臺灣博碩士論文加值系統

B型肝炎病毒(HBV)是廣為流傳在世界的病毒之一。B型肝炎是常見的感染疾病，會造成慢性及急性肝炎，急性B型肝炎在成人有95%以上可以痊癒，而新生兒感染則多數會成為慢性帶原者。慢性B型肝炎病患會有較高的風險轉變為肝硬化及肝癌進而導致死亡，故B型肝炎傳染成為全球傳染病研究的重點之一。然而慢性B型肝炎感染一直沒有很好的動物模式可進行研究。為了研究慢性B型肝炎的機制，本實驗室利用高壓流體注射B型肝炎病毒複製子B6.2在FVB/N老鼠肝臟，發現可以成功建立B型肝炎病毒持續感染的動物模式；但若是將B6.2複製子的表面抗原(Hepatits B surface antigen, HBsAg)在單點Asn-214上突變成為Ser-214的新複製子，B6.2S，以同樣方式高壓流體注射，可使原本可在小鼠持續感染的病毒產生被清除的結果。故本實驗的目的是想要解析這個差異造成的機制。首先，我們將B6.2及B6.2S兩個複製子轉染於人類肝癌細胞株(Huh7細胞、HepG2細胞)或FVB/N老鼠肝臟細胞內，然後觀察HBsAg在細胞內的表現量與分泌至細胞外的量，結果發現B6.2S的表面抗原相較於B6.2的表面抗原，不易分泌至細胞外，以至於在細胞外以及血清中HBsAg表現量偏低。在先前的文獻中曾報導過B型肝炎病毒的s抗原可以抑制宿主Toll-like Receptor (TLR)訊息傳遞，而降低宿主抗病毒之先天性免疫反應，例如細胞激素以及發炎因子的產生下降。故我們假設在B6.2S小鼠中，可能因為s抗原在血清中的表現量較少，故未能有效抑制宿主免疫反應，造成B6.2S最終會被清除。但同時我們也很好奇這兩個只差一個胺基酸的s抗原在抑制先天性免疫反應的能力上是否有不同。所以我們以TLR2，4，9的配體(ligand)去刺激從老鼠肝臟分離出來的非實質細胞(non-parenchymal cells, NPC)，並同時加入純化的這兩種s抗原來檢查細胞激素以及發炎因子的產生是否不同。經由RT-qPCR的確認，我們發現B6.2及B6.2S的s抗原均會抑制TLR配體刺激活化NPC反應，減少IL-6及TNF-α的生成；B6.2S的s抗原的抑制能力還稍稍大於B6.2的s抗原，但沒有統計差異。此結果顯示B6.2及B6.2S這兩種複製子的s抗原皆具有相當的能力抑制先天性免疫反應，故這點不能用來解釋為何B6.2複製子在老鼠體內不會被清除，B6.2複製子會被清除的原因。 

Abstract

Hepatitis B virus (HBV) is one of the most prevalent viruses in the world. The infection of HBV is common in human and can lead to both acute and chronic liver diseases. More than 95% of acutely infected adults recover from the infection, whereas most of the neonatal transmitted infections become persistent. Chronic hepatitis B patients will have a higher risk of cirrhosis and liver cancer, which then lead to death. HBV infection has become a major research focus of global infectious diseases. Studying HBV persistence has long been hampered by the lacking of an immunocompetent animal model. Thus, the mechanism underlying the HBV chronicity has remained unclear. Our lab has established a HBV-persistent animal model in the FVB/N mice by hydrodynamic injection of an HBV replicon, B6.2, into the livers. Using this animal model, we have found that a single amino acid change at Asn-214 of clone B6.2 to become Ser-214, generating a new clone called B.2S, lead to a complete clearance of this new clone. This study aims to investigate the mechanism accounting for the difference between clones B6.2 and B6.2S. We first examined the expression of hepatitis B virus surface protein(HBsAg) of clones B6.2 and B6.2S in two hepatoma cell lines (Huh7 and HepG2) and in FVB/N mouse hepatocytes. Both in vitro and in vivo results demonstrated that, clone B6.2S expressed higher levels of intracellular HBsAg, whereas it secreted much lower levels of extracellular HBsAg, than clone B6.2. Previous studies have showed that the surface antigen of HBV can impair the activation of Toll-like Receptors (TLRs) pathways and inhibit the production of inflammatory cytokines, leading to suppression of host