請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/53646
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 | Chen-Yen Chung | en |
dc.contributor.author | 鍾承諺 | zh_TW |
dc.date.accessioned | 2021-06-16T02:26:55Z | - |
dc.date.available | 2017-09-25 | |
dc.date.copyright | 2015-09-25 | |
dc.date.issued | 2015 | |
dc.date.submitted | 2015-08-04 | |
dc.identifier.citation | 1. Yan, H., et al. Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus, (2012).
2. König, A., et al. Kinetics of the bile acid transporter and hepatitis B virus receptor Na+/taurocholate cotransporting polypeptide (NTCP) in hepatocytes. Journal of Hepatology 61, 867-875 (2014). 3. Mason, W.S., Aldrich, C., Summers, J. & Taylor, J.M. Asymmetric replication of duck hepatitis B virus DNA in liver cells: Free minus-strand DNA. Proceedings of the National Academy of Sciences of the United States of America 79, 3997-4001 (1982). 4. Weiser, B., Ganem, D., Seeger, C. & Varmus, H.E. Closed circular viral DNA and asymmetrical heterogeneous forms in livers from animals infected with ground squirrel hepatitis virus. Journal of Virology 48, 1-9 (1983). 5. Walter, E., Keist, R., Niederöst, B., Pult, I. & Blum, H.E. Hepatitis B virus infection of tupaia hepatocytes in vitro and in vivo. Hepatology 24, 1-5 (1996). 6. Gripon, P., et al. Infection of a human hepatoma cell line by hepatitis B virus. Proceedings of the National Academy of Sciences 99, 15655-15660 (2002). 7. Schulze, A., Gripon, P. & Urban, S. Hepatitis B virus infection initiates with a large surface protein–dependent binding to heparan sulfate proteoglycans. Hepatology 46, 1759-1768 (2007). 8. Yan, H., et al. Molecular Determinants of Hepatitis B and D Virus Entry Restriction in Mouse Sodium Taurocholate Cotransporting Polypeptide. Journal of Virology 87, 7977-7991 (2013). 9. Li, H., et al. HBV life cycle is restricted in mouse hepatocytes expressing human NTCP. Cell Mol Immunol 11, 175-183 (2014). 10. Giersch, K., et al. Hepatitis Delta co-infection in humanized mice leads to pronounced induction of innate immune responses in comparison to HBV mono-infection. Journal of Hepatology. 11. Cooper, A. & Shaul, Y. Clathrin-mediated Endocytosis and Lysosomal Cleavage of Hepatitis B Virus Capsid-like Core Particles. Journal of Biological Chemistry 281, 16563-16569 (2006). 12. Zerial, M. & McBride, H. Rab proteins as membrane organizers. Nat Rev Mol Cell Biol 2, 107-117 (2001). 13. Macovei, A., Petrareanu, C., Lazar, C., Florian, P. & Branza-Nichita, N. Regulation of Hepatitis B Virus Infection by Rab5, Rab7, and the Endolysosomal Compartment. Journal of Virology 87, 6415-6427 (2013). 14. Stross, C., et al. A dileucine motif is involved in plasma membrane expression and endocytosis of rat sodium taurocholate cotransporting polypeptide (Ntcp), (2013). 15. Stross, C., et al. Protein kinase C induces endocytosis of the sodium taurocholate cotransporting polypeptide, (2010). 16. 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. Antimicrobial Agents and Chemotherapy 41, 1715-1720 (1997). 