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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 陳培哲(Pei-Jer Chen) | |
dc.contributor.author | Chang-Ru Wu | en |
dc.contributor.author | 吳章如 | zh_TW |
dc.date.accessioned | 2021-06-16T06:44:38Z | - |
dc.date.available | 2016-10-09 | |
dc.date.copyright | 2014-10-09 | |
dc.date.issued | 2014 | |
dc.date.submitted | 2014-07-28 | |
dc.identifier.citation | 1. Fung, S.K. and A.S. Lok, Hepatitis B virus genotypes: do they play a role in the outcome of HBV infection? Hepatology, 2004. 40(4): p. 790-2.
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Chisari, F.V., et al., Expression of hepatitis B virus large envelope polypeptide inhibits hepatitis B surface antigen secretion in transgenic mice. J Virol, 1986. 60(3): p. 880-7. 15. Ou, J.H. and W.J. Rutter, Regulation of secretion of the hepatitis B virus major surface antigen by the preS-1 protein. J Virol, 1987. 61(3): p. 782-6. 16. Dienstag, J.L., Hepatitis B virus infection. N Engl J Med, 2008. 359(14): p. 1486-500. 17. Garcia, T., et al., Drastic reduction in the production of subviral particles does not impair hepatitis B virus virion secretion. J Virol, 2009. 83(21): p. 11152-65. 18. Li, X.D., et al., Hepatitis C virus protease NS3/4A cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity. Proc Natl Acad Sci U S A, 2005. 102(49): p. 17717-22. 19. Wieland, S., et al., Genomic analysis of the host response to hepatitis B virus infection. Proc Natl Acad Sci U S A, 2004. 101(17): p. 6669-74. 20. Guy, C.S., et al., Intrahepatic expression of genes affiliated with innate and adaptive immune responses immediately after invasion and during acute infection with woodchuck hepadnavirus. J Virol, 2008. 82(17): p. 8579-91. 21. Fletcher, S.P., et al., Identification of an intrahepatic transcriptional signature associated with self-limiting infection in the woodchuck model of hepatitis B. Hepatology, 2013. 57(1): p. 13-22. 22. Dunn, C., et al., Temporal analysis of early immune responses in patients with acute hepatitis B virus infection. Gastroenterology, 2009. 137(4): p. 1289-300. 23. Yan, H., et al., Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus. Elife, 2012. 1: p. e00049. 24. Cheng, J., et al., Recombinant HBsAg inhibits LPS-induced COX-2 expression and IL-18 production by interfering with the NFkappaB pathway in a human monocytic cell line, THP-1. J Hepatol, 2005. 43(3): p. 465-71. 25. Wang, S., et al., Hepatitis B virus surface antigen selectively inhibits TLR2 ligand-induced IL-12 production in monocytes/macrophages by interfering with JNK activation. J Immunol, 2013. 190(10): p. 5142-51. 26. Xu, Y., et al., HBsAg inhibits TLR9-mediated activation and IFN-alpha production in plasmacytoid dendritic cells. Mol Immunol, 2009. 46(13): p. 2640-6. 27. Jan, R.H., et al., Hepatitis B virus surface antigen can activate dendritic cells and modulate T helper type immune response. Microbiol Immunol, 2011. 55(1): p. 51-9. 28. Zeissig, S., et al., Hepatitis B virus-induced lipid alterations contribute to natural killer T cell-dependent protective immunity. Nat Med, 2012. 18(7): p. 1060-8. 29. Heath, W.R., et al., Cross-tolerance: a pathway for inducing tolerance to peripheral tissue antigens. J Exp Med, 1998. 187(10): p. 1549-53. 30. Limmer, A., et al., Efficient presentation of exogenous antigen by liver endothelial cells to CD8+ T cells results in antigen-specific T-cell tolerance. Nat Med, 2000. 