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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76719完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 何佳安 | |
| dc.contributor.author | Ya-Ju Chang | en |
| dc.contributor.author | 張雅茹 | zh_TW |
| dc.date.accessioned | 2021-07-10T21:35:37Z | - |
| dc.date.available | 2021-07-10T21:35:37Z | - |
| dc.date.copyright | 2016-10-14 | |
| dc.date.issued | 2016 | |
| dc.date.submitted | 2016-08-02 | |
| dc.identifier.citation | 1. Ferlay, J. et al. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. International Journal of Cancer. 136, E359-386 (2015).
2. Ferlay, J. et al. Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. International Journal of Cancer. 127, 2893-2917 (2010). 3. Parkin, D.M., Bray, F., Ferlay, J. & Pisani, P. Global cancer statistics, 2002. CA: a Cancer Journal for Clinicians 55, 74-108 (2005). 4. Fattovich, G., Stroffolini, T., Zagni, I. & Donato, F. Hepatocellular carcinoma in cirrhosis: incidence and risk factors. Gastroenterology 127, S35-50 (2004). 5. Cohen, M. et al. Increased emotional distress in daughters of breast cancer patients is associated with decreased natural cytotoxic activity, elevated levels of stress hormones and decreased secretion of Th1 cytokines. International Journal of Cancer. 100, 347-354 (2002). 6. Shi, J., Zhu, L., Liu, S. & Xie, W.F. A meta-analysis of case-control studies on the combined effect of hepatitis B and C virus infections in causing hepatocellular carcinoma in China. British Journal of Cancer 92, 607-612 (2005). 7. Lavanchy, D. Hepatitis B virus epidemiology, disease burden, treatment, and current and emerging prevention and control measures. Journal of Viral Hepatitis 11, 97-107 (2004). 8. Parkin, D.M. The global health burden of infection-associated cancers in the year 2002. International Journal of Cancer. 118, 3030-3044 (2006). 9. Yang, J.D. et al. Cirrhosis is present in most patients with hepatitis B and hepatocellular carcinoma. Clinical Gastroenterology and Hepatology 9, 64-70 (2011). 10. Mittal, S. & El-Serag, H.B. Epidemiology of hepatocellular carcinoma: consider the population. Journal of Clinical Gastroenterology 47 Suppl, S2-6 (2013). 11. Henkler, F. et al. Intracellular localization of the hepatitis B virus HBx protein. The Journal of General Virology 82, 871-882 (2001). 12. Yang, Y. et al. HBV X protein (HBX) interacts with general transcription factor TFIIB both in vitro and in vivo. Chinese Medical Sciences Journal 14, 152-157 (1999). 13. Haviv, I., Shamay, M., Doitsh, G. & Shaul, Y. Hepatitis B virus pX targets TFIIB in transcription coactivation. Molecular and Cellular Biology 18, 1562-1569 (1998). 14. Qadri, I., Maguire, H.F. & Siddiqui, A. Hepatitis B virus transactivator protein X interacts with the TATA-binding protein. Proceedings of the National Academy of Sciences of the United States of America 92, 1003-1007 (1995). 