跳到主要內容

臺灣博碩士論文加值系統

(44.200.86.95) 您好!臺灣時間:2024/05/28 10:16
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

: 
twitterline
研究生:札蘇光士
研究生(外文):Gansukh Choijilsuren
論文名稱:肝素在生理濃度及無PEG環境下可以促進B型肝炎病毒之細胞感染
論文名稱(外文):Heparin at physiological concentration can enhance PEG-free in vitro infection with human hepatitis B virus
指導教授:施嘉和
指導教授(外文):Chiaho Shih
學位類別:博士
校院名稱:國立陽明大學
系所名稱:生化暨分子生物研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:中文
論文頁數:56
中文關鍵詞:肝素B型肝炎病毒HBV 感染HBV in vitro 感染系統
外文關鍵詞:HeparinHBV infectionHBV entryHBV in vitro infection system
相關次數:
  • 被引用被引用:0
  • 點閱點閱:99
  • 評分評分:
  • 下載下載:4
  • 收藏至我的研究室書目清單書目收藏:0
B 型肝炎病毒(Hepatitis B Virus, HBV) 為一種會造成慢性肝炎、肝硬化及肝癌的血液性
病原體。HBV 進入細胞的機制目前並未完全了解,需被進一步的研究。最近藉由在
HepG2 細胞上表現sodium taurocholate cotransporting polypeptide (NTCP)的in vitro 感染
系統中,NTCP 已被證實為一個主要的receptor。然而這個感染系統需要依靠一個與生
理上不相關的化學物質polyethylene glycol (4% PEG) 。在HBV in vitro 感染的系統中,
高濃度的肝素(Heparin)被利用來做為一個抑制感染的抑制劑控制組。令人意外地,在
HepaRG 和HepG2 NTCP 的細胞中,生理濃度情況下的肝素可以藉由一個對Pre-S1
peptide 敏感,同時依靠NTCP 的方式促進HBV 感染。相較於肝素 的N-sulfation,
O-sulfation 對於促進HBV 的感染更為重要。這個建立於HepG2-NTCP-AS 細胞上的感
染系統能夠提供HBV genotype B 及C 以及從HBeAg 陽性及陰性的慢性帶原者血清中
獲得的病毒進行感染。綜合上述的結果,我們的研究提供了一個類似於自然界中HBV
感染人類的系統。此外,研究的結果也提供了一個在血液中肝素及與肝素結合的宿主因
子可能也參與HBV 病毒進入細胞的研究方向。
Hepatitis B virus (HBV) is a blood-borne pathogen responsible for chronic hepatitis, cirrhosis,
and liver cancer. The mechanism of HBV entry into hepatocytes remains to be investigated.
Recently, sodium taurocholate cotransporting polypeptide (NTCP) was discovered as a major
HBV receptor based on an in vitro infection system using NTCP-reconstituted HepG2 cells.
However, this infection system relies on the compound polyethylene glycol (4% PEG), which
is not physiologically relevant to human infection. High concentration of heparin has been
commonly used as an inhibitor control for in vitro infection in the field. Surprisingly, we found
that heparin at physiological concentration can enhance HBV infection in a PreS1-peptide
sensitive, NTCP-dependent manner in both HepaRG and HepG2-NTCP-AS cells. O-sulfation
of heparin is more important for the infection enhancement than N-sulfation. This system
based on the HepG2-NTCP-AS cells can support in vitro infection with HBV genotypes B and
C, as well as using serum samples from HBeAg positive and negative chronic carriers. In
summary, our study provides a PEG-free infection system closely resembling human natural
infection. In addition, it points to a future research direction for heparin and heparin-binding
host factor(s) in the blood, which are potentially involved in viral entry.
Acknowledgments - i
中文摘要 - ii
English Abstract -iii
Table of Contents - iv
List of Figures - vi
List of Tables - viii
Chapter 1 Introduction -1
1.1 Hepatitis B virus biology - 1
1.1.1 Classification of viruses within the Hepadnavirus family - 1
1.1.2 Virus structure - 2
1.1.3 Genome structure and organization - 2
1.1.4 The early events of HBV entry - 3
1.2 HBV in vitro infection systems - 4
1.3 Heparin effect on HBV entry - 5
1.4 Aim of the study - 6
Chapter 2 Results - 7
2.1 Establishment of an HBV in vitro infection system using a
HepG2-NTCP-AS cell line - 7
2.2 Heparin at physiological concentration in human plasma can stimulate
HBV in vitro infection - 8
2.3 Heparin facilitates PEG-free in vitro infection with HBV - 12
2.4 Effect of heparin sulfation for heparin-enhancement on HBV entry
- 13
Chapter 3 Discussion - 15
3.1 Heparin inhibition effect - 15
3.2 Heparin enhancement effect - 16
3.3 Possible mechanisms for heparin enhancement and inhibition - 18
Chapter 4 Methods - 20
4.1 Ethics statement - 20
4.2 Reagents - 20
4.3 Establishment of HepG2-NTCP-AS cell line - 21
4.4 Plasma fractionation - 21
4.5 HBV in vitro infection assay - 22
4.6 Western blot - 23
4.7 Immunofluorescence assay - 23
4.8 Southern blot assay - 24
4.9 HBsAg and HBeAg detection by ELISA - 24
References - 51