6(12): p. 1348-54. 31. Takahashi, K., et al., Immunochemical structure of hepatitis B e antigen in the serum. J Immunol, 1983. 130(6): p. 2903-7. 32. Ou, J.H., O. Laub, and W.J. Rutter, Hepatitis B virus gene function: the precore region targets the core antigen to cellular membranes and causes the secretion of the e antigen. Proc Natl Acad Sci U S A, 1986. 83(6): p. 1578-82. 33. Standring, D.N., et al., A signal peptide encoded within the precore region of hepatitis B virus directs the secretion of a heterogeneous population of e antigens in Xenopus oocytes. Proc Natl Acad Sci U S A, 1988. 85(22): p. 8405-9. 34. Visvanathan, K., et al., Regulation of Toll-like receptor-2 expression in chronic hepatitis B by the precore protein. Hepatology, 2007. 45(1): p. 102-10. 35. Wei, X., et al., Abnormal fecal microbiota community and functions in patients with hepatitis B liver cirrhosis as revealed by a metagenomic approach. BMC Gastroenterol, 2013. 13: p. 175. 36. Li, J., et al., Exosomes mediate the cell-to-cell transmission of IFN-alpha-induced antiviral activity. Nat Immunol, 2013. 14(8): p. 793-803. 37. Dreux, M., et al., Short-range exosomal transfer of viral RNA from infected cells to plasmacytoid dendritic cells triggers innate immunity. Cell Host Microbe, 2012. 12(4): p. 558-70. 38. Xu, L., et al., Liver type I regulatory T cells suppress germinal center formation in HBV-tolerant mice. Proc Natl Acad Sci U S A, 2013. 110(42): p. 16993-8. 39. Lin, Y.J., et al., Hepatitis B virus core antigen determines viral persistence in a C57BL/6 mouse model. Proc Natl Acad Sci U S A, 2010. 107(20): p. 9340-5. 40. Boni, C., et al., Characterization of hepatitis B virus (HBV)-specific T-cell dysfunction in chronic HBV infection. J Virol, 2007. 81(8): p. 4215-25. 41. Lanford, R.E., et al., GS-9620, an oral agonist of Toll-like receptor-7, induces prolonged suppression of hepatitis B virus in chronically infected chimpanzees. Gastroenterology, 2013. 144(7): p. 1508-17, 1517 e1-10. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/57402 | - |
dc.description.abstract | B型肝炎(Hepatitis B)是由B型肝炎病毒(Hepatitis B virus, HBV)所引起的一種具有潛在性危及生命的肝臟感染症。據世界衛生組織(World Health Organization, WHO)估計,全世界目前約有二億四千萬人罹患慢性B型肝炎,因此B型肝炎是一個不容忽視的全球性公共衛生議題。B型肝炎病毒轉譯出的表面抗原(HBsAg)計有大型(large, L)、中型(medium, M)、小型(small, S)等三種抗原型態。HBsAg除了會構成病毒表面的外套膜(envelope)之外,也會自我組裝形成不具感染性的次病毒顆粒(subviral particles),因其不含HBV核酸之故。根據臨床研究發現,在感染HBV的病人血清中,次病毒顆粒產生的數量遠多於具感染性的B型肝炎病毒,約達1,000~10,000倍之多。目前廣為醫界所接受的學說,認為造成慢性B型肝炎感染的主要原因是感染者對HBV病毒所產生的免疫反應不足所導致,部分可能與病毒的免疫抑制功能有關。過去一些研究顯示,HBsAg抗原可能會抑制免疫細胞之功能。本實驗室建構了一種利用高壓注射法(hydrodynamic injection)將B型肝炎病毒核酸送入肝細胞的動物模式,使B型肝炎病毒得以長期持續在小鼠的肝臟細胞內表現,藉以模擬人類感染B型肝炎病毒後慢性帶原的現象。利用此一技術,我們希望藉由觀察小鼠對B型肝炎清除能力的改變,來探討B型肝炎病毒表面抗原的免疫調節功能。
在本研究中,我們利用B型肝炎病毒表面抗原的抗體,將肝臟細胞內以及肝臟細胞外的自由型表面抗原(free HBsAg)短暫地清除,再來偵測血清中的B型肝炎病毒e抗原(HBeAg)與麩丙酮酸轉胺脢(Alanine aminotransferase, ALT)的濃度有無改變。實驗結果顯示,利用抗體中和肝臟細胞外或肝臟細胞內的free HBsAg後,HBsAg抗原在血清中會急遽地下降,但大約4周後又會回復到和對照組相同的濃度,而HBeAg抗原也持續存在。本研究說明了利用抗體短暫地中和小鼠肝臟細胞內或外的自由型表面抗原並不會影響到小鼠對B型肝炎的清除。未來,希望利用各種不同HBsAg抗原的突變體找出其可能的免疫調節功能。 | zh_TW |