15. Qadri, I., Conaway, J.W., Conaway, R.C., Schaack, J. & Siddiqui, A. Hepatitis B virus transactivator protein, HBx, associates with the components of TFIIH and stimulates the DNA helicase activity of TFIIH. Proceedings of the National Academy of Sciences of the United States of America 93, 10578-10583 (1996). 16. Le, T.T. et al. Mutational analysis of human RNA polymerase II subunit 5 (RPB5): the residues critical for interactions with TFIIF subunit RAP30 and hepatitis B virus X protein. Journal of Biochemistry 138, 215-224 (2005). 17. Jaitovich-Groisman, I. et al. Transcriptional regulation of the TFIIH transcription repair components XPB and XPD by the hepatitis B virus x protein in liver cells and transgenic liver tissue. The Journal of Biological Chemistry 276, 14124-14132 (2001). 18. Liu, H. et al. Hepatitis B virus X protein promotes hepatoma cell invasion and metastasis by stabilizing Snail protein. Cancer Science 103, 2072-2081 (2012). 19. Shih, W.L., Kuo, M.L., Chuang, S.E., Cheng, A.L. & Doong, S.L. Hepatitis B virus X protein inhibits transforming growth factor-beta -induced apoptosis through the activation of phosphatidylinositol 3-kinase pathway. The Journal of Biological Chemistry 275, 25858-25864 (2000). 20. Guan, J. et al. Involvement of extracellular signal-regulated kinase/mitogen-activated protein kinase pathway in multidrug resistance induced by HBx in hepatoma cell line. World Journal of Gastroenterology : WJG 10, 3522-3527 (2004). 21. Waris, G., Huh, K.W. & Siddiqui, A. Mitochondrially associated hepatitis B virus X protein constitutively activates transcription factors STAT-3 and NF-kappa B via oxidative stress. Molecular and Cellular Biology 21, 7721-7730 (2001). 22. Lee, Y.H. & Yun, Y. HBx protein of hepatitis B virus activates Jak1-STAT signaling. The Journal of Biological Chemistry 273, 25510-25515 (1998). 23. Godar, S. et al. Growth-inhibitory and tumor- suppressive functions of p53 depend on its repression of CD44 expression. Cell 134, 62-73 (2008). 24. Lee, S.G. & Rho, H.M. Transcriptional repression of the human p53 gene by hepatitis B viral X protein. Oncogene 19, 468-471 (2000). 25. Choi, B.H., Choi, M., Jeon, H.Y. & Rho, H.M. Hepatitis B viral X protein overcomes inhibition of E2F1 activity by pRb on the human Rb gene promoter. DNA and Cell Biology 20, 75-80 (2001). 26. Qadri, I., Ferrari, M.E. & Siddiqui, A. The hepatitis B virus transactivator protein, HBx, interacts with single-stranded DNA (ssDNA). Biochemical characterizations of the HBx-ssDNA interactions. The Journal of Biological Chemistry 271, 15443-15450 (1996). 27. Benn, J. & Schneider, R.J. Hepatitis B virus HBx protein activates Ras-GTP complex formation and establishes a Ras, Raf, MAP kinase signaling cascade. Proceedings of the National Academy of Sciences of the United States of America 91, 10350-10354 (1994). 28. Hohne, M. et al. Malignant transformation of immortalized transgenic hepatocytes after transfection with hepatitis B virus DNA. The EMBO journal 9, 1137-1145 (1990). 