List of Figures

Fig 1. Establishment of an HBV in vitro infection system using a HepG2-NTCP-AS cell line. (Illustration of experimental design) - 26
Fig 2. Flag-tagged NTCP expression was detected by western blot in a HepG2-NTCP-AS cell line - 27
Fig 3. Increasing trend of both HBsAg and HBeAg confirms HepG2-NTCP-AS cells were infected with human HBV-containing serum - 28
Fig 4. HBV replicative intermediates were analysed by Southern blot assay - 29
Fig 5. HBc proteins were detected on HBV infected HepG2-NTCP-AS cells by IFA analysis - 30
Fig 6. HBV from both HBeAg positive and negative patients’ serum is infectious in vitro - 31
Fig 7. Heparin at physiological concentration in human plasma can stimulate HBV in vitro infection. (Illustration of experimental design) - 32
Fig 8. Human plasma, with or without heat pre-treatment, enhanced HBV in vitro infection - 33
Fig 9. Heparin is the principal ingredient in human plasma to the enhancement of HBV infection - 34
Fig 10. Heparin alone without human plasma can still enhance HBV infection - 35
Fig 11. Heparin at physiological concentrations (1 to 5 µg/ml) can enhance HBV infection in a dose-response experiment - 36
Fig 12. Treatment with PreS1 lipopolypeptide inhibited heparin-enhanced infection of HepG2-NTCP-AS cells - 37
Fig 13. Heparin-enhanced HBV infection can be abrogated by the continuous presence of a nucleoside analog 3TC (10uM), from 1 to 9 dpi - 38
Fig 14. Heparin facilitates PEG-free in vitro infection with HBV. (Illustration of experimental design) - 39
Fig 15. Heparin alone can enhance HBV infection in a PEG-independent manner - 40
Fig 16. A combinatory effect of heparin (4.5 µg/ml) and PEG (1.2%) can be detected by Southern blot analysis - 41
Fig 17. In a PEG-free in vitro infection system, low dose heparin (4.5 µg/ml) significantly increased the percentage of HBc-positive HepG2-NTCP-AS cells by confocal microscopy - 42
Fig 18. O-sulfation of heparin is required for its enhancement on HBV infection in HepG2-NTCP-AS cell system - 43
Fig 19. Heparin enhancement on HBV infection using the HepG2-NTCP-AS cells were validated by using a HepaRG infection system -44
Fig 20. Heparin can further enhance HBV infection using HepaRG cells in the presence of 1.2% or 4% PEG - 45
Fig 21. O-sulfation is also important for heparin enhancement of HBV infection in the HepaRG cell system - 46
Fig 22. Hypothetical mechanisms for the effects of heparin concentration on HBV entry via a two-step process - 47

