臺灣博碩士論文加值系統

丙型肝炎病毒的非結構蛋白5A在某些情況下[例如細胞凋亡]，N端1-154的片段會被切割。已經有研究發現，非結構蛋白5A去掉N端154個胺基酸的C端片段具有轉錄活性，然而目前尚不知非結構蛋白5A N端1-154片段的功能。在丙型肝炎病毒複製子細胞中，過度表達非結構蛋白5A確實能夠增加病毒複製，但表達非結構蛋白5A N端1-154則是會抑制病毒複製。胞外體(exosome)約30-100 nm是多顆粒囊體的膜向內凹陷所形成的細胞外顆粒，已有研究指出胞外體能夠幫助丙型肝炎病毒的傳染。胞外體純化自過度表現非結構蛋白5A或非結構蛋白5A N端1-154片段的細胞，能夠發現這兩種蛋白質的存在。而帶有此兩種蛋白質的胞外體，在丙型肝炎病毒複製子細胞中，也會影響丙型肝炎病毒的複製能力。此外，在表達非結構蛋白5A或其N端1-154的人類肝癌細胞(Huh7)，藉由超音波震盪，我們已純化人工合成的顆粒，而這些顆粒對丙型肝炎病毒複製能力的影響，還有待調查。

Hepatitis C virus (HCV) NS5A protein could be cleaved at residue 154 under certain circumstances, e.g., apoptosis. NS5A without its N-terminal 154 amino acid possesses transcriptional activity. The function of the N-terminal 154 a.a. of NS5A (NS5A 1-154) is unknown. Over-expressed NS5A could enhance HCV replication while NS5A 1-154 would repress viral replication in the replicon system. Exosomes, nano-particles (30 -100 nm diameter) derived from cells, are formed by the inward budding of the multivesicular body (MVB) membrane. It is reported that exosomes could facilitate HCV transmission. We have also found that HCV NS5A protein or NS5A 1-154 could be detected in exosomes of Huh7 cells over-expressing these two proteins. Moreover, exosomes with NS5A enhanced HCV replication while exosomes with NS5A 1-154 repressed viral replication in HCV replicon cells. Artificial vesicles with NS5A protein or NS5A 1-154 through the sonication of Huh7 cells over-expressing these two proteins were also developed. Effects of these artificial vesicles on HCV replication are under investigations.

中文摘要............................................................1
Abstract............................................................2
第一章 緒論........................................................4
1.1丙型肝炎病毒的基因及結構....................................4
1.2流行病學................................................... 5
1.3傳染途徑................................................... 6
1.4治療及預防................................................. 6
1.5非結構蛋白5A.............................................. 8
1.6非結構蛋白3............................................... 9
1.7 Caspase 3、NS5A以及NS5A N端短片段胺基酸..................9
1.8胞外體exosome..............................................11
第二章 研究目的...................................................12
第三章 實驗方法...................................................14
3.1質體建立及引子設計........................................ 14
3.1.1 Transformation.......................................16
3.1.2 抽plasmid.......................................... 17
3.2瓊脂膠體電泳(agarose gel electrophoresis)..................18
3.3細胞培養.................................................. 18
3.4細胞保存.................................................. 20
3.5細胞解凍.................................................. 21
3.6細胞繼代.................................................. 21
3.7細胞計數.................................................. 22
3.8 Plasmid transfection...................................... 23
3.9 Lentivirus production & infection......................... 24
3.10胞外體收集............................................... 25
3.11蛋白質萃取............................................... 26
3.12蛋白質定量............................................... 26
3.13 SDS-PAGE(SDS Poly-Acrylamide-Gel-Electrophoresis)........ 27
3.14蛋白質轉漬............................................... 29
3.15流式細胞儀分析........................................... 31
3.16 qPCR(Quantitative Polymerase Chain Reaction)............. 31
3.16.1去氧核醣核酸水解酶處理(deoxyribonuclease).......... 32
3.16.2反轉錄合成cDNA..................................... 33
3.17超音破震盪細胞生成顆粒................................... 35
第四章 實驗結果...................................................36
4.1過度表達轉殖基因與胞外體蛋白質組成分析.................... 36
4.2 HCV replicon中表現NS5A N端短片段對HCV複製的影響.........39
4.3胞外體含有NS5A 1-154對HCV replicon複製能力的影響.........43
4.4利用流式細胞儀分析胞外體上蛋白的拓譜性.................... 46
4.5利用Bafilomycin A1抑制胞外體進入細胞阻止胞外體對HCV複製的影
響.........................................................53
4.6細胞震碎顆粒作為載體影響HCV 複製能力.......................54
4.7利用流式細胞儀分析超音波震盪生成顆粒的蛋白拓譜性...........58
第五章 討論與結論.................................................61
第六章 參考文獻...................................................65

1.Hepatitis C virus syndrome: A constellation of organ- and non-organ specific autoimmune disorders, B-cell non-Hodgkin's lymphoma, and cancer.Ferri C1, Sebastiani M, Giuggioli D, Colaci M, Fallahi P, Piluso A, Antonelli A, Zignego AL. World J Hepatol. 2015 Mar 27;7(3):327-43. doi: 10.4254/wjh.v7.i3.327.

