請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/43910
完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 陳培哲 | |
dc.contributor.author | Mou-Mei Wang | en |
dc.contributor.author | 王慕梅 | zh_TW |
dc.date.accessioned | 2021-06-15T02:32:28Z | - |
dc.date.available | 2009-09-15 | |
dc.date.copyright | 2009-09-15 | |
dc.date.issued | 2009 | |
dc.date.submitted | 2009-08-14 | |
dc.identifier.citation | 1. McMahon, B.J., Epidemiology and natural history of hepatitis B. Semin Liver Dis, 2005. 25 Suppl 1: p. 3-8.
2. Chu, C.M. and Y.F. Liaw, HBsAg seroclearance in asymptomatic carriers of high endemic areas: appreciably high rates during a long-term follow-up. Hepatology, 2007. 45(5): p. 1187-92. 3. Yang, P.L., et al., Hydrodynamic injection of viral DNA: a mouse model of acute hepatitis B virus infection. Proc Natl Acad Sci U S A, 2002. 99(21): p. 13825-30. 4. Moriyama, T., et al., Immunobiology and pathogenesis of hepatocellular injury in hepatitis B virus transgenic mice. Science, 1990. 248(4953): p. 361-4. 5. Larkin, J., et al., Hepatitis B virus transgenic mouse model of chronic liver disease. Nat Med, 1999. 5(8): p. 907-12. 6. Liu, F., Y. Song, and D. Liu, Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA. Gene Ther, 1999. 6(7): p. 1258-66. 7. Milich, D.R. and F.V. Chisari, Genetic regulation of the immune response to hepatitis B surface antigen (HBsAg). I. H-2 restriction of the murine humoral immune response to the a and d determinants of HBsAg. J Immunol, 1982. 129(1): p. 320-5. 8. Milich, D.R., R.E. Louie, and F.V. Chisari, Genetic regulation of the immune response to hepatitis B surface antigen (HBsAg). V. T cell proliferative response and cellular interactions. J Immunol, 1985. 134(6): p. 4194-202. 9. Milich, D.R. and A. McLachlan, The nucleocapsid of hepatitis B virus is both a T-cell-independent and a T-cell-dependent antigen. Science, 1986. 234(4782): p. 1398-401. 10. 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. 11. Suzuki, T., et al., Intravenous injection of naked plasmid DNA encoding hepatitis B virus (HBV) produces HBV and induces humoral immune response in mice. Biochem Biophys Res Commun, 2003. 300(3): p. 784-8. 12. Kao, J.H., Hepatitis B viral genotypes: clinical relevance and molecular characteristics. J Gastroenterol Hepatol, 2002. 17(6): p. 643-50. 13. Kirschberg, O., et al., A multiplex-PCR to identify hepatitis B virus--enotypes A-F. J Clin Virol, 2004. 29(1): p. 39-43. 14. Norder, H., A.M. Courouce, and L.O. Magnius, Complete genomes, phylogenetic relatedness, and structural proteins of six strains of the hepatitis B virus, four of which represent two new genotypes. Virology, 1994. 198(2): p. 489-503. 15. Orito, E., et al., Geographic distribution of hepatitis B virus (HBV) genotype in patients with chronic HBV infection in Japan. Hepatology, 2001. 34(3): p. 590-4. 16. Kidd-Ljunggren, K., Y. Miyakawa, and A.H. Kidd, Genetic variability in hepatitis B viruses. J Gen Virol, 2002. 83(Pt 6): p. 1267-80. 17. Milich, D. and T.J. Liang, Exploring the biological basis of hepatitis B e antigen in hepatitis B virus infection. Hepatology, 2003. 38(5): p. 1075-86. 18. Chen, M., et al., Immune tolerance split between hepatitis B virus precore and core proteins. J Virol, 2005. 79(5): p. 3016-27. 19. Milich, D.R., et al., The secreted hepatitis B precore antigen can modulate the immune response to the nucleocapsid: a mechanism for persistence. J Immunol, 1998. 