29. Murakami, S. Hepatitis B virus X protein: a multifunctional viral regulator. Journal of Gastroenterology 36, 651-660 (2001). 30. Jia, L., Wang, X.W. & Harris, C.C. Hepatitis B virus X protein inhibits nucleotide excision repair. International Journal of Cancer. 80, 875-879 (1999). 31. Fischer, M., Runkel, L. & Schaller, H. HBx protein of hepatitis B virus interacts with the C-terminal portion of a novel human proteasome alpha-subunit. Virus Genes 10, 99-102 (1995). 32. Schuster, R. et al. Conserved transactivating and pro-apoptotic functions of hepadnaviral X protein in ortho- and avihepadnaviruses. Oncogene 21, 6606-6613 (2002). 33. Lara-Pezzi, E. et al. The hepatitis B virus X protein promotes tumor cell invasion by inducing membrane-type matrix metalloproteinase-1 and cyclooxygenase-2 expression. The Journal of Clinical Investigation 110, 1831-1838 (2002). 34. Ou, D.P. et al. Clinical analysis of the risk factors for recurrence of HCC and its relationship with HBV. World Journal of Gastroenterology 11, 2061-2066 (2005). 35. Ou, D.P., Tao, Y.M., Chang, Z.G., Tang, F.Q. & Yang, L.Y. Hepatocellular carcinoma cells containing hepatitis B virus X protein have enhanced invasive potential conditionally. Digestive and liver disease : official journal of the Italian Society of Gastroenterology and the Italian Association for the Study of the Liver 38, 262-267 (2006). 36. Vu, T.H. & Werb, Z. Matrix metalloproteinases: effectors of development and normal physiology. Genes & Development 14, 2123-2133 (2000). 37. Roy, R., Zhang, B. & Moses, M.A. Making the cut: protease-mediated regulation of angiogenesis. Experimental Cell Research 312, 608-622 (2006). 38. Deryugina, E.I. & Quigley, J.P. Matrix metalloproteinases and tumor metastasis. Cancer Metastasis Reviews 25, 9-34 (2006). 39. Mohammed, F.F., Smookler, D.S. & Khokha, R. Metalloproteinases, inflammation, and rheumatoid arthritis. Annals of the Rheumatic Diseases 62 Suppl 2, ii43-47 (2003). 40. Ou, D.P., Tao, Y.M., Tang, F.Q. & Yang, L.Y. The hepatitis B virus X protein promotes hepatocellular carcinoma metastasis by upregulation of matrix metalloproteinases. International Journal of Cancer. 120, 1208-1214 (2007). 41. Itoh, T. et al. Reduced angiogenesis and tumor progression in gelatinase A-deficient mice. Cancer Research 58, 1048-1051 (1998). 42. Mignatti, P. & Rifkin, D.B. Biology and biochemistry of proteinases in tumor invasion. Physiological Reviews 73, 161-195 (1993). 43. Bergers, G. et al. Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis. Nature Cell Biology 2, 737-744 (2000). 44. Stam, J.C., Michiels, F., van der Kammen, R.A., Moolenaar, W.H. & Collard, J.G. Invasion of T-lymphoma cells: cooperation between Rho family GTPases and lysophospholipid receptor signaling. The EMBO Journal 17, 4066-4074 (1998). 45. Folkman, J. Role of angiogenesis in tumor growth and metastasis. Seminars in Oncology 29, 15-18 (2002). 