請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/49557
完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 蔡丰喬(Feng-Chiao Tsai) | |
dc.contributor.author | Pei-Ju Tsai | en |
dc.contributor.author | 蔡沛儒 | zh_TW |
dc.date.accessioned | 2021-06-15T11:34:38Z | - |
dc.date.available | 2016-08-26 | |
dc.date.copyright | 2016-08-26 | |
dc.date.issued | 2016 | |
dc.date.submitted | 2016-08-16 | |
dc.identifier.citation | 1. Hepatitis B. WHO Fact sheet 2016 July 2016; Available from: http://www.who.int/mediacentre/factsheets/fs204/en/.
2. Chang, M.H., Hepatitis B virus infection. Semin Fetal Neonatal Med, 2007. 12(3): p. 160-7. 3. 楊志元 and 陳祈玲, 台灣地區B 型肝炎病毒之血清流行病學研究, in 行政院衛生署疾病管制局100 年度科技研究發展計畫. 2011. 4. 許須美, 台灣B型肝炎疫苗史. 疫情報導, 1998: p. 82-91. 5. Ganem, D. and Prince, A., Hepatitis B virus infection--natural history and clinical consequences. N Engl J Med, 2004. 350(11): p. 1118-29. 6. Kremsdorf, D., Soussan, P., Paterlini-Brechot, P., and Brechot, C., Hepatitis B virus-related hepatocellular carcinoma: paradigms for viral-related human carcinogenesis. Oncogene, 2006. 25(27): p. 3823-33. 7. Cho, J.Y., Paik, Y.H., Sohn, W., Cho, H.C., Gwak, G.Y., Choi, M.S., Lee, J.H., Koh, K.C., Paik, S.W., and Yoo, B.C., Patients with chronic hepatitis B treated with oral antiviral therapy retain a higher risk for HCC compared with patients with inactive stage disease. Gut, 2014. 63(12): p. 1943-50. 8. Diao, J., Garces, R., and Richardson, C., X protein of hepatitis B virus modulates cytokine and growth factor related signal transduction pathways during the course of viral infections and hepatocarcinogenesis. Cytokine Growth Factor Rev., 2001. 12(2-3): p. 189-205. 9. Twu, J.S. and Schloemer, R.H., Transcriptional trans-Activating Function of Hepatitis B Virus. J Virol, 1987. 61(11): p. 3448-53. 10. Spandau, D.F. and Lee, C.H., trans-activation of viral enhancers by the hepatitis B virus X protein. J Virol, 1988. 62(2): p. 427-34. 11. Antonucci, T.K. and Rutter, W.J., Hepatitis B Virus (HBV) Promoters Are Regulated by the HBV Enhancer in a Tissue-Specific Manner. J Virol, 1989. 63(2): p. 579-83. 12. Bulla, G.A. and Siddiqui, A., The Hepatitis B Virus Enhancer Modulates Transcription of the Hepatitis B Virus Surface Antigen Gene from an Internal Location. J Virol, 1988. 62(4): p. 1437-41. 13. Shaul, Y., Rutter, W.J., and Laub, O., A human hepatitis B viral enhancer element. EMBO J, 1985. 4(2): p. 427-30. 14. Summers, J. and Mason, W.S., Replication of the genome of a hepatitis B--like virus by reverse transcription of an RNA intermediate. Cell, 1982. 29(2): p. 403-15. 15. Bouchard, M.J., Wang, L.H., and Schneider, R.J., Calcium signaling by HBx protein in hepatitis B virus DNA replication. Science, 2001. 294(5550): p. 2376-8. 16. Kim, C.M., Koike, K., Saito, I., Miyamura, T., and Jay, G., HBx gene of hepatitis B virus induces liver cancer in transgenic mice. Nature, 1991. 351(6324): p. 317-20. 17. Ma, W.L., Hsu, C.L., Wu, M.H., Wu, C.T., Wu, C.C., Lai, J.J., Jou, Y.S., Chen, C.W., Yeh, S., and Chang, C., Androgen receptor is a new potential therapeutic target for the treatment of hepatocellular carcinoma. Gastroenterology, 2008. 