160(4): p. 2013-21. 20. Chan, H.L., M. Hussain, and A.S. Lok, Different hepatitis B virus genotypes are associated with different mutations in the core promoter and precore regions during hepatitis B e antigen seroconversion. Hepatology, 1999. 29(3): p. 976-84. 21. Chu, C.J., M. Hussain, and A.S. Lok, Hepatitis B virus genotype B is associated with earlier HBeAg seroconversion compared with hepatitis B virus genotype C. Gastroenterology, 2002. 122(7): p. 1756-62. 22. Yuen, M.F., et al., Significance of hepatitis B genotype in acute exacerbation, HBeAg seroconversion, cirrhosis-related complications, and hepatocellular carcinoma. Hepatology, 2003. 37(3): p. 562-7. 23. Locarnini, S., Molecular virology and the development of resistant mutants: implications for therapy. Semin Liver Dis, 2005. 25 Suppl 1: p. 9-19. 24. Yuen, M.F., et al., Role of hepatitis B virus genotypes Ba and C, core promoter and precore mutations on hepatocellular carcinoma: a case control study. Carcinogenesis, 2004. 25(9): p. 1593-8. 25. Chen, C.H., et al., Clinical significance of hepatitis B virus (HBV) genotypes and precore and core promoter mutations affecting HBV e antigen expression in Taiwan. J Clin Microbiol, 2005. 43(12): p. 6000-6. 26. Yeh, S.H., et al., Quantification and genotyping of hepatitis B virus in a single reaction by real-time PCR and melting curve analysis. J Hepatol, 2004. 41(4): p. 659-66. 27. Sugiyama, M., et al., Influence of hepatitis B virus genotypes on the intra- and extracellular expression of viral DNA and antigens. Hepatology, 2006. 44(4): p. 915-24. 28. Ito, K., et al., Characterization of genotype-specific carboxyl-terminal cleavage sites of hepatitis B virus e antigen precursor and identification of furin as the candidate enzyme. J Virol, 2009. 83(8): p. 3507-17. 29. Kobayashi, M., et al., Clinical features of hepatitis B virus genotype A in Japanese patients. J Gastroenterol, 2003. 38(7): p. 656-62. 30. Kobayashi, M., et al., Clinical characteristics of patients infected with hepatitis B virus genotypes A, B, and C. J Gastroenterol, 2002. 37(1): p. 35-9. 31. Livingston, S.E., et al., Clearance of hepatitis B e antigen in patients with chronic hepatitis B and genotypes A, B, C, D, and F. Gastroenterology, 2007. 133(5): p. 1452-7. 32. Ganem, D. and H.E. Varmus, The molecular biology of the hepatitis B viruses. Annu Rev Biochem, 1987. 56: p. 651-93. 33. Summers, J., J.M. Smolec, and R. Snyder, A virus similar to human hepatitis B virus associated with hepatitis and hepatoma in woodchucks. Proc Natl Acad Sci U S A, 1978. 75(9): p. 4533-7. 34. Barker, L.F., et al., Transmission of type B viral hepatitis to chimpanzees. J Infect Dis, 1973. 127(6): p. 648-62. 35. Bertoni, R., et al., Human class I supertypes and CTL repertoires extend to chimpanzees. J Immunol, 1998. 161(8): p. 4447-55. 36. Guidotti, L.G., et al., Viral clearance without destruction of infected cells during acute HBV infection. Science, 1999. 284(5415): p. 825-9. 37. Chisari, F.V., Hepatitis B virus gene expression in transgenic mice. Mol Biol Med, 1989. 6(2): p. 143-9. 38. Gunther, S., et al., A novel method for efficient amplification of whole hepatitis B virus genomes permits rapid functional analysis and reveals deletion mutants in immunosuppressed patients. J Virol, 1995. 69(9): p. 5437-44. 