2.Transposon mouse models to elucidate the genetic mechanisms of hepatitis B viral induced hepatocellular carcinoma.Chiu AP, Tschida BR, Lo LH, Moriarity BS, Rowlands DK, Largaespada DA, Keng VW. World J Gastroenterol. 2015 Nov 14;21(42):12157-70. doi: 10.3748/wjg.v21.i42.12157.

3.Topology of hepatitis C virus envelope glycoproteins.Op De Beeck A, Dubuisson J. Rev Med Virol. 2003 Jul-Aug;13(4):233-41.
4.Hepatitis C IRES: translating translation into a therapeutic target.Jubin R. Curr Opin Mol Ther. 2001 Jun;3(3):278-87.

5.The Genotype of Hepatitis C Virus Has Important Clinical and Therapeutic Implications. Pei-Chen Hsieh, Hsing-Tao Kuo , Wen-Chun Cho, Yung-Liang Liao, and Ching-Nan Lin Department of Pathology, Department of Internal Medicine; Chi-Mei Foundation Hospital, Tainan, Taiwan

6.Hepatitis C virus proteins.Dubuisson J.World J Gastroenterol. 2007 May 7;13(17):2406-15..

7.Global distribution and prevalence of hepatitis C virus genotypes.Messina JP, Humphreys I, Flaxman A, Brown A, Cooke GS, Pybus OG, Barnes E. Hepatology. 2015 Jan;61(1):77-87. doi: 10.1002/hep.27259. Epub 2014 Jul 28.

8.Ryan KJ, Ray CG, eds. (2004). Sherris Medical Microbiology(4th ed.). McGraw Hill. pp. 551–2. ISBN 0838585299

9."Hepatitis C FAQs for Health Professionals". CDC. January 8, 2016. Retrieved 4 February 2016.

10.Acute hepatitis C: prevention and treatment.Ozaras R, Tahan V. Expert Rev Anti Infect Ther. 2009 Apr;7(3):351-61. doi: 10.1586/eri.09.8.

11.Hepatitis C: diagnosis and treatment.Wilkins T, Malcolm JK, Raina D, Schade RR. Am Fam Physician. 2010 Jun 1;81(11):1351-7.

12.Chronic Hepatitis C Virus Advances in Treatment, Promise for the Future. Springer Verlag. 2011. pp. 103–104.ISBN 9781461411918.

13.Status of hepatitis C virus vaccination: Recent update. Abdelwahab KS, Ahmed Said ZN. World J Gastroenterol. 2016 Jan 14;22(2):862-73. doi: 10.3748/wjg.v22.i2.862.

14.Epidemiology of hepatitis C virus infection. Alter MJ. World J Gastroenterol. 2007 May 7;13(17):2436-41.

15.Progress in the development of preventive and therapeutic vaccines for hepatitis C virus. Torresi J, Johnson D, Wedemeyer H. J Hepatol. 2011 Jun;54(6):1273-85. doi: 10.1016/j.jhep.2010.09.040. Epub 2011 Jan 12.

16.An overview of emerging therapies for the treatment of chronic hepatitis C. Ilyas JA, Vierling JM. Clin Liver Dis. 2011 Aug;15(3):515-36. doi: 10.1016/j.cld.2011.05.002.

17.Hepatitis C vaccine hampered by viral complexity, many technical restraints. Randal J. J Natl Cancer Inst. 1999 Jun 2;91(11):906-8.

18.Hepatitis C guidance: AASLD-IDSA recommendations for testing, managing, and treating adults infected with hepatitis C virus. AASLD/IDSA HCV Guidance Panel. Hepatology. 2015 Sep;62(3):932-54. doi: 10.1002/hep.27950. Epub 2015 Aug 4.

19.Clinical practice. Chronic hepatitis C infection. Rosen HR.N Engl J Med. 2011 Jun 23;364(25):2429-38. doi: 10.1056/NEJMcp1006613.