List of Tables

Table 1. Clinical data of serum samples from Taiwanese HBV patients - 49
Table 2. Effects of heparin concentration on HBV in vitro infection - 50
1. Shih, C., et al. Control and Eradication Strategies of Hepatitis B Virus. Trends in
Microbiology 24, 739-749 (2016).
2. Chen, C.J., et al. Risk of hepatocellular carcinoma across a biological gradient of serum
hepatitis B virus DNA level. Jama-J Am Med Assoc 295, 65-73 (2006).
3. Blumberg, B.S. Citation Classic - New Antigen in Leukemia Sera. Cc/Life Sci, 14-14
(1979).
4. Schaefer, S. Hepatitis B virus taxonomy and hepatitis B virus genotypes. World J
Gastroentero 13, 14-21 (2007).
5. Drexler, J.F., et al. Bats carry pathogenic hepadnaviruses antigenically related to
hepatitis B virus and capable of infecting human hepatocytes. P Natl Acad Sci USA 110,
16151-16156 (2013).
6. Chai, N., et al. Properties of subviral particles of hepatitis B virus. Journal of Virology
82, 7812-7817 (2008).
7. Wynne, S.A., Crowther, R.A. & Leslie, A.G.W. The crystal structure of the human
hepatitis B virus capsid. Mol Cell 3, 771-780 (1999).
8. Urban, S. New insights into hepatitis B and hepatitis delta virus entry. Future Virology
3, 253-264 (2008).
9. Delius, H., Gough, N.M., Cameron, C.H. & Murray, K. Structure of the Hepatitis-B
Virus Genome. Journal of Virology 47, 337-343 (1983).
10. 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).
11. Watashi, K., et al. Cyclosporin A and Its Analogs Inhibit Hepatitis B Virus Entry Into
Cultured Hepatocytes Through Targeting a Membrane Transporter, Sodium
Taurocholate Cotransporting Polypeptide (NTCP). Hepatology 59, 1726-1737 (2014).
12. Leistner, C.M., Gruen-Bernhard, S. & Glebe, D. Role of glycosaminoglycans for
binding and infection of hepatitis B virus. Cellular Microbiology 10, 122-133 (2008).
13. Iwamoto, M., et al. Evaluation and identification of hepatitis B virus entry inhibitors
using HepG2 cells overexpressing a membrane transporter NTCP. Biochem Bioph Res
Co 443, 808-813 (2014).
14. Zahn, A. & Allain, J.P. Hepatitis C virus and hepatitis B virus bind to heparin:
purification of largely IgG-free virions from infected plasma by heparin
chromatography. Journal of General Virology 86, 677-685 (2005).
15. Yan, H., et al. Sodium taurocholate cotransporting polypeptide is a functional receptor
for human hepatitis B and D virus. Elife 1(2012).

