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  1. NTU Theses and Dissertations Repository
  2. 生物資源暨農學院
  3. 動物科學技術學系
請用此 Handle URI 來引用此文件: http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/48313
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dc.contributor.advisor朱有田,姜延年
dc.contributor.authorTung-Hsien Tsaien
dc.contributor.author蔡東憲zh_TW
dc.date.accessioned2021-06-15T06:52:12Z-
dc.date.available2016-02-20
dc.date.copyright2011-02-20
dc.date.issued2011
dc.date.submitted2011-02-14
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Baumrucker, C. R. and B. H. Stemberger. 1989. Insulin and insulin-like growth factor-I stimulate DNA synthesis in bovine mammary tissue in vitro. J. Anim. Sci. 67: 3503-3514.
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Farrelly, N., Y. J. Lee, J. Oliver, C. Dive, and C. H. Streuli. 1999. Extracellular matrix regulates apoptosis in mammary epithelium through a control on insulin signaling. J. Cell Biol. 144: 1337-1348.
Flint, D. J., M. Boutinaud, C. B. Whitelaw, G. J. Allan, and A. F. Kolb. 2006. Prolactin inhibits cell loss and decreases matrix metalloproteinase expression in the involuting mouse mammary gland but fails to prevent cell loss in the mammary glands of mice expressing IGFBP-5 as a mammary transgene. J. Mol. Endocrinol. 36: 435-448.
Hennighausen, L. and G. W. Robinson. 2001. Signaling pathways in mammary gland development. Dev. Cell 1: 467-475.
Hwa, V., Y. Oh, and R. G. Rosenfeld. 1999. The insulin-like growth factor-binding protein (IGFBP) superfamily. Endocr. Rev. 20: 761-787.
Imai, Y., A. Moralez, U. Andag, J. B. Clarke, W. H. Busby, Jr., and D. R. Clemmons. 2000. Substitutions for hydrophobic amino acids in the N-terminal domains of IGFBP-3 and -5 markedly reduce IGF-I binding and alter their biologic actions. J. Biol. Chem. 275: 18188-18194.
Jones, J. I. and D. R. Clemmons. 1995. Insulin-like growth factors and their binding proteins: biological actions. Endocr. Rev. 16: 3-34.
Kalus, W., M. Zweckstetter, C. Renner, Y. Sanchez, J. Georgescu, M. Grol, D. Demuth, R. Schumacher, C. Dony, K. Lang, and T. A. Holak. 1998. Structure of the IGF-binding domain of the insulin-like growth factor-binding protein-5 (IGFBP-5): implications for IGF and IGF-I receptor interactions. EMBO. J. 17: 6558-6572.
Marshman, E. and C. H. Streuli. 2002. Insulin-like growth factors and insulin-like growth factor binding proteins in mammary gland function. Breast Cancer Res. 4: 231-239.
Marshman, E., K. A. Green, D. J. Flint, A. White, C. H. Streuli, and M. Westwood. 2003. Insulin-like growth factor binding protein 5 and apoptosis in mammary epithelial cells. J. Cell Sci. 116: 675-682.
McGrath, M. F., R. J. Collier, D. R. Clemmons, W. H. Busby, C. A. Sweeny, and G. G. Krivi. 1991. The direct in vitro effect of insulin-like growth factors (IGFs) on normal bovine mammary cell proliferation and production of IGF binding proteins. Endocrinology 129: 671-678.
Neuenschwander, S., A. Schwartz, T. L. Wood, C. T. Roberts, Jr., L. Hennighausen, and D. LeRoith. 1996. Involution of the lactating mammary gland is inhibited by the IGF system in a transgenic mouse model. J. Clin. Invest. 97: 2225-2232.
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Schedlich, L. J., T. F. Young, S. M. Firth, and R. C. Baxter. 1998. Insulin-like growth factor-binding protein (IGFBP)-3 and IGFBP-5 share a common nuclear transport pathway in T47D human breast carcinoma cells. J. Biol. Chem. 273: 18347-18352.
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Sorrell, A. M., J. H. Shand, E. Tonner, M. Gamberoni, P. A. Accorsi, J. Beattie, G. J. Allan, and D. J. Flint. 2006. Insulin-like growth factor-binding protein-5 activates plasminogen by interaction with tissue plasminogen activator, independently of its ability to bind to plasminogen activator inhibitor-1, insulin-like growth factor-I, or heparin. J. Biol. Chem. 281: 10883-10889.
Ständker, L., P. Wobst, S. Mark, and W. G. Forssmann. 1998. Isolation and characterization of circulating 13-kDa C-terminal fragments of human insulin-like growth factor binding protein-5. FEBS. Lett. 441: 281-286.
Strange, R., F. Li, S. Saurer, A. Burkhardt, and R. R. Friis. 1992. Apoptotic cell death and tissue remodelling during mouse mammary gland involution. Development 115: 49-58.
Talhouk, R. S., M. J. Bissell, and Z. Werb. 1992. Coordinated expression of extracellular matrix-degrading proteinases and their inhibitors regulates mammary epithelial function during involution. J. Cell Biol. 118: 1271-1282.
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Xu, Q., B. Yan, S. Li, and C. Duan. 2004. Fibronectin binds insulin-like growth factor-binding protein 5 and abolishes its ligand-dependent action on cell migration. J. Biol. Chem. 279: 4269-4277.
Zhao, Y., P. Yin, L. A. Bach, and C. Duan. 2006. Several acidic amino acids in the N-domain of insulin-like growth factor-binding protein-5 are important for its transactivation activity. J. Biol. Chem. 281: 14184-14191.