135(3): p. 947-55, 955 e1-5. 18. Chiu, C.M., Yeh, S.H., Chen, P.J., Kuo, T.J., Chang, C.J., Chen, P.J., Yang, W.J., and Chen, D.S., Hepatitis B virus X protein enhances androgen receptor-responsive gene expression depending on androgen level. Proc Natl Acad Sci U S A, 2007. 104(8): p. 2571-8. 19. Yang, W.J., Chang, C.J., Yeh, S.H., Lin, W.H., Wang, S.H., Tsai, T.F., Chen, D.S., and Chen, P.J., Hepatitis B virus X protein enhances the transcriptional activity of the androgen receptor through c-Src and glycogen synthase kinase-3beta kinase pathways. Hepatology, 2009. 49(5): p. 1515-24. 20. Klein, N.P., Bouchard, M.J., Wang, L.H., Kobarg, C., and Schneider, R.J., Src kinases involved in hepatitis B virus replication. EMBO J, 1999. 18(18): p. 5019-27. 21. Sanjay, A., Houghton, A., Neff, L., DiDomenico, E., Bardelay, C., Antoine, E., Levy, J., Gailit, J., Bowtell, D., Horne, W.C., and Baron, R., Cbl Associates with Pyk2 and Src to Regulate Src Kinase Activity, αvβ3 Integrin-Mediated Signaling, Cell Adhesion, and Osteoclast Motility. J Cell Biol, 2001. 152(1): p. 181-95. 22. Kohno, T., Matsuda, E., Sasaki, H., and Sasaki, T., Protein-tyrosine kinase CAKβ/PYK2 is activated by binding Ca2+/calmodulin to FERM F2 α2 helix and thus forming its dimer. Biochem J, 2008. 410(3): p. 513-23. 23. Rahmani, Z., Huh, K.W., Lasher, R., and Siddiqui, A., Hepatitis B virus X protein colocalizes to mitochondria with a human voltage-dependent anion channel, HVDAC3, and alters its transmembrane potential. J Virol, 2000. 74(6): p. 2840-6. 24. Shirakata, Y. and Koike, K., Hepatitis B virus X protein induces cell death by causing loss of mitochondrial membrane potential. J Biol Chem, 2003. 278(24): p. 22071-8. 25. Geng, X., Harry, B.L., Zhou, Q., Skeen-Gaar, R.R., Ge, X., Lee, E.S., Mitani, S., and Xue, D., Hepatitis B virus X protein targets the Bcl-2 protein CED-9 to induce intracellular Ca2+ increase and cell death in Caenorhabditis elegans. Proc Natl Acad Sci U S A, 2012. 109(45): p. 18465-70. 26. Altschuld, R.A., Hohl, C.M., Castillo, L.C., Garleb, A.A., Starling, R.C., and Brierley, G.P., Cyclosporin inhibits mitochondrial calcium efflux in isolated adult rat ventricular cardiomyocytes. Am J Physiol, 1992. 262(6 Pt 2): p. H1699-704. 27. Alonso, M.T., Barrero, M.J., Michelena, P., Carnicero, E., Cuchillo, I., García, A.G., García-Sancho, J., Montero, M., and Alvarez, J., Ca2+-induced Ca2+ Release in Chromaffin Cells Seen from inside the ER with Targeted Aequorin. J Cell Biol, 1999. 144(2): p. 241-54. 28. Kim, J.H., Lee, S.R., Li, L.H., Park, H.J., Park, J.H., Lee, K.Y., Kim, M.K., Shin, B.A., and Choi, S.Y., High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. PLoS One, 2011. 6(4): p. e18556. 29. Grynkiewicz, G., Poenie, M., and Tsien, R.Y., A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem, 1985. 260(6): p. 3440-50. 30. Kong, S.K. and Lee, C.Y., The Use of Fura 2 for Measurement of Free Calcium Concentration. Biochem Educ, 1995. 