17. Ramsby, M. & Makowski, G. Differential Detergent Fractionation of Eukaryotic Cells. Cold Spring Harbor Protocols 2011, prot5592 (2011). 18. Ni, Y., et al. Hepatitis B and D Viruses Exploit Sodium Taurocholate Co-transporting Polypeptide for Species-Specific Entry into Hepatocytes. Gastroenterology 146, 1070-1083.e1076 (2014). 19. Iwamoto, M., et al. Evaluation and identification of hepatitis B virus entry inhibitors using HepG2 cells overexpressing a membrane transporter NTCP. Biochemical and Biophysical Research Communications 443, 808-813 (2014). 20. Lin, Y.-J., Wu, H.-L., Chen, D.-S. & Chen, P.-J. Hepatitis B Virus Nucleocapsid but Not Free Core Antigen Controls Viral Clearance in Mice. Journal of Virology 86, 9266-9273 (2012). 21. Yan, R., et al. Spinoculation Enhances HBV Infection in NTCP-Reconstituted Hepatocytes. PLoS ONE 10, e0129889 (2015). 22. Mellor, H. & Parker, P.J. The extended protein kinase C superfamily. Biochemical Journal 332, 281-292 (1998). 23. Brandon, N.J., et al. Subunit-Specific Association of Protein Kinase C and the Receptor for Activated C Kinase with GABA Type A Receptors. The Journal of Neuroscience 19, 9228-9234 (1999). 24. Cheng, X., Buckley, D. & Klaassen, C.D. Regulation of hepatic bile acid transporters Ntcp and Bsep expression. Biochemical pharmacology 74, 1665-1676 (2007). 25. Anwer, M.S., et al. Dephosphorylation of Ser-226 Facilitates Plasma Membrane Retention of Ntcp. Journal of Biological Chemistry 280, 33687-33692 (2005). 26. Marsh, M. & Helenius, A. Virus Entry: Open Sesame. Cell 124, 729-740 (2006). 27. Constantinescu, S.N., Cernescu, C.D. & Popescu, L.M. Effects of protein kinase C inhibitors on viral entry and infectivity. FEBS Letters 292, 31-33 (1991). 28. Matlin, K.S., Reggio, H., Helenius, A. & Simons, K. Infectious entry pathway of influenza virus in a canine kidney cell line. The Journal of Cell Biology 91, 601-613 (1981). 29. Lakadamyali, M., Rust, M.J. & Zhuang, X. Endocytosis of influenza viruses. Microbes and infection / Institut Pasteur 6, 929-936 (2004). 30. Arora, D.J.S. & Gasse, N. Influenza virus hemagglutinin stimulates the protein kinase C activity of human polymorphonuclear leucocytes. Arch. Virol. 143, 2029-2037 (1998). 31. Sieczkarski, S.B., Brown, H.A. & Whittaker, G.R. Role of Protein Kinase C βII in Influenza Virus Entry via Late Endosomes. Journal of Virology 77, 460-469 (2003). 32. Wittkop, L., et al. Inhibition of protein kinase C phosphorylation of hepatitis B virus capsids inhibits virion formation and causes intracellular capsid accumulation. Cellular Microbiology 12, 962-975 (2010). 33. Wang, Y., Zhang, J., Yi, X.-j. & Yu, F.-S.X. Activation of ERK1/2 MAP kinase pathway induces tight junction disruption in human corneal epithelial cells. Experimental Eye Research 78, 125-136 (2004). 34. Stuart, R.O. & Nigam, S.K. Regulated assembly of tight junctions by protein kinase C. Proceedings of the National Academy of Sciences of the United States of America 92, 6072-6076 (1995). 35. Schulze, A., Mills, K., Weiss, T.S. & Urban, S. Hepatocyte polarization is essential for the productive entry of the hepatitis B virus. Hepatology 55, 373-383 (2012). 