46. Bjorklund, M. & Koivunen, E. Gelatinase-mediated migration and invasion of cancer cells. Biochimica et Biophysica Acta 1755, 37-69 (2005). 47. Lamb, R.F. et al. AP-1-mediated invasion requires increased expression of the hyaluronan receptor CD44. Molecular and Cellular Biology 17, 963-976 (1997). 48. Lara-Pezzi, E. et al. The hepatitis B virus X protein (HBx) induces a migratory phenotype in a CD44-dependent manner: possible role of HBx in invasion and metastasis. Hepatology 33, 1270-1281 (2001). 49. Natoli, G. et al. Ras- and Raf-dependent activation of c-jun transcriptional activity by the hepatitis B virus transactivator pX. Oncogene 9, 2837-2843 (1994). 50. Hofmann, M. et al. A link between ras and metastatic behavior of tumor cells: ras induces CD44 promoter activity and leads to low-level expression of metastasis-specific variants of CD44 in CREF cells. Cancer Research 53, 1516-1521 (1993). 51. Thomas, L., Etoh, T., Stamenkovic, I., Mihm, M.C., Jr. & Byers, H.R. Migration of human melanoma cells on hyaluronate is related to CD44 expression. The Journal of Investigative Dermatology 100, 115-120 (1993). 52. Haramaki, M. et al. Expression of CD44 in human hepatocellular carcinoma cell lines. Hepatology 21, 1276-1284 (1995). 53. Mathew, J. et al. CD44 is expressed in hepatocellular carcinomas showing vascular invasion. The Journal of Pathology 179, 74-79 (1996). 54. Lokeshwar, V.B. & Bourguignon, L.Y. The lymphoma transmembrane glycoprotein GP85 (CD44) is a novel guanine nucleotide-binding protein which regulates GP85 (CD44)-ankyrin interaction. The Journal of Biological Chemistry 267, 22073-22078 (1992). 55. Ponta, H., Sherman, L. & Herrlich, P.A. CD44: from adhesion molecules to signalling regulators. Nature reviews. Molecular cell biology 4, 33-45 (2003). 56. Goodison, S., Urquidi, V. & Tarin, D. CD44 cell adhesion molecules. Molecular Pathology 52, 189-196 (1999). 57. Fujita, N. et al. Expression of CD44 in normal human versus tumor endometrial tissues: possible implication of reduced expression of CD44 in lymph-vascular space involvement of cancer cells. Cancer Research 54, 3922-3928 (1994). 58. Orian-Rousseau, V. et al. Hepatocyte growth factor-induced Ras activation requires ERM proteins linked to both CD44v6 and F-actin. Molecular Biology of the Cell 18, 76-83 (2007). 59. Li, Z. et al. CD44v/CD44s expression patterns are associated with the survival of pancreatic carcinoma patients. Diagnostic Pathology 9, 79 (2014). 60. Cudmore, S., Reckmann, I. & Way, M. Viral manipulations of the actin cytoskeleton. Trends in Microbiology 5, 142-148 (1997). 61. Pearce-Pratt, R., Malamud, D. & Phillips, D.M. Role of the cytoskeleton in cell-to-cell transmission of human immunodeficiency virus. Journal of Virology 68, 2898-2905 (1994). 62. Chen, H.S. et al. The woodchuck hepatitis virus X gene is important for establishment of virus infection in woodchucks. Journal of Virology 67, 1218-1226 (1993). 63. Kimura, K., Nagaki, M., Saio, M., Moriwaki, H. & Kakimi, K. Role of CD44 in CTL-induced acute liver injury in hepatitis B virus transgenic mice. Journal of Gastroenterology 44, 218-227 (2009). 