innate immunity. Hence, we speculate that the lower levels of extracellular HBsAg of clone B6.2S may cause less inhibition on host innate immunity, thus resulting in viral clearance. On the other hand, we were also curious about whether the surface antigens of B6.2 and B6.2S, differing only by one amino acid, might antagonize innate immunity differently. Therefore, we examined the cytokines gene (IL-6 and TNF-α) expression in the nonparachymal cells (NPC), which were isolated from FVB/N mice, in response to the treatment of TLR2, TLR4, and TLR9 ligands, in the presence or absence of B6.2-HBsAg or B6.2S-HBsAg. The results of RT-qPCR analysis showed that both B6.2-HBsAg and B6.2S-HBsAg exhibited inhibitory effects on the three TLR signaling pathways. Although B6.2S-HBsAg seemed to inhibit TLR signaling slightly more than B6.2-HBsAg, the difference was not statistically significant. Collectively, these results suggest that both B6.2-HBsAg and B6.2S-HBsAg have comparable inhibitory effects on host innate immunity, which thus cannot be the reason explaining the different persistent rates of both HBV clones.

目錄
致謝……i
中文摘要……ii
Abstract……iii
目錄……iv
圖目錄……v
第一章 緒論……1
一、 B型肝炎簡介……1
二、 B型肝炎之病毒學……6
三、 實驗室前人的研究……13
四、 實驗目的……15
第二章 實驗材料與方法……16
一、 實驗材料……16
二、 實驗方法……18
第三章 實驗結果……24
一、 B6.2S複製子較B6.2複製子分泌較少量之s抗原至細胞外……24
二、 較高量之細胞外s抗原及B6.2S之s抗原均容易造成HBV複製子的持續存在……24
三、 探討兩個s抗原(S-B6.2及S-B6.2S)，對TLR配體(ligand)刺激活化非實質性細胞(non-parenchymal cells, NPC)之影響……25
第四章 討論……28
參考文獻……32
圖表……37
附圖……43

1. World Health Organization. Hepatitis B. WHO, 2017. http://www.who.int/mediacentre/factsheets/fs204/en/.
2. Schweitzer A, Horn J, Mikolajczyk RT, Krause G, Ott JJ. Estimations of worldwide prevalence of chronic hepatitis B virus infection: a systematic review of data published between 1965 and 2013. Lancet. 2015;386(10003):1546-55.
3. WHO. Hepatitis B vaccines. Wkly Epidemiol Rec 2009;2009:405–20.
4. WHO 2015. WHO/UNICEF coverage estimates 2014 revision. July 2015. Immunization Vaccines and Biologicals, (IVB), Geneva, World Health Organization. Vaccine-preventable diseases monitoring system. http://www. who.int/gho/immunization/hepatitis/en/, accessed March 2016.
5. Ott JJ, Horn J, Krause G, Mikolajczyk RT. Time trends of chronic HBV infection over prior decades - A global analysis. J Hepatol 2017;66(1):48-54.
6. 林思鳳。台灣地區B型肝炎病毒之血清流行病學研究。行政院衛生署疾病管制局100 年度科技研究發展計畫，計畫編號：DOH100-DC-2041。
7. Sung JL. Hepatitis B virus infection and its sequelae in Taiwan. Gastroenterol Jpn 1984; 19(4): 363-6.
8. Chen DS, Sung JL. Hepatitis B virus infection on Taiwan. N Engl J Med 1977; 297(12): 668-9.
9. 衛生福利部疾病管制署。急性病毒性B型肝炎-專業人士版，2014-01-17。http://www.cdc.gov.tw/professional/info.aspx?treeid=4c19a0252bbef869&nowtreeid=4dc827595f55c334&tid=A84462013CFA5C1A.