36. Mee, C.J., et al. Polarization Restricts Hepatitis C Virus Entry into HepG2 Hepatoma Cells. Journal of Virology 83, 6211-6221 (2009). 37. Pan, W., et al. Genetic polymorphisms in Na+-taurocholate co-transporting polypeptide (NTCP) and ileal apical sodium-dependent bile acid transporter (ASBT) and ethnic comparisons of functional variants of NTCP among Asian populations. Xenobiotica 41, 501-510 (2011). 38. Zhou, X., et al. Structural basis of the alternating-access mechanism in a bile acid transporter. Nature 505, 569-573 (2014). 39. Yan, H., et al. Viral Entry of Hepatitis B and D Viruses and Bile Salts Transportation Share Common Molecular Determinants on Sodium Taurocholate Cotransporting Polypeptide. Journal of Virology 88, 3273-3284 (2014). 40. Peng, L., et al. The p.Ser267Phe variant in SLC10A1 is associated with resistance to chronic hepatitis B. Hepatology 61, 1251-1260 (2015). | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/53646 | - |
dc.description.abstract | 慢性B 型肝炎病毒帶原者在全球約有2.4億,這些帶原者有很高的危險因子會發展為肝癌,因此仍然是世界上尚未解決的感染性疾病。細胞感染模式的建立對於研究B肝病毒及其衛星病毒D肝病毒的生長循環、病程及新抗病毒藥的開發而言相當重要。過去二十多年來,人類及樹鼩的初代肝細胞常被使用來研究自然的B肝病毒感染模式。然而在實驗上廣泛被使用之人類肝癌細胞株像是Huh7 及HepG2並無法自然的被B肝病毒所感染。另一方面,B肝病毒在肝細胞上的功能性受器在之前的研究都沒有被找到,因此長久下來也受限了B肝病毒學的發展。在2012年由中國李文輝教授領導的研究團隊發表的文章中,他們使用了光化白胺酸修飾的短片段B肝大型表面抗原去找出樹鼩初代肝細胞上與其作用的關鍵蛋白。最後找到了鈉離子-牛磺膽酸共轉運蛋白( Sodium Taurocholate Co-transporting Polypeptide, NTCP )可能是B肝的細胞表面受器之一,並成功的使得表達人類NTCP的人類肝癌細胞被B肝及D肝病毒感染。根據這兩年的文獻我們發現,NTCP表達的肝癌細胞株雖然可以支持病毒的感染但是感染的效率比起先前之初代細胞自然感染模式差距相當大。在此篇論文中,我們建立了數株表達人類NTCP之肝癌細胞株,另外加入了日本NIID的Wakita 教授團隊所提供的G2-NTCP-C4細胞株,並且利用細胞株誘導產生的B型肝炎病毒顆粒進行細胞感染來作為我們主要實驗的模式。根據前的研究發現到B肝病毒主要會透過Clathrin及其受器蛋白控制的胞吞作用進入肝細胞,此現象與NTCP本身調節細胞表面數量的內吞作用一致。NTCP的內吞作用已知是受到細胞內蛋白激酶所調控,在細胞模式中加入蛋白激酶的刺激劑 PMA 可以在短時間觀察到NTCP內吞現象的發生。因此我們進而以G2-NTCP-C4細胞株來探討在B型肝病毒接種的期間使用蛋白激酶刺激劑PMA是否會提升感染的效率。我們初步的結果發現,在G2-NTCP-C4細胞中接種B型肝炎病毒處理PMA的實驗裡,接種病毒前處理的組別於感染後5至7天在細胞培養液中偵測到相對高量的B肝病毒表面抗原以及e抗原,然而感染後第9天與控制組並無明顯提升。此外,接種HBV後24小時再進行PMA處理的實驗中,以高病毒量感染細胞的組別在感染後第9天釋放出高量的表面抗原及e抗原。從western blot以及免疫螢光的結果顯示,在感染後第9天的細胞內前處理以及後處理PMA的組別表現出較高量的HBV核心抗原蛋白。 | zh_TW |
dc.description.abstract | Infection with Hepatitis B virus (HBV) is still a major health problem worldwide . Approximately 240 million people chronically infected with HBV have a greater risk of developing hepatocellular carcinoma(HCC). Cell culture systems are required to study the replication cycle of HBV and HDV infection, pathogenesis and the mechanisms of antiviral drugs. From two decades study, primary human (PHH) and tupaia hepatocytes(PTH) has been used to study HBV natural infection. Yan et al. 1 in 2012 year used HBV Large S protein-derived photo-leucine modified peptide to identify sodium taurocholate co-transporting polypeptide as a functional HBV and HDV entry receptor. Expression of full length of human NTCP in HepG2 and Huh-7 cells renders these cells susceptible to HBV and HDV2. However, recent studys demonstrated that HBV entry efficiency in these NTCP-reconstituted cells , compared to natural infection still unffective.