39. Guidotti, L.G., et al., High-level hepatitis B virus replication in transgenic mice. J Virol, 1995. 69(10): p. 6158-69. 40. Chen, M.T., et al., A function of the hepatitis B virus precore protein is to regulate the immune response to the core antigen. Proc Natl Acad Sci U S A, 2004. 101(41): p. 14913-8. 41. Scaglioni, P.P., M. Melegari, and J.R. Wands, Biologic properties of hepatitis B viral genomes with mutations in the precore promoter and precore open reading frame. Virology, 1997. 233(2): p. 374-81. 42. Moriyama, K., et al., Reduced precore transcription and enhanced core-pregenome transcription of hepatitis B virus DNA after replacement of the precore-core promoter with sequences associated with e antigen-seronegative persistent infections. Virology, 1996. 226(2): p. 269-80. 43. Cerny, A., C. Ferrari, and F.V. Chisari, The class I-restricted cytotoxic T lymphocyte response to predetermined epitopes in the hepatitis B and C viruses. Curr Top Microbiol Immunol, 1994. 189: p. 169-86. 44. Spandau, D.F. and C.H. Lee, trans-activation of viral enhancers by the hepatitis B virus X protein. J Virol, 1988. 62(2): p. 427-34. 45. Xu, Z., et al., Enhancement of hepatitis B virus replication by its X protein in transgenic mice. J Virol, 2002. 76(5): p. 2579-84. 46. Melegari, M., S.K. Wolf, and R.J. Schneider, Hepatitis B virus DNA replication is coordinated by core protein serine phosphorylation and HBx expression. J Virol, 2005. 79(15): p. 9810-20. 47. Colgrove, R., G. Simon, and D. Ganem, Transcriptional activation of homologous and heterologous genes by the hepatitis B virus X gene product in cells permissive for viral replication. J Virol, 1989. 63(9): p. 4019-26. 48. Chen, H.S., et al., The woodchuck hepatitis virus X gene is important for establishment of virus infection in woodchucks. J Virol, 1993. 67(3): p. 1218-26. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/43910 | - |
dc.description.abstract | B型肝炎病毒基因型B和C是包括我們台灣在內,亞洲常見的兩個基因型。根據臨床上的觀察,感染B型肝炎病毒基因型C會併發較嚴重的肝病,並有高危險率發展成肝癌。除此之外,在慢性感染過程當中,B型肝炎病毒基因型C的病人會有較高的比率在病毒核及核心抗原的啟動子發生突變,以及較高的比率發生核抗原反轉的延遲。因此,這兩項因素被認為是造成肝細胞癌的主因。
為了要探討B型肝炎病毒基因型B和C造成如此不同的免疫反應,我們使用實驗室已建立的小鼠免疫耐受模式,此模式是選用C57BL/6 小鼠以高壓注射法(Hydrodynamic injection)將帶有B型肝炎病毒基因並具有複製能力的質體pAAV/HBV1.2從尾靜脈導入老鼠體內。根據研究的結果,我們發現在打入B型肝炎病毒基因型B或C的小鼠表現差不多量的表面抗原和核抗原。此外,這兩個抗原的清除率在打入基因型B或C的小鼠上也是差不多的。至於產上抗體的情形,除了基因複製體 (clone) B7 以外,打入其他基因複製體的老鼠有百分之五十以上在核抗原消失的一個月後都尚未產生核抗體,這暗示著B型肝炎病毒基因型B和C在老鼠身上都產生延遲核抗原反轉的情形。為了觀察另一個影響核抗原反轉的因素,我們建立了帶有核抗原啟動子雙點突變的基因複製體。同樣以高壓注射法打入原生型和突變型的複製體做比較,我們發現打入啟動子突變型複製體的小鼠會產生較低的核抗原。此外,只有突變型複製體又帶有B型肝炎病毒基因型C的序列會在老鼠身上發生核抗原反轉延遲現象。令我們感到有趣的是,在表面抗原方面,打入突變型複製在體誘發老鼠大量表現後,表面抗原隨之快速被清除。 總結上述的實驗結果,我們發現在已建立的小鼠耐受模式下,B型肝炎病毒基因型B和C誘導產生相似的免疫反應於C57BL/6小鼠體內。對於研究的主要觀察目標-核抗原的反轉情形,在B型肝炎病毒基因型B和C的小鼠內都看到相同延遲的現象,並且只有B型肝炎病毒基因型C的啟動子突變型才有觀察到如同原生型的延遲反轉現象。然而這些結果並不能驗證臨床上病人感染B型肝炎病毒基因型B和C所看到的現象。未來我們所要設法改善的是要延長B型肝炎病毒在小鼠中的表現,比照臨床慢性感染的情形;並且要避免相同基因型的複製體間所造成的變異,如此才可避免我們比較B型肝炎病毒基因型B和C之間造成的誤差。 | zh_TW |
dc.description.abstract | HBV genotypes B and C are two common genotypes in Asian region, including Taiwan. According to clinical observation, genotype C infection is associated with more severe liver diseases and higher risks of hepatocellular carcinoma development. Besides, genotype C carriers have higher frequency of basal core promoter (BCP) mutation and delayed HBeAg seroconversion during chronic HBV infection. Therefore, it is suggested that the genotype C and BCP mutation are two main factors contributing to HCC development.