23(2): p. 97-98. 31. Case, R.M., Eisner, D., Gurney, A., Jones, O., Muallem, S., and Verkhratsky, A., Evolution of calcium homeostasis: from birth of the first cell to an omnipresent signalling system. Cell Calcium, 2007. 42(4-5): p. 345-50. 32. Strehler, E.E. and Zacharias, D.A., Role of alternative splicing in generating isoform diversity among plasma membrane calcium pumps. Physiol Rev, 2001. 81(1): p. 21-50. 33. Palmer, A.E., Giacomello, M., Kortemme, T., Hires, S.A., Lev-Ram, V., Baker, D., and Tsien, R.Y., Ca2+ indicators based on computationally redesigned calmodulin-peptide pairs. Chem Biol, 2006. 13(5): p. 521-30. 34. Stornaiuolo, M., Lotti, L.V., Borgese, N., Torrisi, M.R., Mottola, G., Martire, G., and Bonatti, S., KDEL and KKXX retrieval signals appended to the same reporter protein determine different trafficking between endoplasmic reticulum, intermediate compartment, and Golgi complex. Mol Biol Cell, 2003. 14(3): p. 889-902. 35. Ikebe, M. and Hartshorne, D.J., Phosphorylation of smooth muscle myosin at two distinct sites by myosin light chain kinase.pdf. J Biol Chem, 1985. 260(18): p. 10027-31. 36. Stephenson, G.M. and Stephenson, D.G., Endogenous MLC2 phosphorylation and Ca(2+)-activated force in mechanically skinned skeletal muscle fibres of the rat. Pflugers Arch, 1993. 424(1): p. 30-8. 37. Feske, S., CRAC channelopathies. Pflugers Arch, 2010. 460(2): p. 417-35. 38. Vervloessem, T., Yule, D.I., Bultynck, G., and Parys, J.B., The type 2 inositol 1,4,5-trisphosphate receptor, emerging functions for an intriguing Ca(2)(+)-release channel. Biochim Biophys Acta, 2015. 1853(9): p. 1992-2005. 39. de Meis, L., Oliveira, G.M., Arruda, A.P., Santos, R., Costa, R.M., and Benchimol, M., The thermogenic activity of rat brown adipose tissue and rabbit white muscle Ca2+-ATPase. IUBMB Life, 2005. 57(4-5): p. 337-45. 40. Lemasters, J.J., Theruvath, T.P., Zhong, Z., and Nieminen, A.L., Mitochondrial calcium and the permeability transition in cell death. Biochim Biophys Acta, 2009. 1787(11): p. 1395-401. 41. Srinivasan, B., Mitochondrial permeability transition pore: an enigmatic gatekeeper. NHS&T, 2012. 1(3): p. 47-51. 42. Mott, J.L., Zhang, D., Freeman, J.C., Mikolajczak, P., Chang, S.W., and Zassenhaus, H.P., Cardiac disease due to random mitochondrial DNA mutations is prevented by cyclosporin A. Biochem Biophys Res Commun, 2004. 319(4): p. 1210-5. 43. Rogers, T.B., Inesi, G., Wade, R., and W.J., L., Use of thapsigargin to study Ca2+ homeostasis in cardiac cells. Biosci Rep, 1995. 15(5): p. 341-9. 44. Korge, P. and Weiss, J.N., Thapsigargin directly induces the mitochondrial permeability transition. Eur J Biochem, 1999. 265(1): p. 273-80. 45. Schmidt, T.P., Goetz, C., Huemer, M., Schneider, G., and Wessler, S., Calcium binding protects E-cadherin from cleavage by Helicobacter pylori HtrA. Gut Pathogens, 2016. 8(1). 46. Hoesch, R.E., Yienger, K., Weinreich, D., and Kao, J.P., Coexistence of Functional IP3 and Ryanodine Receptors in Vagal Sensory Neurons and Their Activation by ATP. J Neurophysiol, 2002. 