In this study, we based on previous reports indicating that HBV and NTCP internalization were both regulated by clathrin-mediated endocytosis. First of all, we treated the cells with protein kinase C alpha activator, phorbol-ester (PMA) to do activate PKC and make NTCP translocation before and after HBV inoculation with HBV inoculum. Our data show that, pretreatment of PMA in NTCP-expressing cells had higher HBsAg , and HBsAg level in supernatant during 5~7 day post infection (d.p.i), but rebound to control level at 9 d.p.i. In contrast to pretreatment group, treating PMA after 24 hour inoculation, high multiplicity of infection (M.O.I) of inoculums group had significance increase HBsAg and HBeAg level at 9 d.p.i.. From the western blot and immunofluorescence data showed that, both PMA-treated groups had higher intracellular HBcAg expression. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T02:26:55Z (GMT). No. of bitstreams: 1 ntu-104-R02445117-1.pdf: 3837823 bytes, checksum: 76592ace772a3e8c873a7d144f88df89 (MD5) Previous issue date: 2015 | en |
dc.description.tableofcontents | 致謝 ii
國立臺灣大學碩士學位論文口試委員會審定書 VII 中文摘要 VIII ABSTRACT X LIST OF ABBREVIATIONS XII CHAPTER 1: INTRODUCTION 1 1.1 Hepatitis B virus infection 1 1.2 Previous study of HBV infection in different cell culture systems 2 1.3 Sodium Taurocholate Cotranspoting polypeptide is a functional HBV and HDV receptor 3 1.4 HBV restriction in the early phase of virus entry within NTCP-expressing Murine hepatoma cell lines 4 1.5 HBV infection in early stage of entry is depended on Clatherin-dependent enodocytosis 5 1.6 Rab5 and Rab7 control the early trafficking of HBV 7 1.7 Roles of Protein kinase C alpha in regulation of NTCP internaliztion 8 1.8 Aims of thesis research 9 CHAPTER 2: MATERIAL AND METHODS 11 2.1 Cell culture system 11 2.2 Establishment of NTCP-expression stable cell lines 12 2.3 HBV virus particles production 12 2.4 Quantification of HBV copies number by qPCR 13 2.5 HBV infection on NTCP-expressing cells 13 2.6 Electrochemiluminescence immunoassay for HBV viral HBs and HBe antigens from culture supernatant of infected cells 14 2.7 Western blotting 14 2.8 Immunofluorescence assay 15 2.9 Protein Kinase C alpha activation assay 16 2.10 Plasmamembrane and cytoplasm form of NTCP protein extraction 16 CHAPTER 3: RESULTS 18 3.1 Establishment of hNTCP-expressing HepG2 cells 18 3.2 Quantification of virion particle in HBV inoculum 19 3.3 HBV susceptibility of NTCP-expressing cells 20 3.4 Protein kinase C alpha activation trigger NTCP internalization of NTCP from plasma membrane in NTCP-expressing cell lines 21 3.5 Effects of Protein kinase C alpha activation in early stage of HBV infection in NTCP-expressing cells 22 CHATER 4: DISCUSSIONS 25 CHATER 5: FIGURES 33 Figure 1. CMV-NTCP-His plasmid construct 33 Figure 2. Expression of hNTCP in antibiotics-resistant cells 34 Figure 3. HBsAg and HBeAg secretion in HBV inoculated G2-NTCP-His CY26 and G2-NTCP-His CY31 35 Figure 4. HBV native particle gel assay 39 Figure 5. Immunofluorscence of NTCP localization after PMA treatment in G2-NTCP-C4 cells 41 Figure 6. Western blot of cytosolic NTCP, membrane NTCP, organelle NTCP in G2-NTCP-C4 after Protein kinase C alpha activation 42 Figure 7. Western blot of cytosolic NTCP, membrane NTCP, in G2-NTCP-C4 after low dose and high dose treatment of PMA activating Protein kinase C alpha 44 Figure 8: Secreted HBsAg and HBeAg level in the supernatants of Protein kinase C alpha activation in early stage of HBV infection in Time point of PMA treatment 47 Figure 9. PMA treatment before HBV inoculation enhance HBsAg and HBeAg secretion at 5~9 day post infection 50 Figure 10. Pretreatment of PMA increased the HBcAg protein level in G2-NTCP-C4 cells at 9 day post infection 51 Figure 11. Immunofluoresence of HBcAg expression in G2-NTCP-C4 cells with PMA treatment 52 Figure 12. PMA treatment not enhance HBsAg and HBeAg in NTCP-reconstituted CY26 cells 53 REFERENCE 55 | |
dc.language.iso | en | |
dc.title | 以表現鈉離子-牛磺膽酸共轉運蛋白之肝癌細胞株探討B型肝炎病毒感受性之分析 | zh_TW |
dc.title | Establishment of Hepatoma Cell Line with Sodium Taurocholate Co-transporting Polypeptide for Efficient HBV Infection | en |
dc.type | Thesis | |
dc.date.schoolyear | 103-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 施嘉和(Chia-Ho Shih),吳慧琳(Hui-Lin Wu) | |
dc.subject.keyword | B型肝炎,鈉離子-牛磺膽酸共轉運蛋白,蛋白激?, | zh_TW |
dc.subject.keyword | HBV,NTCP,PKC, | en |
dc.relation.page | 57 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2015-08-04 | |
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 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-104-1.pdf 目前未授權公開取用 | 3.75 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。