In order to explore the different immune responses between HBV genotypes B and C, we used the established immunocompetent mouse model, in which the C57BL/6 mice were introduced with the replication-competent plasmid pAAV/HBV1.2 by hydrodynamic injection. We found that C57BL/6 mice express the similar levels of serum HBsAg and HBeAg after injection of HBV genotypes B and C. In addition, the clearance rate of serum HBsAg and HBeAg were also similar in mice injected by HBV genotypes B and C. As to antibody production, with the exception of clone B7, over 50% mice had not generated serum anti-HBe more than one month since the HBeAg was cleared, indicating the delayed HBeAg seroconversion occurred in both HBV genotypes B and C. To investigate the other factor for HBeAg seroconversion, we constructed the clone with basal core promoter (BCP) double mutation. After hydrodynamic injection, comparing with the wild type, the mice injected by BCP mutants showed lower level of serum HBeAg. Moreover, the delayed HBeAg seroconversion was only observed when the mice were injected by the mutant clone of HBV genotype C. Interestingly, serum HBsAg level, on the other hand, declined rapidly after boosting in mice injected by BCP mutants of both genotypes B and C. Taken together, the overall expressions of HBV were not different significantly between HBV genotypes B and C in the C57BL/6 mice model, indicating their similar immune response induced after hydrodynamic injection. Moreover, regard to HBeAg seroconversion, mice injected by HBV genotypes B or C showed delayed HBeAg seroconversion, whereas only the mice injected by BCP mutant of genotype C could reproduce the same phenomenon relevant to the wild type. Therefore, these data suggested that we could not address the clinical observation of HBV genotypes B and C in this developed mouse model. Further studies are needed to prolong the HBV persistent expression of genotypes B and C in the mice and avoid clone-dependent variation for comparison. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T02:32:28Z (GMT). No. of bitstreams: 1 ntu-98-R96445116-1.pdf: 1802012 bytes, checksum: 63be06fe61319804f5cea9c9528479ea (MD5) Previous issue date: 2009 | en |
dc.description.tableofcontents | 1.Introduction…………………………………………………………………. 1
1.1 Natural history of HBV infection…………………………………................. 1 1.2 Hydrodynamic HBV animal study…………………………………………... 1 1.3 HBV genotype……………………………………………………………….. 3 1.4 Molecular biology of HBeAg………………………………………………... 5 1.5 Genotype B and C in HBeAg seroconversion………………………………... 6 1.6 Basal Core Promoter and Precore mutation during HBeAg seroconversion………………………………………………………………... 7 1.7 To reproduce the clinical feature of HBV genotypes B and C by the hydrodynamic-base mouse model……………………………………………. 9 2. Material and Methods……………………………………………............ 11 2.1 Patients……………………………………………………………………....... 11 2.2 HBV genotype………………………………………………………………... 11 2.3 Constructs…………………………………………………………………….. 12 2.4 Cell culture and transfection………………………………………………….. 12 2.5 Animal study………………………………………………………………...... 13 2.6 DIG-label HBV probe……………………………………………................... 13 2.7 Southern blotting…………………………………………………................... 14 2.8 Northern blotting…………………………………………………................... 15 2.9 Western blotting……………………………………………………………… 16 2.10 Detection of extracellular HBV DNA………………………………………. 17 2.11 Detection of serum HBV antigen and antibody…………………………….. 17 2.12 BCP T1762/A1764 mutant clone…………………………………………… 18 3. Result………………………………………………………………………… 20 3.1 Comparison of the replication, transcription and protein expression levels of HBV genotypes B and C in cell culture…………………………………………….. 20 3.1.1 Generation of six replication-competent constructs of HBV genotypes B and C…………………………………………………………. 20 3.1.2 Characterization of four major HBV transcripts in transfected Huh7 cells by Northern blotting…………………………………………………………. 21 3.1.3 Characterization of HBV replication intermediates in transfected Huh7 cells by Southern blotting…………………………………………………………. 22 3.1.4 Extracellular expression of HBV DNA in culture media of transfected Huh7 cells by real-time PCR……………………………………………………. 22 3.1.5 Intracellular expression of HBV antigens in transfected Huh7 cells by Western blotting…………………………………………………………………… 23 3.1.6 Extracellular expression of HBV antigens in culture media of transfected Huh7 cells by an EIA assay……………………………………………………... 