64. Ghigna, C. et al. Cell motility is controlled by SF2/ASF through alternative splicing of the Ron protooncogene. Molecular Cell 20, 881-890 (2005). 65. Warzecha, C.C., Sato, T.K., Nabet, B., Hogenesch, J.B. & Carstens, R.P. ESRP1 and ESRP2 are epithelial cell-type-specific regulators of FGFR2 splicing. Molecular Cell 33, 591-601 (2009). 66. Yanagisawa, M. et al. A p120 catenin isoform switch affects Rho activity, induces tumor cell invasion, and predicts metastatic disease. The Journal of Biological Chemistry 283, 18344-18354 (2008). 67. Warzecha, C.C. et al. An ESRP-regulated splicing programme is abrogated during the epithelial-mesenchymal transition. The EMBO Journal 29, 3286-3300 (2010). 68. Ishii, H. et al. Epithelial splicing regulatory proteins 1 (ESRP1) and 2 (ESRP2) suppress cancer cell motility via different mechanisms. The Journal of Biological Chemistry 289, 27386-27399 (2014). 69. Shapiro, I.M. et al. An EMT-driven alternative splicing program occurs in human breast cancer and modulates cellular phenotype. PLoS Genetics 7, e1002218 (2011). 70. Arzumanyan, A. et al. Epigenetic repression of E-cadherin expression by hepatitis B virus x antigen in liver cancer. Oncogene 31, 563-572 (2012). 71. Reinke, L.M., Xu, Y. & Cheng, C. Snail represses the splicing regulator epithelial splicing regulatory protein 1 to promote epithelial-mesenchymal transition. The Journal of Biological Chemistry 287, 36435-36442 (2012). 72. Chanmee, T., Ontong, P., Kimata, K. & Itano, N. Key Roles of Hyaluronan and Its CD44 Receptor in the Stemness and Survival of Cancer Stem Cells. Front Oncol 5, 180 (2015). 73. Beasley, R.P., Hwang, L.Y., Lin, C.C. & Chien, C.S. Hepatocellular carcinoma and hepatitis B virus. A prospective study of 22 707 men in Taiwan. Lancet 2, 1129-1133 (1981). 74. Hwang, G.Y. et al. Detection of the hepatitis B virus X protein (HBx) antigen and anti-HBx antibodies in cases of human hepatocellular carcinoma. Journal of Clinical Microbiology 41, 5598-5603 (2003). 75. de-Medina, T., Haviv, I., Noiman, S. & Shaul, Y. The X protein of hepatitis B virus has a ribo/deoxy ATPase activity. Virology 202, 401-407 (1994). 76. Kekule, A.S., Lauer, U., Weiss, L., Luber, B. & Hofschneider, P.H. Hepatitis B virus transactivator HBx uses a tumour promoter signalling pathway. Nature 361, 742-745 (1993). 77. Benn, J. & Schneider, R.J. Hepatitis B virus HBx protein deregulates cell cycle checkpoint controls. Proceedings of the National Academy of Sciences of the United States of America 92, 11215-11219 (1995). 78. Lv, M. et al. Identification of BST-2/tetherin-induced hepatitis B virus restriction and hepatocyte-specific BST-2 inactivation. Scientific Reports 5, 11736 (2015). 79. Takada, S., Kido, H., Fukutomi, A., Mori, T. & Koike, K. Interaction of hepatitis B virus X protein with a serine protease, tryptase TL2 as an inhibitor. Oncogene 9, 341-348 (1994). 