Hydrodynamically Injected Mouse Model | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 105-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 陶秘華(Mi-Hua Tao),葉秀慧(Shiou-Hwei Yeh) | |
| dc.subject.keyword | B型肝炎病毒基因型,B型肝炎病毒D基因型,B型肝炎病毒A基因型,高壓流體注射小鼠模式,表面抗原,核心抗原, | zh_TW |
| dc.subject.keyword | HBV genotypes,HBV genotype D,HBV genotype A,hydrodynamic injection mouse model,core protein,surface protein, | en |
| dc.relation.page | 68 | |
| dc.identifier.doi | 10.6342/NTU201704052 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2017-08-21 | |
| 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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