dc.description.abstract | Hepatitis B is a potentially life-threatening persistent liver infection caused by the hepatitis B virus (HBV). According to the latest estimates of World Health Organization (WHO), more than 240 million people have chronic hepatitis B infection in the world. Therefore, it is a major global health problem. The HBV viral envelope consists of three related hepatitis B surface Antigens (HBsAg), the large (L), medium (M) and small (S) ones. In addition to the formation of viral envelope, the HBsAg can also be secreted as the non-infectious subviral particles, in which no viral genome exists. Based on clinical findings, the subviral particles are typically expressed 1,000~10,000 times higher than infectious virions in the sera of patients with HBV infection. Currently, it is widely accepted that chronic HBV infection is due to ineffective host antiviral immune response against HBV infection, which is thought to be partly caused by immune-suppressive viral molecules. Previous studies suggested that HBsAg, the viral envelope protein, could inhibit the function of immune cells. In the past, our laboratory has successfully established a technique called “hydrodynamic injection” to bring the nucleic acids of HBV into mouse hepatocytes, in which HBV proteins and viral particles can express and last for a long time, to mimic the human chronic HBsAg carriers. By using this technique, we hope to investigate the role of HBsAg in immune regulation through the observation of the clearance of HBsAg in mice.
In this study, hydrodynamic injection of HBV DNA-expressing plasmid in CBA mice was conducted, and then anti-HBsAg antibody with two different concentrations was injected to block extracellular free HBsAg particles 24 hours later. In addition, HBV DNA plasmid along with anti-HBsAg antibody were hydrodynamically co-injected into hepatocytes. The levels of serum HBeAg and alanine aminotransferase(ALT) were determined after antibody injection. The results showed that the serum HBsAg levels were immediately depleted in both experiments. However, after four weeks the HBsAg levels bound back to control group level in both groups. The mice remained persistently seropositive for HBsAg for fourteen weeks in the extracellular group. The results suggested that a transient inhibition of extracellular free HBsAg particles and hydrodynamically co-injected with anti-HBsAg antibody along with HBV plasmid will not affect HBV persistence in CBA mice. In the future, we hope continue the study to understand more clearly the immune regulation mechanism in which the HBsAg involves by use of HBV mutants. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T06:44:38Z (GMT). No. of bitstreams: 1 ntu-103-R01445109-1.pdf: 2427916 bytes, checksum: d0dd3a14f2c7465c97a9e08719e62cfc (MD5) Previous issue date: 2014 | en |
dc.description.tableofcontents | 國立台灣大學(碩)博士學位論文口試委員審定書 I
致謝 II 中文摘要 III Abstract V 1 Introduction 1 1.1 Background information of HBV infection 1 1.2 Animal models for HBV infection 2 1.3 HBV hydrodynamic mouse model 4 1.4 Molecular biology of HBV surface antigen (HBsAg) 5 1.5 Innate immunity in HBV infection 7 1.6 Innate immune functions of HBV surface antigen 9 1.7 Molecular biology of HBV e antigen 10 1.8 The hypothesis of the study 11 2 Materials and methods 13 2.1 HBV plasmid construct 13 2.2 Hydrodynamic HBV transfection mouse model 13 2.3 Monoclonal antibodies 14 2.4 Detecting of cytokine 14 2.5 Cell culture and transfection 15 2.6 Northern blotting 16 2.7 Western blotting 16 2.8 Frozen sections 17 2.9 Immunohistochemistry stain 18 3 Results 20 3.1 Transient neutralization of extracellular free HBV surface antigen