80. Truant, R., Antunovic, J., Greenblatt, J., Prives, C. & Cromlish, J.A. Direct interaction of the hepatitis B virus HBx protein with p53 leads to inhibition by HBx of p53 response element-directed transactivation. Journal of Virology 69, 1851-1859 (1995). 81. Cheong, J.H., Yi, M., Lin, Y. & Murakami, S. Human RPB5, a subunit shared by eukaryotic nuclear RNA polymerases, binds human hepatitis B virus X protein and may play a role in X transactivation. The EMBO Journal 14, 143-150 (1995). 82. Murakami, S. Hepatitis B virus X protein: structure, function and biology. Intervirology 42, 81-99 (1999). 83. Cha, M.Y., Kim, C.M., Park, Y.M. & Ryu, W.S. Hepatitis B virus X protein is essential for the activation of Wnt/beta-catenin signaling in hepatoma cells. Hepatology 39, 1683-1693 (2004). 84. Werb, Z. ECM and cell surface proteolysis: regulating cellular ecology. Cell 91, 439-442 (1997). 85. Chung, T.W., Lee, Y.C. & Kim, C.H. Hepatitis B viral HBx induces matrix metalloproteinase-9 gene expression through activation of ERK and PI-3K/AKT pathways: involvement of invasive potential. FASEB journal 18, 1123-1125 (2004). 86. Zhang, X. et al. Up-regulated microRNA-143 transcribed by nuclear factor kappa B enhances hepatocarcinoma metastasis by repressing fibronectin expression. Hepatology 50, 490-499 (2009). 87. Lara-Pezzi, E. et al. Effect of the hepatitis B virus HBx protein on integrin-mediated adhesion to and migration on extracellular matrix. Journal of Hepatol 34, 409-415 (2001). 88. Lara-Pezzi, E., Roche, S., Andrisani, O.M., Sanchez-Madrid, F. & Lopez-Cabrera, M. The hepatitis B virus HBx protein induces adherens junction disruption in a src-dependent manner. Oncogene 20, 3323-3331 (2001). 89. Yang, S.Z. et al. Experimental study of epithelial-mesenchymal transition induced by HBx protein in liver cancer cell. Zhonghua yi xue za zhi 90, 818-821 (2010). 90. Teng, J., Wang, X., Xu, Z. & Tang, N. HBx-dependent activation of Twist mediates STAT3 control of epithelium-mesenchymal transition of liver cells. Journal of Cellular Biochemistry 114, 1097-1104 (2013). | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76719 | - |
| dc.description.abstract | B型肝炎病毒(Hepatitis B virus, HBV)會造成肝癌發生的致病因素之一，且會促進肝癌細胞的移動及侵襲(Liver cancer cell migration and invasion)的發生。研究指出HBV X蛋白表現的肝癌細胞株可觀察到CD44家族成員的表現量提高，而CD44標準型(CD 44 standard, CD44s)存在於所有正常細胞，表現量提高會促進與細胞外基質的交互作用促進細胞骨架的重組及形態的改變而影響生長，而CD44變異型(CD44 variant, CD44v)則是與腫瘤的轉移有很大的關係。因此HBV X蛋白在肝癌移動及轉移的過程中是否可藉由改變癌細胞膜上CD44s及CD44v的表現量變化而促進肝癌轉移是本篇研究想探討的重點，此外也希望能了解在肝癌轉移的過程中HBV X蛋白是透過怎麼樣訊息傳遞機制去調控CD44s及CD44v的表現。本篇研究使用穩定表現HBV X蛋白的肝癌細胞株進行實驗，由西方墨點法觀察到HBV X蛋白會促進N-cadherin及抑制E-cadherin表現造成EMT現象的發生，而從幾丁質酶譜法(Gelatin zymography)可看到MMP2/9的酶切能力提高，並且也利用西方墨點法及流式細胞儀確定了HBV X蛋白會促進CD44及CD44v6的表現，由這些結果可觀察到HBV X蛋白會提高肝癌細胞的轉移能力。而從細胞電生理的癌細胞移動/侵襲實驗也確認了HBV X蛋白會提高肝癌移行/侵襲能力。我們的實驗結果也觀察到在HBV X蛋白調控肝癌轉移時RNA剪切過程(Splicing)的關鍵因子ESRP1和ESRP2的表現。更進一步發現，HBV X蛋白促進肝癌轉移是透過轉錄因子Snail而去抑制ESRP1和提高ESRP2的表現進而去促進CD44v6的產生而增加肝癌轉移的能力，或許未來能藉由調控ESRP1和ESRP2的表現來阻止B型肝炎病毒衍生的肝癌轉移的發生。 | zh_TW |