88(3): p. 1212-9. 47. Chami, M., Ferrari, D., Nicotera, P., Paterlini-Brechot, P., and Rizzuto, R., Caspase-dependent alterations of Ca2+ signaling in the induction of apoptosis by hepatitis B virus X protein. J Biol Chem, 2003. 278(34): p. 31745-55. 48. Bird, G.S., DeHaven, W.I., Smyth, J.T., and Putney, J.W., Jr., Methods for studying store-operated calcium entry. Methods, 2008. 46(3): p. 204-12. 49. Yang, B. and Bouchard, M.J., The hepatitis B virus X protein elevates cytosolic calcium signals by modulating mitochondrial calcium uptake. J Virol, 2012. 86(1): p. 313-27. 50. Geng, X., Huang, C., Qin, Y., McCombs, J.E., Yuan, Q., Harry, B.L., Palmer, A.E., Xia, N.S., and Xue, D., Hepatitis B virus X protein targets Bcl-2 proteins to increase intracellular calcium, required for virus replication and cell death induction. Proc Natl Acad Sci U S A, 2012. 109(45): p. 18471-6. 51. Rong, Y.P., Bultynck, G., Aromolaran, A.S., Zhong, F., Parys, J.B., De Smedt, H., Mignery, G.A., Roderick, H.L., Bootman, M.D., and Distelhorst, C.W., The BH4 domain of Bcl-2 inhibits ER calcium release and apoptosis by binding the regulatory and coupling domain of the IP3 receptor. Proc Natl Acad Sci U S A, 2009. 106(34): p. 14397-402. 52. Chae, H.J., Kim, H.R., Xu, C., Bailly-Maitre, B., Krajewska, M., Krajewski, S., Banares, S., Cui, J., Digicaylioglu, M., Ke, N., Kitada, S., Monosov, E., Thomas, M., Kress, C.L., Babendure, J.R., Tsien, R.Y., Lipton, S.A., and Reed, J.C., BI-1 regulates an apoptosis pathway linked to endoplasmic reticulum stress. Mol Cell, 2004. 15(3): p. 355-66. 53. Robinson, K.S., Clements, A., Williams, A.C., Berger, C.N., and Frankel, G., Bax inhibitor 1 in apoptosis and disease. Oncogene, 2011. 30(21): p. 2391-400. 54. Xu, Q. and Reed, J.C., Bax inhibitor-1, a mammalian apoptosis suppressor identified by functional screening in yeast. Mol Cell, 1998. 1(3): p. 337-46. 55. Youn, T.J., Piao, H., Kwon, J.S., Choi, S.Y., Kim, H.S., Park, D.G., Kim, D.W., Kim, Y.G., and Cho, M.C., Effects of the calcineurin dependent signaling pathway inhibition by cyclosporin A on early and late cardiac remodeling following myocardial infarction. Eur J Heart Fail, 2002. 4(6): p. 713-8. 56. Abell, E., Ahrends, R., Bandara, S., Park, B.O., and Teruel, M.N., Parallel adaptive feedback enhances reliability of the Ca2+ signaling system. Proc Natl Acad Sci U S A, 2011. 108(35): p. 14485-90. 57. Xiao, J., Liang, D., Zhao, H., Liu, Y., Zhang, H., Lu, X., Liu, Y., Li, J., Peng, L., and Chen, Y.H., 2-Aminoethoxydiphenyl borate, a inositol 1,4,5-triphosphate receptor inhibitor, prevents atrial fibrillation. Exp Biol Med (Maywood), 2010. 235(7): p. 862-8. 58. Sei, Y., Gallagher, K.L., and Daly, J.W., Multiple effects of caffeine on Ca2+ release and influx in human B lymphocytes. Cell Calcium, 2001. 29(3): p. 149-60. 59. Saleem, H., Tovey, S.C., Molinski, T.F., and Taylor, C.W., Interactions of antagonists with subtypes of inositol 1,4,5-trisphosphate (IP3) receptor. Br J Pharmacol, 2014. 171(13): p. 3298-312. 