Zhu, X., N. Ling, and S. Shimasaki. 1993. Cloning of the rat insulin-like growth factor binding protein-5 gene and DNA sequence analysis of its promoter region. Biochem. Biophys. Res. Commun. 190: 1045-1052.
dc.identifier.urihttp://tdr.lib.ntu.edu.tw/jspui/handle/123456789/48313-
dc.description.abstract類胰島素生長因子 (insulin-like growth factors, IGFs) 在哺乳類動物生理和發育中扮演著重要的角色,調控細胞的生長、分化和凋亡。其生化活性受到與其具有高親和力結合蛋白 (insulin-like growth factor binding proteins, IGFBPs) 的影響。在小鼠乳腺中,第五型類胰島素結合蛋白 (IGFBP-5) 於退乳時期會大量表現,藉由與IGFs的結合抑制下游驅動的細胞存活訊息,同時與tissue plasminogen activator (t-PA) 作用活化plasminogen成為plasmin,促進小鼠乳腺退乳。由於IGFBP-5於乳腺各時期及物種間的表現量並不相同,其功能也不盡相同。而IGFBP-5在山羊乳腺中所扮演的角色尚未明瞭,此研究之目的想探討山羊IGFBP-5對於不朽化山羊乳腺細胞株 (caprine mammary cell, CMC) 的影響。
首先,從山羊乳腺初級培養細胞當中將山羊IGFBP-5的cDNA選殖出來,將其轉譯出的胺基酸序列與其他物種做比對,在山羊、綿羊、牛、豬、人類、小鼠與大鼠間,相似度非常高。進一步將山羊IGFBP-5的cDNA架接至可於哺乳類動物細胞表現的載體上,轉染至CMC後,利用免疫螢光染色分析,山羊IGFBP-5主要表現在細胞質,且與內質網 (ER) 和高基氏體 (Golgi) 座落在相同的位置。為測定山羊IGFBP-5對於β-酪蛋白基因啟動子活性的影響,將山羊β-酪蛋白基因啟動子架接至具有螢火蟲冷光素酶基因之質體,另外將thymidine kinase 啟動子架接至具有珊瑚蟲冷光素酶基因之質體上,矯正細胞轉型效率,將上述兩個質體與山羊IGFBP-5 共同轉染至CMC,經泌乳素刺激後48 小時,利用雙重冷光酶系統分析β-酪蛋白基因啟動子之活性,發現外源性表現山羊IGFBP-5 於CMC 能降低由泌乳素誘導山羊β-酪蛋白基因啟動子之活性倍數。
為了克服細胞轉型效率不同的問題,利用pET system 表現山羊IGFBP-5 重組蛋白。首先將山羊IGFBP-5 架接至pET 質體中,並轉型至大腸桿菌宿主BL21(DE3)。利用IPTG 誘導山羊IGFBP-5 重組蛋白的表現,於添加後5 小時表現量最高,隨後以lysozyme和超音波震盪的方式破菌獲得粗萃的蛋白質,進一步利用Ni-NTA His Bind Resin 純化目標山羊IGFBP-5 重組蛋白,並找出純化最佳條件 [4 mL 粗萃蛋白與1 mL resin 結合反應1 小時,將混合物移入column 中,依序以4 mL Wsh Buffer(20 mM imidazole) 清洗column 1 次, 1 mL Wsh Buffer (40 mM imidazole) 清洗columnn 4 次。1 mL Wsh Buffer (50 mM imidazole) 清洗columnn 3 次。滴完後以0.5 mL Elute Buffer (80 和250 mM imidazole) 濾出目標山羊IGFBP-5 重組蛋白各4次]。純化的山羊IGFBP-5 重組蛋白以西方吸漬法分析,分子量約為34 kDa,且山羊IGFBP-5 重組蛋白可抑制由IGF-1 所誘導的下游Akt 磷酸化的情形,證實所產製出的山羊IGFBP-5 重組蛋白具有生化活性。
將山羊乳腺初級培養細胞和CMC,培養在富含胞外基質的Matrigel 當中,細胞會體外形成3D 的乳腺泡 (acini),於培養過程同時添加山羊IGFBP-5 重組蛋白(10 nM、100 nM),培養10 天後發現處理組的乳腺泡形成的數量較少,乳腺泡大小也比較小,經由螢光染色觀察後發現處理組的空腔形成情況並不明顯。
本試驗證實山羊IGFBP-5 會抑制泌乳素對於β-酪蛋白基因啟動子之活性的調控。此外,利用pET system 表現並純化重組的山羊IGFBP-5 蛋白,初步證明此蛋白具有生化活性,處理體外3D 培養的細胞,會影響乳腺泡的形成和大小。未來可更進一步利用重組的山羊IGFBP-5 來探討IGFBP-5 於乳腺發育過程中所可能扮演的角色及其所參與之分子機制。
zh_TW
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Previous issue date: 2011
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dc.description.tableofcontents目錄
頁次
目錄 i
圖次 ii
壹、 中文摘要 1
貳、 英文摘要 3
參、文獻檢討 5
肆、材料與方法 14
伍、結果 32
陸、討論 37
柒、結論 40
捌、參考文獻 41
dc.language.isozh-TW
dc.title山羊第五型類胰島素生長因子結合蛋白於乳腺細胞之功能研究zh_TW
dc.titleThe functional study of caprine IGFBP-5 in mammary cellsen
dc.typeThesis
dc.date.schoolyear99-1
dc.description.degree碩士
dc.contributor.oralexamcommittee李宜儒,楊瀅臻
dc.subject.keyword山羊,乳腺,第五型類胰島素生長因子結合蛋白,zh_TW
dc.subject.keywordcaprine,mammary,IGFBP-5,en
dc.relation.page62
dc.rights.note有償授權
dc.date.accepted2011-02-14
dc.contributor.author-college生物資源暨農學院zh_TW
dc.contributor.author-dept動物科學技術學研究所zh_TW
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