10. Ganem, D. Hepatitis B virus infection - natural history and clinical consequences. N Engl J Med 2004; 350(11): 1118-29.
11. Liaw YF, Chu CM. Hepatitis B virus infection. Lancet 2009; 373(9663): 582-92.
12. http://www.wikidoc.org/index.php/File:HBV_serum_markers.png
13. Liang TJ. Hepatitis B: The virus and disease. Hepatology 2009; 49(5 Suppl): S13–S21.
14. Walter E, Keist R, Niederöst B, Pult I, Blum HE. Hepatitis B virus infection of tupaia hepatocytes in vitro and in vivo. Hepatology 1996; 24(1):1-5.
15. Sandhu P, Haque M, Humphries-Bickley T, Ravi S, Song J. Hepatitis B Virus immunopathology, model systems, and current therapies. Front Immunol 2017;8:436. doi: 10.3389/fimmu.2017.00436.
16. Dane DS, Cameron CH, Briggs M. Virus-like particles in serum of patients with Australia-antigen-associated hepatitis. Lancet 1970; 1(7649): 695–8.
17. Renae Walsh, Stephen Locarnini. Hepatitis B Precore Protein: Pathogenic Potential and Therapeutic Promise. Yonsei Med J 2012; 53(5): 875–85.
18. Minor MM, Slagle BL. Hepatitis B Virus HBx Protein interactions with the Ubiquitin Proteasome System. Viruses 2014; 6(11): 4683-702.
19. Das A, Maini MK. Innate and adaptive immune responses in hepatitis B virus infection. Dig Dis 2010; 28(1): 126-32.
20. Molla S. Review on human immune response against hepatitis B virus (HBV) infection. J Emerg Dis Virol 2016; 2(3): doi http://dx.doi. org/10.16966/2473-1846.117
21. Summers J, O'Connell A, Millman I. Genome of hepatitis B virus: restriction enzyme cleavage and structure of DNA extracted from Dane particles. Proc Natl Acad Sci USA 1975; 72(11): 4597-601.
22. Ou JH, Laub O, Rutter WJ. 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): 1578-82.
23. Mack DH, Bloch W, Nath N, Sninsky JJ. Hepatitis B virus particles contain a polypeptide encoded by the largest open reading frame: a putative reverse transcriptase. J Virol 1988; 62(12): 4786-90.
24. Seeger C, Mason WS. Mason, Hepatitis B virus biology. Microbiol Mol Biol Rev 2000; 64(1): 51-68.
25. Medzhitov R, Janeway C Jr. Innate immunity. N Engl J Med 2000; 343(5): 338-44.
26. Faure-Dupuy S, Lucifora J, Durantel D. Interplay between the Hepatitis B virus and innate immunity: from an understanding to the development of therapeutic concepts. Viruses 2017; 9(5). pii: E95. doi: 10.3390/v9050095.
27. Boehme KW, Compton T. Innate sensing of viruses by toll-like receptors. J Virol 2004; 78(15): 7867-73.
28. Ganem D, Varmus HE. The molecular biology of the hepatitis B viruses. Annu Rev Biochem 1987; 56: 651-93.
29. Revill P, Yuan Z. New insights into how HBV manipulates the innate immune response to establish acute and persistent infection. Antivir Ther 2013; 18(1): 1-15.
30. Foster GR, Ackrill AM, Goldin RD, Kerr IM, Thomas HC, Stark GR. Expression of the terminal protein region of hepatitis B virus inhibits cellular responses to interferons alpha and gamma and double-stranded RNA. Proc Natl Acad Sci USA 1991; 88(7): 2888-92.
31. Yu S, Chen J, Wu M, Chen H, Kato N, Yuan Z. Hepatitis B virus polymerase inhibits RIG-I- and Toll-like receptor 3-mediated beta interferon induction in human hepatocytes through interference with interferon regulatory factor 3 activation and dampening of the interaction between TBK1/IKKepsilon and DDX3. J Gen Virol 2010; 91(Pt 8): 2080-90.