60. Pande, J., Szewczyk, M.M., and Grover, A.K., Allosteric inhibitors of plasma membrane Ca pumps: Invention and applications of caloxins. World J Biol Chem, 2011. 2(3): p. 39-47. 61. Sweeney, Z.K., Minatti, A., Button, D.C., and Patrick, S., Small-molecule inhibitors of store-operated calcium entry. ChemMedChem, 2009. 4(5): p. 706-18. 62. Kim, J.H., Kang, S., Kim, J., and Ahn, B.Y., Hepatitis B Virus Core Protein Stimulates the Proteasome-Mediated Degradation of Viral X Protein. Journal of Virology, 2003. 77(13): p. 7166-7173. 63. Hu, Z.Y., Zhang Z.S., Doo, E., Coux, O., Goldberg, A.L., and Liang, T.J., Hepatitis B Virus X Protein Is both a Substrate and a Potential Inhibitor of the Proteasome Complex. J Virol, 1999. 73(9): p. 7231-7240. 64. Lai, K.P., Law, A.Y., Lau, M.C., Takei, Y., Tse, W.K., and Wong, C.K., Osmotic stress transcription factor 1b (Ostf1b) promotes migration properties with the modulation of epithelial mesenchymal transition (EMT) phenotype in human embryonic kidney cell. Int J Biochem Cell Biol, 2013. 45(8): p. 1921-6. 65. Levrero, M. and Zucman-Rossi, J., Mechanisms of HBV-induced hepatocellular carcinoma. J Hepatol, 2016. 64(1 Suppl): p. S84-S101. 66. Decorsiere, A., Mueller, H., van Breugel, P.C., Abdul, F., Gerossier, L., Beran, R.K., Livingston, C.M., Niu, C., Fletcher, S.P., Hantz, O., and Strubin, M., Hepatitis B virus X protein identifies the Smc5/6 complex as a host restriction factor. Nature, 2016. 531(7594): p. 386-9. 67. Lilienbaum, A. and Israel, A., From Calcium to NF- B Signaling Pathways in Neurons. Molecular and Cellular Biology, 2003. 23(8): p. 2680-2698. 68. Khalaf, H., Jass, J., and Olsson, P.E., The role of calcium, NF-κB and NFAT in the regulation of CXCL8 and IL-6 expression in Jurkat T-cells. Int J Biochem Mol Biol, 2013. 4(3): p. 150-6. 69. Takeuchi, K., Sato, S.I., Abe, K., Kimura, M., Abe, T.A., Yoshinaga, K., and Inaba, H., Intracellular compartmentalization of fura-2 dye demonstrated by laser-excitation fluorescence microscopy: a problem in measuring cytosolic free calcium concentration using fura-2 fluorescence in vascular smooth muscle cells. Tohoku J Exp Med, 1989. 159(1): p. 23-35. 70. Chen, T.W., Wardill, T.J., Sun, Y., Pulver, S.R., Renninger, S.L., Baohan, A., Schreiter, E.R., Kerr, R.A., Orger, M.B., Jayaraman, V., Looger, L.L., Svoboda, K., and Kim, D.S., Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature, 2013. 499(7458): p. 295-300. 71. Tralhao, J.G., Roudier, J., Morosan, S., Giannini, C., Tu, H., Goulenok, C., Carnot, F., Zavala, F., Joulin, V., Kremsdorf, D., and Brechot, C., Paracrine in vivo inhibitory effects of hepatitis B virus X protein (HBx) on liver cell proliferation: an alternative mechanism of HBx-related pathogenesis. Proc Natl Acad Sci U S A, 2002. 99(10): p. 6991-6. 72. Chazotte, B., Labeling nuclear DNA with hoechst 33342. Cold Spring Harb Protoc, 2011. 2011(1): p. pdb prot5557. 73. Hancock, J.F., Cadwallader, K., Paterson, H., and Marshall, C.J., A CAAX or a CAAL motif and a second signal are sufficient for plasma membrane targeting of ras proteins. EMBO J, 1991. 10(13): p. 4033-9. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/49557 | - |
