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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 | Chia-Hui Lin | en |
dc.contributor.author | 林珈卉 | zh_TW |
dc.date.accessioned | 2021-06-15T12:30:46Z | - |
dc.date.available | 2016-08-26 | |
dc.date.copyright | 2016-08-26 | |
dc.date.issued | 2016 | |
dc.date.submitted | 2016-08-04 | |
dc.identifier.citation | 1.Schaefer, S., Hepatitis B virus: significance of genotypes. J Viral Hepat, 2005.12(2): p. 111-24.
2.Beasley, R.P., Hepatitis B virus. The major etiology of hepatocellular carcinoma.Cancer, 1988. 61(10): p. 1942-56. 3.Seeger, C. and W.S. Mason, Hepatitis B virus biology. Microbiol Mol Biol Rev, 2000. 64(1): p. 51-68. 4.Blumberg, B.S., et al., A serum antigen (Australia antigen) in Down's syndrome, leukemia, and hepatitis. Ann Intern Med, 1967. 66(5): p. 924-31. 5.Schaefer, S., Hepatitis B virus taxonomy and hepatitis B virus genotypes. World J Gastroenterol, 2007. 13(1): p. 14-21. 6.Dane, D.S., C.H. Cameron, and M. Briggs, Virus-like particles in serum of patients with Australia-antigen-associated hepatitis. Lancet, 1970. 1(7649): p. 695-8. 7.Heermann, K.H., et al., Immunogenicity of the gene Sand Pre-S domains in hepatitis B virions and HBsAg filaments. Intervirology, 1987. 28(1): p. 14-25. 8.Urban, S., et al., Strategies to inhibit entry of HBV and HDV into hepatocytes.Gastroenterology, 2014. 147(1): p. 48-64. 9.Huang, H.C., et al., Entry of hepatitis B virus into immortalized human primary hepatocytes by clathrin-dependent endocytosis. J Virol, 2012. 86(17): p. 9443- 53. 10.Beck, J. and M. Nassal, Hepatitis B virus replication. World J Gastroenterol, 2007. 13(1): p. 48-64. 11.Watanabe, T., et al., Involvement of host cellular multivesicular body functions in hepatitis B virus budding. Proc Natl Acad Sci U S A, 2007. 104(24): p. 10205- 10. 12.Allweiss, L. and M. Dandri, Experimental in vitro and in vivo models for the study of human hepatitis B virus infection. J Hepatol, 2016. 64(1 Suppl): p. S17- 31. 13.Yan, H., et al., Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus. Elife, 2012. 1: p. e00049. 14.Ni, Y., et al., Hepatitis B and D viruses exploit sodium taurocholate co- transporting polypeptide for species-specific entry into hepatocytes.Gastroenterology, 2014. 146(4): p. 1070-83. 15.Le Seyec, J., et al., Infection process of the hepatitis B virus depends on the presence of a defined sequence in the pre-S1 domain. J Virol, 1999. 73(3): p. 2052-7. 16.Glebe, D., et al., Mapping of the hepatitis B virus attachment site by use of infection-inhibiting preS1 lipopeptides and tupaia hepatocytes. Gastroenterology, 2005. 129(1): p. 234-45. 17.Drexler, J.F., et al., Bats carry pathogenic hepadnaviruses antigenically related to hepatitis B virus and capable of infecting human hepatocytes. Proc Natl Acad Sci U S A, 2013. 110(40): p. 16151-6. 18.De Falco, S., et al., N-terminal myristylation of HBV preS1 domain enhances receptor recognition. J Pept Res, 2001. 57(5): p. 390-400. 19.Le Seyec, J., et al., Role of the pre-S2 domain of the large envelope protein in hepatitis B virus assembly and infectivity. J Virol, 1998. 72(7): p. 5573-8. 20.Le Duff, Y., M. Blanchet, and C. Sureau, The pre-S1 and antigenic loop infectivity determinants of the hepatitis B virus envelope proteins arefunctionally independent. J Virol, 2009. 83(23): p. 12443-51. 21.Abou-Jaoude, G. and C. Sureau, Entry of hepatitis delta virus requires the conserved cysteine residues of the hepatitis B virus envelope protein antigenic loop and is blocked by inhibitors of thiol-disulfide exchange. J Virol, 2007. 81(23): p. 13057-66. 22.Konig, A., et al., Kinetics of the bile acid transporter and hepatitis B virus receptor Na+/taurocholate cotransporting polypeptide (NTCP) in hepatocytes. J Hepatol, 2014. 61(4): p. 867-75. 23.Iwamoto, M., et al., Evaluation and identification of hepatitis B virus entry inhibitors using HepG2 cells overexpressing a membrane transporter NTCP. Biochem Biophys Res Commun, 2014. 443(3): p. 808-13. 24.Peng, L., et al., The p.Ser267Phe variant in SLC10A1 is associated with resistance to chronic hepatitis B. Hepatology, 2015. 61(4): p. 1251-60. 