32. Wang H, Ryu WS. Hepatitis B virus polymerase blocks pattern recognition receptor signaling via interaction with DDX3: implications for immune evasion. PLoS Pathog 2010; 6(7): e1000986.
33. Wei C, Ni C, Song T, Liu Y, Yang X, Zheng Z, Jia Y, Yuan Y, Guan K, Xu Y, Cheng X, Zhang Y, Yang X, Wang Y, Wen C, Wu Q, Shi W, Zhong H. The hepatitis B virus X protein disrupts innate immunity by downregulating mitochondrial antiviral signaling protein. J Immunol 2010; 185(2): 1158-68.
34. Kumar M, Jung SY, Hodgson AJ, Madden CR, Qin J, Slagle BL. Hepatitis B virus regulatory HBx protein binds to adaptor protein IPS-1 and inhibits the activation of beta interferon. J Virol 2011; 85(2): 987-95.
35. Rosmorduc O, Sirma H, Soussan P, Gordien E, Lebon P, Horisberger M, Bréchot C, Kremsdorf D. Inhibition of interferon-inducible MxA protein expression by hepatitis B virus capsid protein. J Gen Virol 1999; 80 ( Pt 5): 1253-62.
36. Fernandez M, Quiroga JA, Carreno V. Hepatitis B virus downregulates the human interferon-inducible MxA promoter through direct interaction of precore/core proteins. J Gen Virol 2003; 84(Pt 8): 2073-82.
37. Li N, Zhang L, Chen L, Feng W, Xu Y, Chen F, Liu X, Chen Z, Liu W. MxA inhibits hepatitis B virus replication by interaction with hepatitis B core antigen. Hepatology 2012; 56(3): 803-11.
38. Wu J, Meng Z, Jiang M, Pei R, Trippler M, Broering R, Bucchi A, Sowa JP, Dittmer U, Yang D, Roggendorf M, Gerken G, Lu M, Schlaak JF. Hepatitis B virus suppresses toll-like receptor-mediated innate immune responses in murine parenchymal and nonparenchymal liver cells. Hepatology 2009; 49(4): 1132-40.
39. Wu S, Kanda T, Imazeki F, Arai M, Yonemitsu Y, Nakamoto S, Fujiwara K, Fukai K, Nomura F, Yokosuka O. Hepatitis B virus e antigen downregulates cytokine production in human hepatoma cell lines. Viral Immunol 2010; 23(5): 467-76.
40. Xu Y, Hu Y, Shi B, Zhang X, Wang J, Zhang Z, Shen F, Zhang Q, Sun S, Yuan Z. HBsAg inhibits TLR9-mediated activation and IFN-alpha production in plasmacytoid dendritic cells. Mol Immunol 2009; 46(13): 2640-6
41. Shi B, Ren G, Hu Y, Wang S, Zhang Z, Yuan Z. HBsAg inhibits IFN-alpha production in plasmacytoid dendritic cells through TNF-alpha and IL-10 induction in monocytes. PLoS One 2012; 7(9): e44900.
42. Op den Brouw ML, Binda RS, van Roosmalen MH, Protzer U, Janssen HL, van der Molen RG, Woltman AM. Hepatitis B virus surface antigen impairs myeloid dendritic cell function: a possible immune escape mechanism of hepatitis B virus. Immunology 2009; 126(2): 280-9.
43. Zou ZQ, Wang L, Wang K, Yu JG. Innate immune targets of hepatitis B virus infection. World J Hepatol 2016; 8(17): 716–25.
44. Ma Z, Zhang E, Yang D, Lu M. Contribution of Toll-like receptors to the control of hepatitis B virus infection by initiating antiviral innate responses and promoting specific adaptive immune responses. Cell Mol Immunol. 2015; 12(3): 273–82.
45. Wang L, Wang K, Zou ZQ. Crosstalk between innate and adaptive immunity in hepatitis B virus infection. World J Hepatol 2015;7(30):2980-91.
46. Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immunity. Cell 2006; 124(4): 783-801.