dc.description.abstract | 慢性B型肝炎病毒(HBV)感染是造成肝細胞癌的主因,但是我們尚未完全解明HBV引發肝細胞癌的機制。科學家發現HBV產生的HBx可以透過調控細胞內的鈣離子,促進HBV的複製,並造成細胞死亡。科學家還發現HBx轉基因鼠容易得到肝癌,由此研究推論HBx是引發肝細胞癌的重要原因。於是我們研究HBx究竟如何調控細胞內鈣離子訊號,且藉此推敲HBx引發肝細胞癌的機制。我們透過拍攝HepG2和Huh7兩種肝癌細胞的活細胞鈣離子螢光影像進行觀察與分析。透過分析細胞質鈣離子指示劑fura-2訊號,我們發現HBx會提升細胞質鈣離子濃度。除了觀察細胞質鈣離子濃度,我們利用T1ER和4mtD3cpv兩種genetic encoded Ca2+ indicators (GECI)分別測量內質網、粒線體的鈣離子濃度。轉染T1ER的細胞顯示表現HBx會提高內質網鈣離子儲藏量,但HBx對HepG2和Huh7粒線體鈣離子恆定的影響並不一致。我們通過鈣離子指示劑發現HBx會增加細胞質和內質網鈣離子恆定濃度。我們還利用西方墨點法計算肌球蛋白輕鏈2磷酸化比例,肌球蛋白輕鏈2活性會因為鈣離子濃度上升而增加,分析結果顯示不論是單純過量表現HBx或是讓細胞表現完整HBV基因組,肌球蛋白輕鏈2磷酸化程度都上升了,表示HBx引發的細胞鈣離子失衡可能影響癌細胞生理。
我們進一步確認HBx會不會透過改變鈣離子幫浦或鈣離子通道活性使鈣離子濃度增加。我們加入毒胡蘿蔔素(thapsigargin)和EGTA觀察鈣離子流變化,分析結果顯示,HBx除了降低內質網釋出鈣離子的速率,還會減緩鈣離子幫浦將鈣離子送出細胞的能力。使用thapsigargin也令內質網流失鈣離子,於是我們外加氯化鈣引起store-operated calcium entry (SOCE),發現HBx會讓SOCE活性上升。我們將攜帶完整HBV基因組的質體轉染到細胞內,發現雖然HBx讓HepG2內質網釋放鈣離子的速率及細胞膜排除鈣離子的幫浦活性下降,但是沒有在Huh7細胞看到這些趨勢。值得一提的是,我們在HepG2和Huh7細胞都觀察到表現HBV基因組會讓SOCE活性增加。為了確認上述實驗結果,我們正以慢病毒製作表現GECI或HBx/HBV的細胞株。我們會利用這些研究材料推敲HBx干預胞內鈣離子恆定的機制,也要檢驗這些鈣離子擾動是否會引發肝細胞癌的及影響病情進展。我們的研究將闡釋HBx對鈣離子訊息及恆定的影響,並為B型肝炎治療和預防帶來新的可能性。 | zh_TW |
dc.description.abstract | Chronic infection of hepatitis B virus (HBV) infection is one of the leading causes of hepatocellular carcinoma (HCC), but how HBV viral proteins cause HCC remains unknown. Previous researches revealed that one of the viral proteins, HBx, induced liver cancer in mouse models. Further investigations implied that HBx might modulate intracellular Ca2+ to promote the replication of HBV viruses and death of hepatocytes. We therefore study how HBx regulates intracellular Ca2+ signaling and its functional significance in HCC. Using live-cell fluorescence Ca2+ imaging on fura-2 loaded HepG2 and Huh7 cells, we noticed that HBx increased cytosolic Ca2+ concentration. Besides cytosolic [Ca2+], we measured [Ca2+] in endoplasmic reticulum (ER) and mitochondria, by applying genetic encoded Ca2+ indicators (GECI), T1ER and 4mtD3cpv, which could reflect [Ca2+] in ER and mitochondria, respectively. T1ER transfected cells showed that HBx increased [Ca2+] inside ER. However, HepG2 and Huh7 showed inconsistent changes of mitochondrial [Ca2+] with HBx expression. So we have found that HBx can increase [Ca2+] in cytosol and ER. Furthermore, western blots showed that Ca2+-dependent phosphorylation of myosin light chain 2 (p-MLC2) increased upon HBx expression, indicating that HBx-induced Ca2+ aberrancy may affect cancer cell physiology.
We further examine whether HBx changed Ca2+ channel or pump activities resulting in [Ca2+] increase. With the addition of thapsigargin and EGTA, we found that the HBx-transfected groups had reduced Ca2+ release from ER to cytosol, and decreased Ca2+ pumping from cytosol to the extracellular space. When thapsigargin depleted intra-ER [Ca2+], we administrated CaCl2, allowing cells to replenish Ca2+ through store- operated calcium entry (SOCE). Such experiments demonstrated that HBx upregulated SOCE. In HepG2 but not Huh7 transfected with wild type