23 3.2 Comparison of the replication, transcription and protein expression levels of HBV genotypes B and C in the established immunocompetent mouse model……... 24 3.2.1 The levels of replication, transcription and protein expression in the livers and sera of C57BL/6 mice after hydrodynamic injection of HBV genotypes B and C…………………………………………………………………………. 24 3.2.2 Six clones of HBV genotypes B and C were divided into three groups for comparison of HBsAg and HBeAg long-term expression in sera of C57BL/6 mice………………………………………………………………………. 25 3.2.3 HBeAg seroconversion in C57BL/6 mice injected by HBV genotypes B and C…………………………………………………………. 27 3.2.4 C57BL/6 mice injected by mix clones of HBV genotypes B and C respectively to follow the result of three groups’ comparisons………………………….. 27 3.3 Comparison of the differences of genome and amino acid sequence between HBV genotypes B and C……………………………………………………………. 28 3.3.1 The differences of genome and amino acid sequence in each open reading frame between HBV Genotypes B and C were analyzed by Mega4 program………………………………………………………………….. 28 3.3.2 Analysis of genome and amino acid difference of six clones in this study.. 29 3.3.3 Evaluation of DNA sequence divergence polymorphism of general HBV genotypes B and C……………………………………………………….. 30 3.4 The effect of basal core promoter mutation on HBeAg seroconversion in C57BL/6 mice after hydrodynamic injection……………………………….. 30 3.4.1 Establishment of basal core promoter mutant constructs of HBV genotype B and C……………………………………………………………………… 30 3.4.2 The replication, transcription and protein expression levels of wild-type HBV and their basal core promoter mutants in cell culture…………………….. 31 3.4.3 Comparison of wild type and basal core promoter mutants for HBeAg serconversion in C57BL/6 mice after hydrodynamic injection…………... 32 Discussion………………………………………………………………………… 34 References………………………………………………………………………… 42 Figures…………………………………………………………………………….. 46 Figure 1. Construction of HBV genotypes B and C pAAV/HBV1.2…………….. 46 Figure 2. HBV transcripts and replication intermediates in the Huh7 cell……….... 47 Figure 3. Intracellular and extracellular expression of HBV antigens……………... 48 Figure 4. HBV genotypes B and C expression in liver tissues and sera of injected C57BL/6 mice………………………………………………………….. 49 Figure 5. Long-term expression of serum HBsAg, HBeAg and anti-HBe of the C57BL/6 mice injected by HBV genotypes B and C DNA………….. 51 Figure 6. C57BL/6 mice injected by mix clones of HBV genotypes B and C to follow the three groups’ comparisons above………………………………… 54 Figure 7. The divergence polymorphism of whole genome sequence aligned between general HBV genotypes B and C………………………………………. 55 Figure 8. Construction of the basal core promoter mutant clone………………… 56 Figure 9. The HBV RNA, protein and DNA expression of wild type and basal core promoter mutants in Huh7 cell………………………………………… 56 Figure 10. Comparison of HBV expression in C57BL/6 mice receiving wild type and basal core promoter mutants by hydrodynamic injection…………….. 59 Tables…………………………………………………………………………….... 60 Table 1. Genotypes and clinical characteristics of the six chronic hepatitis B carriers……………………………………………………… 60 Table 2. The difference of nucleotide and amino acid sequence in each open reading frame between HBV genotypes B and C………………………………... 61 Table 3. Pairwise nucleotide difference (%) between different HBV clones…….. 62 Table 4. Pairwise amino acid difference (%) between different HBV clones…..... 63 Appendix………………………………………………………………………….. 65 Figure A1. The comparison of HBV persistent between FVB and C57BL/6 mice injected by the same HBV plasmid.………………………………… 65 Figure A2. The comparison of HBV persistent between male and female FVB mice.………………………………………………………………… 66 | |
dc.language.iso | en | |
dc.title | 以C57BL/6老鼠研究B型肝炎病毒基因型B和C所引起之免疫反應模式 | zh_TW |
dc.title | The pattern of immune response between HBV genotypes B and C in the C57BL/6 mouse model | en |
dc.type | Thesis | |
dc.date.schoolyear | 97-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 葉秀慧,王弘毅 | |
dc.subject.keyword | B型肝炎病毒基因型,核抗原反轉現象,高壓注射法,核抗原啟動子突變,C57BL/6小鼠, | zh_TW |
dc.subject.keyword | HBV genotype,HBeAg seroconversion,Hydrodynamic injection,BCP mutant,C57BL/6 mice, | en |
dc.relation.page | 66 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2009-08-14 | |
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 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-98-1.pdf 目前未授權公開取用 | 1.76 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。