20.Hepatitis C virus entry: molecular mechanisms and targets for antiviral therapy. Zeisel MB, Barth H, Schuster C, Baumert TF. Front Biosci (Landmark Ed). 2009 Jan 1;14:3274-85.

21.Splicing diversity of the human OCLN gene and its biological significance for hepatitis C virus entry. Kohaar I, Ploss A, Korol E, Mu K, Schoggins JW, O'Brien TR, Rice CM, Prokunina-Olsson L. J Virol. 2010 Jul;84(14):6987-94. doi: 10.1128/JVI.00196-10. Epub 2010 May 12.

22.Formation and function of hepatitis C virus replication complexes require residues in the carboxy-terminal domain of NS4B protein. Aligo J, Jia S, Manna D, Konan KV. Virology. 2009 Oct 10;393(1):68-83. doi: 10.1016/j.virol.2009.07.033. Epub 2009 Aug 22.

23.Phosphorylation of hepatitis C virus NS5A nonstructural protein: a new paradigm for phosphorylation-dependent viral RNA replication? Huang Y, Staschke K, De Francesco R, Tan SL. Virology. 2007 Jul 20;364(1):1-9. Epub 2007 Apr 2.

24.Hepatitis C virus NS5A: tales of a promiscuous protein. Macdonald A, Harris M. J Gen Virol. 2004 Sep;85(Pt 9):2485-502.

25.Hepatitis C Virus: Molecular Pathways and Treatments Edited by: Dr. Oumaima Stambouli Published by OMICS Group eBooks 731 Gull Ave, Foster City. CA 94404, USA

26.Nonstructural protein 3 of the hepatitis C virus encodes a serine-type proteinase required for cleavage at the NS3/4 and NS4/5 junctions. Bartenschlager R, Ahlborn-Laake L, Mous J, Jacobsen H. J Virol. 1993 Jul;67(7):3835-44.

27.Hepatitis C virus markers in infection by hepatitis C virus: In the era of directly acting antivirals. Coppola N, Pisaturo M, Zampino R, Macera M, Sagnelli C, Sagnelli E. World J Gastroenterol. 2015 Oct 14;21(38):10749-59. doi: 10.3748/wjg.v21.i38.10749.

28.An overview of HCV molecular biology, replication and immune responses. Ashfaq UA, Javed T, Rehman S, Nawaz Z, Riazuddin S. Virol J. 2011 Apr 11;8:161. doi: 10.1186/1743-422X-8-161.

29.Overview of ENSG00000164305_CASP3 marker .http://www.orthomam.univ-montp2.fr/orthomam/data/cds/detailMarkers/ENSG00000164305_CASP3.xml

30.Apoptosis: an overview. Wyllie AH. Br Med Bull. 1997;53(3):451-65.

31.Zinc is a potent inhibitor of the apoptotic protease, caspase-3. A novel target for zinc in the inhibition of apoptosis. Perry DK, Smyth MJ, Stennicke HR, Salvesen GS, Duriez P, Poirier GG, Hannun YA. J Biol Chem. 1997 Jul 25;272(30):18530-3.

32.Cleavage of the hepatitis C virus NS5A protein by caspase-3 in the interferon sensitivity-determining region in a sequence-dependent manner. Hidajat R, Nagano-Fujii M, Deng L, Hotta H. Kobe J Med Sci. 2004;50(5-6):153-66.

33.The hepatitis C virus core protein interacts with NS5A and activates its caspase-mediated proteolytic cleavage. Goh PY, Tan YJ, Lim SP, Lim SG, Tan YH, Hong WJ. Virology. 2001 Nov 25;290(2):224-36.

34.A virocidal amphipathic {alpha}-helical peptide that inhibits hepatitis C virus infection in vitro. Cheng G, Montero A, Gastaminza P, Whitten-Bauer C, Wieland SF, Isogawa M, Fredericksen B, Selvarajah S, Gallay PA, Ghadiri MR, Chisari FV. Proc Natl Acad Sci U S A. 2008 Feb 26;105(8):3088-93. doi: 10.1073/pnas.0712380105. Epub 2008 Feb 19.

35.Exosomes and HIV Gag bud from endosome-like domains of the T cell plasma membrane. Booth AM, Fang Y, Fallon JK, Yang JM, Hildreth JE, Gould SJ. J Cell Biol. 2006 Mar 13;172(6):923-35.

36.Classification, functions, and clinical relevance of extracellular vesicles. van der Pol E, Böing AN, Harrison P, Sturk A, Nieuwland R. Pharmacol Rev. 2012 Jul;64(3):676-705. doi: 10.1124/pr.112.005983. Epub 2012 Jun 21.