HBV genome, we also observed decreased Ca2+ flow from ER to cytosol, and downregulated plasma membrane pump activity. Interestingly, both HepG2 and Huh7 expressing HBV genome have increased SOCE activities. To verify the above results, we are making stable cell lines expressing GECI and/or HBx/HBV genome. Using these tools we will elucidate the mechanism how HBx disrupts intracellular Ca2+ homeostasis. We will also investigate whether those Ca2+ changes contribute to the initiation and progression of HCC. Our work will clarify the role of HBx on Ca2+ signaling, thus may shed light on HBV treatment and HCC prevention. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T11:34:38Z (GMT). No. of bitstreams: 1 ntu-105-R03443016-1.pdf: 9750994 bytes, checksum: 175e5107d0129bd6c859828255cf475e (MD5) Previous issue date: 2016 | en |
dc.description.tableofcontents | 圖目錄 iv
表目錄 vi Abstract vii 中文摘要 ix 第一章 研究動機 1 第一節 背景介紹 1 1、B型肝炎患者容易得到肝細胞癌 1 2、HBx會導致肝細胞癌 2 3、HBx會活化Src kinase 3 4、HBx透過活化Pyk2刺激Src訊息傳遞路徑 3 5、HBx能影響細胞鈣離子恆定增進病毒複製能力 4 第二節 研究目的 4 第三節 實驗思考流程 5 第二章 結果 6 第一節 製作含有HBx、P2A linker、螢光蛋白的質體 6 第二節 讓細胞表現HBx 10 第三節 染fura-2以偵測胞內鈣離子 11 第四節 HBx對於細胞鈣離子平衡狀態的影響 12 1、HBx讓細胞質鈣離子增加 12 2、粒線體的鈣離子恆定 14 3、內質網的鈣離子恆定 14 4、小結:HBx讓細胞質和內質網的鈣離子濃度上升 15 第五節 HBx讓p-MLC2的活性增加 18 第六節 HBx對胞內鈣離子流動的影響 21 1、粒線體的MPT變化在兩種細胞中不一致 23 2、內質網與細胞膜上鈣離子通道的變化 25 3、測量內質網釋放鈣離子的速率 27 4、IP3R受HBx影響 27 5、細胞排出鈣離子的速率 30 6、HBx讓SOCE活性增加 30 7、小結:HBx會影響內質網釋出鈣離子和鈣離子進出細胞的速率 33 第七節 轉染HBV是否會看到一樣的現象 34 1、內質網鈣離子釋出的速率並無顯著差異 34 2、細胞排出鈣離子的速率亦無顯著差異 34 3、SOCE因HBx顯著上升 34 第三章 討論 36 第一節 HBx如何影響細胞鈣離子恆定 36 第二節 如何印證HBx對各鈣離子幫浦是否有影響 38 第三節 為何轉染HBV看不到相同結果 40 第四節 如何增進p-MLC2定量結果的代表性 41 第五節 驗證HBx如何透過改變鈣離子恆定影響細胞生理 42 第六節 實驗上待克服的難題 43 1、如何消弭指示劑本身的影響 43 2、如何增加HBx的表現效率,減少未表現HBx的相消效果 43 第七節 以GECI驗證HBx對鈣離子恆定的影響 44 第四章 未來展望 47 第五章 材料與方法 48 第一節 製作含有HBx、P2A linker、螢光蛋白的質體 48 第二節 細胞培養 48 第三節 轉染質體 49 第四節 以fura-2測定細胞質鈣離子含量 50 第五節 以GECI測定胞器的鈣含量 50 第六節 萃取細胞蛋白質及定量 51 第七節 萃取細胞DNA並測定標的存在 53 第八節 透過慢病毒讓細胞表現GECI及HBx 54 第六章 附錄 57 第一節 附表 57 第二節 分析影像用到的程式碼 59 1、分析細胞fura-2訊號 59 2、視野下所有細胞隨時間變化的鈣離子訊號平均值 63 3、比較每組處理之細胞質鈣離子濃度 63 4、計算內質網釋出鈣離子的速率 66 5、計算細胞膜鈣離子幫浦活性 70 6、比較SOCE活性 74 7、分析轉染4mtD3cpv或T1ER細胞之鈣離子訊號 78 8、計算表現GECI細胞之胞器鈣離子濃度 83 9、計算粒線體鈣離子幫浦活性 86 第七章 參考資料 91 | |
dc.language.iso | zh-TW | |
dc.title | HBx對細胞鈣離子恆定影響之研究 | zh_TW |
dc.title | The Influence of HBx on Cellular Ca2+ Homeostasis | en |
dc.type | Thesis | |
dc.date.schoolyear | 104-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 張偉嶠(Wei-Chiao Chang),葉秀慧(Shiou-Hwei Yeh) | |
dc.subject.keyword | B 型肝炎病毒,HBx,鈣離子,fura-2,genetic encoded calcium indicator,store-operated calcium entry, | zh_TW |
dc.subject.keyword | hepatitis B virus,HBx,Ca2+,fura-2,genetic encoded calcium indicator,store-operated calcium entry, | en |
dc.relation.page | 97 | |
dc.identifier.doi | 10.6342/NTU201602932 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2016-08-17 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 藥理學研究所 | zh_TW |
顯示於系所單位: | 藥理學科所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-105-1.pdf 目前未授權公開取用 | 9.52 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。