with anti-HBsAg Ab in CBA/Caj mice 20 3.1.1 Determination of serum HBsAg levels in CBA/Caj mice after E6F6 injection 20 3.1.2 Determination of serum HBeAg, anti-core Ab, and anti-e Ab levels in CBA/Caj mice after E6F6 injection 20 3.2 HBV-carrier CBA mice treated with antibiotics and E6F6 21 3.3 Hydrodynamical co-injection of HBV plasmids along with anti- HBsAg antibody E6F6 22 3.3.1 Determination of serum HBsAg level in CBA/Caj mice after E6F6 injection 22 3.3.2 Determination of serum HBeAg, ALT, anti-core Ab, and anti-e Ab levels in CBA/Caj mice after E6F6 injection 24 3.3.3 Long-term neutralization of extracellular free HBV surface antigen with anti-HBsAg Ab in CBA/Caj mice 25 3.3.4 Determination of cytokines during the period of HBsAg was neutralized by E6F6 25 3.4 Neutralization of extracellular free HBV surface antigen with anti-HBsAg Ab after hydrodynamic injection of e-null HBV mutant 26 3.5 Phenotypes in Huh7 cells of HBV surface antigen mutants 27 3.5.1 RNA expression of HBV surface antigen mutants 27 3.5.2 Protein expression of HBV surface antigen mutants 27 3.5.3 Extracellular secretion of hepatitis B surface antigen mutants 28 4 Discussion 29 4.1 Transient neutralization of free circulating hepatitis B surface antigens does not increase the HBV clearance rate in CBA mice 29 4.2 Delivery of anti-S antibody into hepatocytes by hydrodynamic injection does not affect HBV persistence rate in CBA mice 30 4.3 HBV e antigen may interfere with the HBsAg production by unknown mechanism 33 5 References 36 6 Figures 39 Figure 1 Neutralization of extracellular HBsAg with anti- HBsAg antibody E6F6 did not affect HBV persistence in CBA mice transfected by hydrodynamic injection of HBV plasmids 39 Figure 2 Antibiotic cocktail treatment reduced the HBsAg level in CBA-HBV-carrier mice 43 Figure 3 Hydrodynamically co-injection of HBV plasmids along with anti- HBsAg antibody E6F6 did not affect HBV persistence in CBA mice 46 Figure 4 Anti-HBcAg antibody enters into hepatocytes by hydrodynamic injection 50 Figure 5 HBsAg not affect IFN-γ and IL-10 production in CBA 53 Figure 6 HBV e antigen not affect HBV clearance in CBA mice 56 Figure 7 The protein expression of HBsAg mutants in vitro 58 Figure 8 Secreted HBsAg of HBV surface antigen mutants at 96 hours post-transfection in Huh7 cells 60 7 Tables 61 Table 1. Mutants of plasmid pAAV-HBV1.2 designed at different positions of nucleotide substitutions and insertions 61 Table 2. Primers of real-time PCR 63 8 Appendix 64 Appendix 1 HBV persistent rate and period in the different mouse strains 64 Appendix 2 The HBV plasmid construct 65 | |
dc.language.iso | en | |
dc.title | 以CBA小鼠模式探討中和肝細胞內外次病毒表面抗原顆粒對B型肝炎病毒持續存在之影響 | zh_TW |
dc.title | Effects of neutralization of extracellular and intracellular HBsAg subviral particles for HBV persistence in CBA mice model | en |
dc.type | Thesis | |
dc.date.schoolyear | 102-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 楊宏志,顧家綺 | |
dc.subject.keyword | 慢性B型肝炎,先天性免疫,B型肝炎表面抗原,B型肝炎e抗原,高壓注射小鼠模式, | zh_TW |
dc.subject.keyword | Chronic hepatitis B,Innate immunity,Hepatitis B virus surface antigen,Hepatitis B virus e antigen,hydrodynamic injection mouse model, | en |
dc.relation.page | 65 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2014-07-28 | |
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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