47. Ma Z, Zhang E, Yang D, Lu M. Contribution of Toll-like receptorsto the control of hepatitis B virus infection by initiating antiviralinnate responses and promoting specific adaptive immune responses. Cell Mol Immunol 2015; 12(3): 273-82.
48. Lu X, Xu Q, Bu X, Ma X, Zhang F, Deng Q, Zhang Y, Ding J. Relationship between expression of toll-like receptors 2/4 in dendritic cells and chronic hepatitis B virus infection. Int J Clin Exp Pathol 2014; 7(9): 6048-55.
49. Wu J, Meng Z, Jiang M, Zhang E, Trippler M, Broering R, Bucchi A, Krux F, Dittmer U, Yang D, Roggendorf M, Gerken G, Lu M, Schlaak JF. Toll-like receptor-induced innate immune responses in non-parenchymal liver cells are cell type-specific. Immunology 2010; 129(3): 363-74.
50. Reynolds JM, Dong C. Toll-like receptor regulation of effector T lymphocyte function. Trends Immunol 2013; 34(10): 511-9.
51. Geng D, Zheng L, Srivastava R, Asprodites N, Velasco-Gonzalez C, Davila E. When Toll-like receptor and T-cell receptor signals collide: a mechanism for enhanced CD8 T-cell effector function. Blood 2010; 116(18): 3494-504.
52. Tabiasco J, Devêvre E, Rufer N, Salaun B, Cerottini JC, Speiser D, Romero P. Human effector CD8+ T lymphocytes express TLR3 as a functional coreceptor. J Immunol 2006; 177(12): 8708-13.
53. Babu S, Blauvelt CP, Kumaraswami V, Nutman TB. Cutting edge: diminished T cell TLR expression and function modulates the immune response in human filarial infection. J Immunol 2006; 176(7): 3885-9.
54. Mandraju R, Murray S, Forman J, Pasare C. Differential ability of surface and endosomal TLRs to induce CD8 T cell responses in vivo. J Immunol 2014; 192(9): 4303-15.
55. Chen SH, Wu HL, Kao JH, Hwang LW. Persistent hepatitis B viral replication in a FVB/N mouse model: impact of host and viral factors. PLoS One 2012; 7(5): e36984.
56. 陳詩蕙。於FVB/N小鼠建立B型肝炎病毒慢性帶原模式以研究宿主與病毒的影響因素。國立臺灣大學微生物學研究所博士論文，2012。
57. Morikawa K, Shimazaki T, Takeda R, Izumi T, Umumura M, Sakamoto N. Hepatitis B: progress in understanding chronicity, the innate immune response, and cccDNA protection. Ann Transl Med 2016;4(18):337.
58. Isogawa M, Robek MD, Furuichi Y, Chisari FV. Toll-like receptor signaling inhibits hepatitis B virus replication in vivo. J Virol 2005; 79(11): 7269-72.
59. Wu J, Lu M, Meng Z, Trippler M, Broering R, Szczeponek A, Krux F, Dittmer U, Roggendorf M, Gerken G, Schlaak JF. Toll-like receptor-mediated control of HBV replication by nonparenchymal liver cells in mice. Hepatology 2007; 46(6): 1769-78.
60. Thompson AJ, Colledge D, Rodgers S, Wilson R, Revill P, Desmond P, Mansell A, Visvanathan K, Locarnini S. Stimulation of the interleukin-1 receptor and Toll-like receptor 2 inhibits hepatitis B virus replication in hepatoma cell lines in vitro. Antivir Ther 2009; 14(6): 797-808.
61. Chang JJ, Lewin SR. Immunopathogenesis of hepatitis B virus infection. Immunol Cell Biol 2007; 85(1): 16-23.
62. Wang S, Chen Z, Hu C, Qian F, Cheng Y, Wu M, Shi B, Chen J, Hu Y, Yuan Z. 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): 5142-51.
63. 楊硯捷。探討BCL6參與在B型肝炎病毒持續複製老鼠模式中所扮演之角色以及B型肝炎病毒之s抗原對免疫反應之影響。國立陽明大學微生物及免疫學研究所碩士論文，2014。