| dc.description.abstract | Hepatitis B virus (HBV) is one of the main aetiological agents for hepatocellular carcinoma (HCC), which elevates mortality rate due to its high incidence of metastasis. The genome X of HBV produces transcriptional trans-activating protein kinase, which is associated with hepatocarcinogenesis. Among cancer stem cell (CSC) markers, CD44 is associated with migration, proliferation, and invasion. In particular, CD44 variant 6 (CD44v6) is critical for metastasis. Therefore HBV x proteim (HBx protein) may contribute to the acquisition of metastatic properties of HCC by increasing their ability to bind hyaluronan acid (HA) through expressing different CD44 in the outer margin of the tumors. The current study aims to elucidate the role of HBx in regulating CD44v6 during HCC metastatic processes. We used HBx-transfected HCC cell lines to confirm the role of HBx in HCC invasion by enhancing epithelial-mesenchymal transition (EMT), matrix metalloproteinase (MMP) 2/9 activity, and CD44v6 expression by immunoblotting and flow cytometry. The metastatic ability of HBx-induced HCC cell lines was investigated using real-time cell analyzer to monitor migration/invasion ability. Our results confirmed that HBx protein is able to promote the metastatic ability in HCC cell lines. Also, we found that there was an regulated protein expression of epithelial splicing regulatory protein (ESRP) 1/2 on HBx-induced HCC cell lines. Furthermore, we also found that HBx regulates metastatic modification of CD44 variants and downstream signaling through the splicing factors -ESRP1 and ESRP2, which function as specific modulators in CD44 splicing, and in enhancing metastatic ability. Additionally, HBx alters the expression and function of ESRP1 and ESRP2, which may be a key element for regulating ECM interaction and degradation on CD44v6-induced metastasis. Regulation of ESRP1 and ESRP2 might be a potential approach to arrest the progression of HBV-related metastasis. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-10T21:35:37Z (GMT). No. of bitstreams: 1 ntu-105-D00b22001-1.pdf: 5533569 bytes, checksum: ccccb3a48c197a826a4f4c64b77352d6 (MD5) Previous issue date: 2016 | en |
| dc.description.tableofcontents | 中文摘要 i
英文摘要 ii 第一章•緒論 1 1.1 HBV是促進肝癌發生之危險因子 1 1.2 HBV X蛋白在病毒感染所扮演的角色 2 1.3 HBV X基因及蛋白在細胞核及細胞質中的作用 3 1.4 HBV X蛋白與轉移的關係 5 1.5 HBV X蛋白與細胞膜上醣蛋白CD44的關係 7 1.6 CD44功能及與腫瘤的關係 8 1.7 HBV X蛋白促進EMT的發生對於細胞的影響 9 1.8 HBV X蛋白如何透過Snail調控EMT 11 第二章•研究動機 14 2.1 實驗動機 14 2.2 實驗探討之方向及訊息傳遞機制 16 第三章•實驗材料與方法 17 3.1 實驗藥品、抗體與儀器 17 3.2 實驗方法 26 3.2.1 細胞培養(Cell culture) 26 3.2.2 細胞穩定表現HBV X蛋白(HBV X protein expression) 29 3.2.3 RNA干擾(RNA interference, RNAi) 30 3.2.4 傷口愈合實驗(Wound-Healing assay) 31 3.2.5 xCELLigence即時細胞分析系統(Real-time cell analyzer) 31 3.2.6 幾丁質酶譜法(Gelatin zymography) 32 3.2.7 mRNA分析(mRNA analysis) 33 3.2.8 即時聚合酶鏈鎖反應(Real-time polymerase chain, real-time PCR) 34 3.2.9 基因表現分析affymetrix array (Gene