37.Exosomes: from biogenesis and secretion to biological function. Keller S, Sanderson MP, Stoeck A, Altevogt P. Immunol Lett. 2006 Nov 15;107(2):102-8. Epub 2006 Oct 17.

38.Vescle formation during reticulocyte maturation. Association of plasma membrane activities with released vesicles (exosomes). Johnstone RM, Adam M, Hammond JR, Orr L, Turbide C. J Biol Chem. 1987 Jul 5;262(19):9412-20.

39.Exosomes: a common pathway for a specialized function. van Niel G, Porto-Carreiro I, Simoes S, Raposo G. J Biochem. 2006 Jul;140(1):13-21.

40.Exosome Biogenesis, Regulation, and Function in Viral Infection. Alenquer M, Amorim MJ Viruses. 2015 Sep 17;7(9):5066-83. doi: 10.3390/v7092862.

41.Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Valadi H, Ekström K, Bossios A, Sjöstrand M, Lee JJ, Lötvall JO. Nat Cell Biol. 2007 Jun;9(6):654-9. Epub 2007 May 7.

42.Exosomes from hepatitis C infected patients transmit HCV infection and contain replication competent viral RNA in complex with Ago2-miR122-HSP90. Bukong TN, Momen-Heravi F, Kodys K, Bala S, Szabo G. PLoS Pathog. 2014 Oct 2;10(10):e1004424. doi: 10.1371/journal.ppat.1004424. eCollection 2014.

43.Growth of human hepatoma cells lines with differentiated functions in chemically defined medium. Nakabayashi H, Taketa K, Miyano K, Yamane T, Sato J. Cancer Res. 1982 Sep;42(9):3858-63.

44.Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Lohmann V, Körner F, Koch J, Herian U, Theilmann L, Bartenschlager R. Science. 1999 Jul 2;285(5424):110-3.

45.The chimpanzee model of hepatitis C virus infections. Lanford RE, Bigger C, Bassett S, Klimpel G. ILAR J. 2001;42(2):117-26.

46.Isolation, identification, and characterization of novel nanovesicles. Zhang HG, Cao P, Teng Y, Hu X, Wang Q, Yeri AS, Zhuang X, Samykutty A, Mu J, Deng ZB, Zhang L, Mobley JA, Yan J, Van Keuren-Jensen K, Miller D .Oncotarget. 2016 May 12. doi: 10.18632/oncotarget.9325. [Epub ahead of print].

47.Nonstructural protein 5A is incorporated into hepatitis C virus low-density particle through interaction with core protein and microtubules during intracellular transport. Lai CK, Saxena V, Tseng CH, Jeng KS, Kohara M, Lai MM. PLoS One. 2014 Jun 6;9(6):e99022. doi: 10.1371/journal.pone.0099022. eCollection 2014.

48.Prerequisites for the analysis and sorting of extracellular vesicle subpopulations by high-resolution flow cytometry. Groot Kormelink T, Arkesteijn GJ, Nauwelaers FA, van den Engh G, Nolte-'t Hoen EN, Wauben MH. Cytometry A. 2016 Feb;89(2):135-47. doi: 10.1002/cyto.a.22644. Epub 2015 Feb 16.

49.Simplified protocol for flow cytometry analysis of fluorescently labeled exosomes and microvesicles using dedicated flow cytometer. Pospichalova V, Svoboda J, Dave Z, Kotrbova A, Kaiser K, Klemova D, Ilkovics L, Hampl A, Crha I, Jandakova E, Minar L, Weinberger V, Bryja V. J Extracell Vesicles. 2015 Mar 31;4:25530. doi: 10.3402/jev.v4.25530. eCollection 2015.

50.Fluorescent dyes for lymphocyte migration and proliferation studies. Parish CR. Immunol Cell Biol. 1999 Dec;77(6):499-508.

51.New fluorescent dyes for lymphocyte migration studies. Analysis by flow cytometry and fluorescence microscopy. Weston SA, Parish CR. J Immunol Methods. 1990 Oct 4;133(1):87-97.

52.Virus-mimetic nanovesicles as a versatile antigen-delivery system. Zhang P, Chen Y, Zeng Y, Shen C, Li R, Guo Z, Li S, Zheng Q, Chu C, Wang Z, Zheng Z, Tian R, Ge S, Zhang X, Xia NS, Liu G, Chen X. Proc Natl Acad Sci U S A. 2015 Nov 10;112(45):E6129-38. doi: 10.1073/pnas.1505799112. Epub 2015 Oct 26.