16. Galle, P.R., et al. In-Vitro Experimental-Infection of Primary Human Hepatocytes with
Hepatitis-B Virus. Gastroenterology 106, 664-673 (1994).
17. Ochiya, T., et al. An Invitro System for Infection with Hepatitis-B Virus That Uses
Primary Human-Fetal Hepatocytes. P Natl Acad Sci USA 86, 1875-1879 (1989).
18. Gripon, P., et al. Hepatitis-B Virus-Infection of Adult Human Hepatocytes Cultured in
the Presence of Dimethyl-Sulfoxide. Journal of Virology 62, 4136-4143 (1988).
19. Gripon, P., Diot, C. & Guguenguillouzo, C. Reproducible High-Level Infection of
Cultured Adult Human Hepatocytes by Hepatitis-B Virus - Effect of
Polyethylene-Glycol on Adsorption and Penetration. Virology 192, 534-540 (1993).
20. Gripon, P., et al. Infection of a human hepatoma cell line by hepatitis B virus. P Natl
Acad Sci USA 99, 15655-15660 (2002).
21. Watashi, K., Urban, S., Li, W.H. & Wakita, T. NTCP and Beyond: Opening the Door to
Unveil Hepatitis B Virus Entry. International Journal of Molecular Sciences 15,
2892-2905 (2014).
22. Watashi, K. & Wakita, T. Hepatitis B Virus and Hepatitis D Virus Entry, Species
Specificity, and Tissue Tropism. Csh Perspect Med 5(2015).
23. Karsten, U., et al. Direct Comparison of Electric Field-Mediated and Peg-Mediated
Cell-Fusion for the Generation of Antibody-Producing Hybridomas. Hybridoma 7,
627-633 (1988).
24. Norwood, T.H., Zeigler, C.J. & Martin, G.M. Dimethyl-Sulfoxide Enhances
Polyethylene Glycol-Mediated Somatic-Cell Fusion. Somat Cell Genet 2, 263-270
(1976).
25. Sugiyama, K., Yamamoto, K., Kamata, O. & Katsuda, N. Heparin of Mast-Cells .2. The
Mechanism of Heparin Release. Jpn J Pharmacol 30, P165-P165 (1980).
26. Engelberg, H. Plasma Heparin Levels in Normal Man. Circulation 23, 578-& (1961).
27. Cavari, S., Stramaccia, L. & Vannucchi, S. Endogenous Heparinase-Sensitive
Anticoagulant Activity in Human Plasma. Thrombosis Research 67, 157-165 (1992).
28. Volpi, N., Cusmano, M. & Venturelli, T. Qualitative and Quantitative Studies of
Heparin and Chondroitin Sulfates in Normal Human Plasma. Bba-Gen Subjects 1243,
49-58 (1995).
29. Francis, H. & Meininger, C.J. A review of mast cells and liver disease: What have we
learned? Digest Liver Dis 42, 529-536 (2010).
30. Sasisekharan, R. & Venkataraman, G. Heparin and heparan sulfate: biosynthesis,
structure and function. Current Opinion in Chemical Biology 4, 626-631 (2000).
31. Peysselon, F. & Ricard-Blum, S. Heparin-protein interactions: From affinity and
kinetics to biological roles. Application to an interaction network regulating
angiogenesis. Matrix Biology 35, 73-81 (2014).
32. Li, W.H. & Urban, S. Entry of hepatitis B and hepatitis D virus into hepatocytes: Basic
insights and clinical implications. Journal of Hepatology 64, S32-S40 (2016).
33. Best, C.H. Preparation of heparin and its use in the first clinical cases. Circulation 19,
79-86 (1959).
34. Li, J.S., et al. Unusual Features of Sodium Taurocholate Cotransporting Polypeptide as
a Hepatitis B Virus Receptor. Journal of Virology 90, 8302-8313 (2016).
35. Sureau, C. & Salisse, J. A conformational heparan sulfate binding site essential to
infectivity overlaps with the conserved hepatitis B virus A-determinant. Hepatology 57,
985-994 (2013).
36. Nugent, M.A. Heparin sequencing brings structure to the function of complex
oligosaccharides. P Natl Acad Sci USA 97, 10301-10303 (2000).
37. Wadstrom, T. & Ljungh, A. Glycosaminoglycan-binding microbial proteins in tissue
adhesion and invasion: key events in microbial pathogenicity. Journal of Medical
Microbiology 48, 223-233 (1999).
38. Liu, J. & Thorp, S.C. Cell surface heparan sulfate and its roles in assisting viral
infectious. Medicinal Research Reviews 22, 1-25 (2002).
39. Vandenberghe, L.H., et al. Heparin binding directs activation of T cells against
adeno-associated virus serotype 2 capsid. Nature Medicine 12, 967-971 (2006).
40. Levy, H.C., et al. Heparin binding induces conformational changes in
Adeno-associated virus serotype 2. Journal of Structural Biology 165, 146-156 (2009).
41. Yang, C.C., Huang, E.Y., Li, H.C., Su, P.Y. & Shih, C. Nuclear Export of Human
Hepatitis B Virus Core Protein and Pregenomic RNA Depends on the Cellular
NXF1-p15 Machinery. PLoS One 9(2014).
42. Chou, S.F., Tsai, M.L., Huang, J.Y., Chang, Y.S. & Shih, C.H. The Dual Role of an
ESCRT-0 Component HGS in HBV Transcription and Naked Capsid Secretion. PLoS
Pathogens 11(2015).
43. Su, P.Y., et al. HBV maintains electrostatic homeostasis by modulating negative
charges from phosphoserine and encapsidated nucleic acids. Sci Rep-Uk 6(2016).
44. Alexander, W. Heparin revisions: a call for heightened vigilance and monitoring. P T
34, 634-635 (2009).
連結至畢業學校之論文網頁點我開啟連結
註: 此連結為研究生畢業學校所提供,不一定有電子全文可供下載,若連結有誤,請點選上方之〝勘誤回報〞功能,我們會盡快修正,謝謝!
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top