25.Meier, A., et al., Myristoylated PreS1-domain of the hepatitis B virus L-protein mediates specific binding to differentiated hepatocytes. Hepatology, 2013. 58(1): p. 31-42. 26.Gripon, P., C. Diot, and C. Guguen-Guillouzo, Reproducible high level infection of cultured adult human hepatocytes by hepatitis B virus: effect of polyethylene glycol on adsorption and penetration. Virology, 1993. 192(2): p. 534-40. 27.Ladner, S.K., et al., Inducible expression of human hepatitis B virus (HBV) in stably transfected hepatoblastoma cells: a novel system for screening potential inhibitors of HBV replication. Antimicrob Agents Chemother, 1997. 41(8): p. 1715-20. 28.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. 29.Zhou, T., et al., Hepatitis B virus e antigen production is dependent upon covalently closed circular (ccc) DNA in HepAD38 cell cultures and may serve as a cccDNA surrogate in antiviral screening assays. Antiviral Res, 2006. 72(2): p. 116-24. 30.Schulze, A., et al., Fine mapping of pre-S sequence requirements for hepatitis B virus large envelope protein-mediated receptor interaction. J Virol, 2010. 84(4): p.1989-2000 | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/50142 | - |
dc.description.abstract | B 型肝炎病毒為急性肝炎(acute hepatitis)或慢性肝炎(chronic hepatitis)的主原 因之一,全世界約有 20 億人口感染過 B 型肝炎病毒,其中有多於 3 億 5 千萬人 為 B 肝表面抗原(HBsAg)陽性的慢性帶原者,而慢性肝炎有極高的比例演變成肝 硬化(cirrhosis)或是肝癌 hepatocellular carcinoma (HCC),其死亡率高達 25%。重 要的是,B 型肝炎慢性患者均無法靠目前的藥物痊癒。B 型肝炎感染肝細胞有許 多機制目前都還不是很清楚,我們必需要建立一個穩定的 B 型肝炎感染系統,用 以研究這些未知的機制並找到慢性 B 肝可能的解藥。過去以來,B 型肝炎在細胞 模式上的感染一直都困難重重,要提升感染率就必須要額外添加 PEG 及 DMSO。 直到發現 HBV 受體 NTCP,這對細胞模式上的感染來說是相當大的進展。而最 近也發現除了 NTCP 以外應該還有其他宿主蛋白會幫助 HBV 進入細胞之後的進 程,使 HBV 可以成功感染肝細胞。因此我們希望可以朝著找到其他影響 HBV 感 染的宿主因子,首先第一步就是要建立一個穩定的細胞模式感染系統。而我們所 使用的是廣為人知的 HepAD38 細胞株,這是一個會自己產生 HBV 的細胞株。 我們以西方墨點法來分析 HepAD38 細胞內的蛋白,發現看不到任何表面蛋白的 訊號,為了找到原因,我們以西方、北方墨點法及細胞培養上的改變去分析 HepAD38 的表面蛋白。最後發現,在 HepAD38 誘導後繼代培養對於 HepAD38 表面抗原之表現有極大的影響,並且發現表面抗原的抗體 13H10 無法辨識到 D 基因型的 HBV。我們也試了幾支不同的抗體,找到了 E11E4 和 Bioss 是可以辨 識 D 基因型 HBV 的表面抗原。簡單來說,沒有繼代對於 HepAD38 表面蛋白的 產生來說是有幫助的,加上使用可以辨認 D 基因型表面蛋白的抗體,使之在西 方墨點法上可以測得 D 基因型表面蛋白的表現。 | zh_TW |
dc.description.abstract | Among the 2 billion global populations once infected with Hepatitis B virus (HBV), 350 million people remain HBV positive carriers. These chronic Hepatitis B (CHB) patients have high risks of developing more severe forms of liver diseases, such as liver cirrhosis and hepatocellular carcinoma (HCC), with a mortality rate of 25%. More importantly, these HBV long-term carriers stay uncured, with relapses andreoccurrences, even under current anti-viral treatments. In order to find possible cure to HBV, establishing a stable HBV infection cell model system is required to investigate the many unknown HBV pathways. However, over the past few years, HBV infection has faced many limitations in cell culture. Although HBV infection can be achieved by adding the two essential components PEG and DMSO, the infection rate was greatly improved after the discovery of the HBV receptor, NTCP. Such discovery took a huge leap forward not only for HBV infection in cell culture, but also for other HBV-related studies. Recently, some research pointed out that other host factors, in addition to NCTP, may contribute to sequential viral pathways, specifically related to viral-host interactions, in both hepatocytes and culture cell lines after HBV entry. Therefore, we aim to examine other possible factors that can affect HBV infection. First of all, to establish a stable infection system in cell culture, HepAD38, a widely known cell line that can produce HBV after induction in tet-off system, was used to serve as the viral source for this study. To confirm that HepAD38 can synthesize functional infectious particles as indicated in previous literature, the protein extract from the cell line was examined using Western Blot, but the results showed no surface protein. A series of experiments were carried out to investigate why there was no surface protein in western blot. I hypothesize that the antibody affinity to recognize HBsAg epitopes, and the cell culture methods are possible factors to impact surface antigen protein detection. To investigate such matter, western blot, northern