expression microarray analysis) 34 3.2.10 免疫沉澱法( Immunoprecipitation, IP) 35 3.2.11 蛋白質萃取及定量 (Protein extraction and normalization) 36 3.2.12 西方墨點法(Immunoblotting) 38 3.2.13 染色質免疫沉澱 (Chromatin immunoprecipitation assay, ChIP) 44 3.2.14 免疫組織化學染色(Immunohistochemistry staining) 45 3.2.15 統計(Statistics) 47 第四章•實驗結果 48 4.1 HepG2細胞表現HBV X蛋白會顯著促進細胞的移動及侵襲作用 48 4.2 HepG2細胞表現HBV X蛋白時可觀察到細胞CD44s和 CD44v6表現量增加 50 4.3 HBV X蛋白會調控CD44剪切相關因子(ESRP1和ESRP2)的表現 51 4.4 HBV X蛋白調控CD44剪切作用經由改變剪切因子的表現而促進CD44v6的表現 52 4.5 HBV X蛋白促進CD44v6-依賴性轉移是經由Sanil去調節ESRP1和ESRP2的表現 53 4.6 抑制CD44剪切作用能有效抑制HBV X蛋白調控的惡性轉移 55 4.7 HBV X蛋白會促進EMT的現象，並且促進MMPs的作用以及EMT進而增加肝癌細胞的惡性轉移潛力 56 4.8 在臨床檢體發現，肝腫瘤表現HBV X蛋白會促進ESRP2的表現 58 第五章•討論 60 實驗結果圖表 63 Fig.1 HepG2x細胞經由Doxycycline處理後，可穩定在細胞中表現HBV X蛋白 67 Fig.2 HBV X蛋白在Doxycycline誘導處理下表現於肝癌細胞株中 69 Fig.3 HBV X蛋白表現於肝癌細胞中會促進肝癌細胞的移動能力 71 Fig.4隨HBV X蛋白表現增加可即時觀測到肝癌細胞移動能力增加 72 Fig.5 HBV X蛋白表現增加可觀察到肝癌細胞侵襲能力的提升 74 Fig.6 HBV X蛋白會促進CD44表現出含有不同外顯子的變異型 77 Fig.7 隨HBV X蛋白表現量提高， CD44s mRNA和CD44v6 mRNA表現也會增加 78 Fig. 8隨HBV X蛋白表現量增加，細胞CD44s 和 CD44v6 mRNA表現量改變 81 Fig.9 隨HBV X蛋白表現量增加，細胞CD44s and CD44v6 蛋白表現量增加，並顯著促進CD44v6表現 83 Fig.10 隨HBV X蛋白表現量增加，細胞內Esrp1和Esrp2 mRNA表現量改變 85 Fig.11 隨HBV X蛋白表現量增加，細胞ESRP1和ESRP2 蛋白表現量改變 87 Fig.12 HBV X蛋白會合到Esrp1和Esrp2基因的啟動子序列區域改變轉錄活性 90 Fig.13 觀察HBV X蛋白表現之肝癌細胞抑制Esrp1或Esrp2基因時，細胞CD44s和CD44v6表現 92 Fig.14、抑制Esrp1或Esrp2基因後觀察HBx促進之細胞移動及侵襲的能力的改變 94 Fig.15 隨HBV X蛋白表現量增加，細胞內轉錄因Snail表現量增加 96 Fig.16 細胞內表現之HBV X 蛋白會與轉錄因子Snail直接結合 97 Fig.17 抑制Snail表現時，會改變HBV X蛋白調控之ESRP1和ESRP2蛋白表現的功能 99 Fig.18 抑制Snail可觀察到細胞侵襲能力有被限制 101 Fig.19 HBV X蛋白會促進MMP2和MMP9蛋白表現 103 Fig.20 HBV X蛋白可透過CD44v6促進MMP2酶切能力增加 104 Fig.21 HBV X蛋白表現會促進細胞EMT現象 106 Fig.22 HBV X蛋白可透過CD44v6表現促進Snail的表現 107 Fig.23 觀察HCC病患有HBV感染及無感染之ESRPs蛋白之變化 112 Fig.24本篇研究訊息機制推論圖 113 參考文獻 115 | |
| dc.language.iso | zh-TW | |
| dc.subject | 肝癌轉移 | zh_TW |
| dc.subject | B型肝炎病毒 | zh_TW |
| dc.subject | HBx蛋白 | zh_TW |
| dc.subject | CD44及CD44v6 | zh_TW |
| dc.subject | ESRP1和ESRP2 | zh_TW |
| dc.subject | ESRP1 and ESRP2 | en |
| dc.subject | HBV | en |
| dc.subject | HCC metastasis | en |
| dc.subject | HBx protein | en |
| dc.subject | CD44 and CD44v6 | en |
| dc.title | 探討B型肝炎病毒之X蛋白促進肝癌轉移之機制:藉由調控上皮剪接調控蛋白1及2活化CD44v6的表現 | zh_TW |
| dc.title | Exploration of HBV X protein on the promotion of Hepatocellular carcinoma (HCC) metastasis: Through the activation of CD44 splice isoform switching by regulating splicing factor ESRP1/ESRP2 | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 104-2 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 徐士蘭,吳立真,鄭建中,陳平 | |
| dc.subject.keyword | B型肝炎病毒,HBx蛋白,CD44及CD44v6,ESRP1和ESRP2,肝癌轉移, | zh_TW |
| dc.subject.keyword | HBV,HBx protein,CD44 and CD44v6,ESRP1 and ESRP2,HCC metastasis, | en |
| dc.relation.page | 127 | |
| dc.identifier.doi | 10.6342/NTU201601829 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2016-08-03 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科技學系 | zh_TW |
| 顯示於系所單位： | 生化科技學系 | |
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