blot, and alternative ways to culture cells were used in this study. The results showed that the surface antigen antibody 13H10 was unable to recognize the epitope on HBV genotype D whereas other surface antigen antibodies, E11E4 and Bioss, yielded promising outcomes. Moreover, in cell culture, whether HepAD38 was passaged after induction has a huge influence on the expression of surface antigen. Passaging HepAD38 after induction could reduce surface protein quantity by almost ten folds in comparison to the ones without. This implies that the first Western Blot results showing no surface protein could be due to its inadequate protein amount collected. Overall, the results in this study suggest that only two surface antigen antibodies, E11E4 and Bioss, and cell culture methods determine whether the surface protein of genotype D would be recognized. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T12:30:46Z (GMT). No. of bitstreams: 1 ntu-105-R03445117-1.pdf: 1703199 bytes, checksum: cb3efca23c40a9da78e0bdbf25e62a4b (MD5) Previous issue date: 2016 | en |
dc.description.tableofcontents | 誌謝 .......................................... i
中文摘要 ....................................... ii 英文摘要 ........................................ iii 目錄 .......................................... v 圖次 ........................................... vii 表次 ............................................ viii 壹、序論 ........................................ 1 1.1 B型肝炎病毒簡介 .............................. 1 1.2 B型肝炎病毒構造與基因 ......................... 2 1.3 B型肝炎病毒生活史 ............................ 3 1.4 B型肝炎感染 ................................. 3 1.4.1 HBV感染模式 ....................... 3 1.4.2 HBV感染肝細胞的決定因子 ............ 4 1.4.3 HBV非活體之細胞培養感染系統 ......... 6 1.5 研究目的 .................................... 7 貳、實驗材料與方法 ............................... 8 2.1 細胞株與細胞培養(cell line and cell culture) . 8 2.2 質體轉染(plasmid transfection) .............. 8 2.3 蛋白質萃取西方墨點法(protein extraction and western blot) .......................................... 8 2.4 RNA萃取及北方墨點法(RNA extraction and northern blot) ................................................ 10 2.5 病毒製造 .................................... 10 2.6 病毒沉澱與濃縮 ............................... 10 2.7 細胞培養液表面抗原和e抗原定性 .................. 11 2.8 血清HBV DNA萃取與HBV DNA定量 ................. 11 參、結果 ......................................... 12 3.1 HepAD38 細胞內的表面蛋白可能無法被抗體辨識 ...... 12 3.2 HepAD38確定有被誘導生成轉錄pgRNA ............... 12 3.3細胞外液表面蛋白的測量 .......................... 13 3.4不同培養液與血清對HepAD38表面蛋白的分泌不造成影響 . 14 3.5不同抗體對於HBV基因型D的表面蛋白之辨識 ........... 14 3.6繼代會使HepAD38分泌的表面蛋白降低 ............... 15 肆、討論 ......................................... 16 附圖 ............................................. 18 附表 ............................................. 35 伍、參考文獻 ...................................... 37 圖次 圖一、B 型肝炎病毒...................................19 圖二、B 型肝炎次病毒顆粒..............................20 圖三、B 型肝炎病毒基因體..............................21 圖四、B 型肝炎病毒生活史..............................22 圖五、HBV 的表面蛋白.................................23 圖六、HepAD38 穩定表現的質體-peteHBV.................24 圖七、HBV 表面蛋白在細胞內外之表現.....................25 圖八、HepAD38 細胞內 HBV RNA 之表現...................26 圖九、繼代與否影響 HepAD38 分泌表面蛋白................27 圖十、不同培養液與不同胎牛血清對 HepAD38 分泌 HBs 的影響.28 圖十一、不同培養液與不同胎牛血清對 HepAD38 外型的影響....29 圖十二、不同抗體對 D 基因型 HBV 表面蛋白之辨認...........30 圖十三、繼代與沒繼代細胞濃縮外液之表面蛋白與細胞蛋白之表現.31 圖十四、質體 AAV/HBV1.2...............................32 圖十五、1.3X-HBV 質體.................................33 圖十六、不同基因型之大型表面抗原之序列...................34 表次 表一、繼代與不繼代影響細胞分泌表面蛋白...................36 | |
dc.language.iso | zh-TW | |
dc.title | 生產B型肝炎病毒之細胞株(HepAD38)特性分析 | zh_TW |
dc.title | Characterization of HBV-producing HepAD38 cell line | en |
dc.type | Thesis | |
dc.date.schoolyear | 104-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 吳慧琳(Hui-Lin Wu),陶秘華(Mi-Hua Tao) | |
dc.subject.keyword | B型肝炎感染,細胞株HepAD38, | zh_TW |
dc.subject.keyword | HepAD38,HBV,HBV infection, | en |
dc.relation.page | 41 | |
dc.identifier.doi | 10.6342/NTU201601877 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2016-08-04 | |
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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