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研究生:陳秀玲
研究生(外文):Hsiu-Ling Cheng
論文名稱:人類肝細胞生長因子的工程改造及其活性分析
論文名稱(外文):Engineered human hepatocyte growth factor for pharmaceutical studies
指導教授:何世屏
指導教授(外文):Shiping He
學位類別:碩士
校院名稱:國立中山大學
系所名稱:生物科學系研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:中文
論文頁數:76
中文關鍵詞:肝細胞生長因子細胞測試重組蛋白重組基因
外文關鍵詞:recombinant geneHGFrecombinant proteincell assay
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肝細胞生長因子 (HGF) 是一種分泌型、在生理上屬多功能性的蛋白質。HGF通過與Met相結達到信號傳達的目的,可以誘導目標細胞進行有絲分裂或形態發生,提高細胞的活動力和侵入性等作用影響細胞的遷移,如此可促使癌細胞的轉移。HGF的α亞基包含了N端區域及四個kringle區域,其與受體結合的關鍵區域位於α亞基上。為了闡明α亞基的多個區域對受體結合及信號傳達的影響,本研究以kringle為單位設計了四個重組基因區域,分別為NK1、NK2、NK3和NK4,並以大腸桿菌為宿主,分別進行四個重組蛋白的表現並利用GST親和性層析管柱來進行重組蛋白的純化。所獲得的重組蛋白皆與GST形成融合蛋白,將重組蛋白進行細胞學測試。研究證明,四個缺失型重組蛋白對人類乳癌細胞的增生機轉有一定的影響,其中GST-NK1與GST-NK2的抑制效果比GST-NK3與GST-NK4明顯。這些結果說明由大腸桿菌所生產的GST-NKs融合蛋白對乳癌細胞具有抑制其生長的作用。本研究也為未來實測細胞增生與細胞轉移等提供了基因表現、純化的基本技術及研究路徑。
Hepatocyte growth factor (HGF) is a multifunctional protein, which secrets via Golgi complex after synthesized, and is hydrolyzed into an active heterodimer containing an α and a β chain by extracellular protease. It is known that HGF functions through surface domain of Met, and thus induces mitosis and metastasis. The interaction domain of HGF is believed to be located in the α-chain. In order to study these findings structurally and functionally, we designed and constructed four different recombinant coding regions of the gene (NK1, NK2, NK3, and NK4) which was then successfully expressed in E. coli. Purification of these four different recombinant proteins with glutathione-agarose column showed that all of the four constructs had been successfully expressed with some degradations. Cell proliferation assay showed that the recombinant proteins inhibited the growth of breast cancer cells to some extent. The assay also showed that GST-NK1 and GST-NK2 were better inhibitors than GST-NK3 and GST-NK4 to the cancer cells. It is concluded that E. coli expression is an appropriate system for achieving functional HGF.
中文摘要……………………………………………………………… 5
英文摘要……………………………………………………………… 6
第一章:概論………………………………………………………… 7
第二章:材料與方法…………………………………………………18
第三章:生長因子基因的重新改造…………………………………39
第四章:重組生長因子的表達及純化………………………………54
第五章:重組生長因子的細胞學鑑定………………………………62
第六章:結論…………………………………………………………71
參考文獻………………………………………………………………72
Blundell, T.L., Burke, D. F., Chirgadze, D., Dhanaraj, V., Hyvönen, M., Innis, C. A., Parisini, E., Pellegrini, L., Sayed, M. and Sibanda, B. L. (2000). Protein-protein interactions in receptor activation and intracellular signaling. Biol. Chem. 381, 955-959.

Bussolino, F., Di Renzo, M. F., Ziche, M., Bocchietto, E., Olivero, M., Naldini, L., Gaudino, G., Tamagnone, L., Coffer, A. and Comoglio, P. M. (1992). Hepatocyte growth factor is a potent angiogenic factor which stimulates endothelial cell motility and growth. J. Cell Biol. 119, 629-641.

Date, K., Matsumoto, K., Shimura, H., Tanaka, M. and Nakamura, T. (1997). HGF/NK4 is a specific antagonist for pleiotrophic actions of hepatocyte growth factor. FEBS Lett. 420, 1-6.

Gohda, E., Tsubouchi, H., Nakayama, H., Hirono, S., Takahashi, K., Koura, M., Hashimoto, S. and Daikuhara, Y. (1986). Human hepatocyte growth factor in plasma from patients with fulminant hepatic failure. Exp. Cell Res. 166, 139-150.

Gherardi, E., Youles, M. E., Miguel, R. N., Blundell, T. L., Iamele, L., Gough, J., Bandyopadhyay, A., Hartmann, G. and Butler, P. J. (2003). Functional map and domain structure of MET, the product of the c-met protooncogene and receptor for hepatocyte growth factor / scatter factor. Proc. Nat. Acad. Sci. 100, 12039-12044.

Gmyrek, G. A., Walburg, M., Webb, C. P., Yu, H. M., You, X., Vaughan, E. D. Woude, G. F. V. and Knudsen, B. S. (2001). Normal and malignant protease epithelial cells differ in their response to hepatocyte growth factor/scatter factor. Am. J. Pathol. 159, 579-590.

Jiang, W. G., Hiscox, S. E., Parr, C., Martin, T. A., Marsumoto, K., Nakamura, T. and Mansel, R. E. (1999). Antagonistic effect of NK4, a novel hepatocyte growth factor variant, on in Vitro angiogenesis of human vascular endothelial cells. Clin. Cancer Res. 5, 3695-3703.

Jiang, Y., Xu, W., Lu, J., He, F. and Yang, X. (2001). Invasiveness of hepatocellular carcinoma cell lines: contribution of hepatocyte growth factor, c-met, and transcription factor Ets-1. Biochem. Biophy. Res. Comm. 286, 1123-1130.

Kermorgant, S., Aparicio, T., Dessirier, V., J.M.Lewin, M. and Lehy, T. (2001). Hepatocyte growth factor induces colonic cancer cell invasiveness via enhanced motility and protease overproduction. Evidence for PI3 kinase and PKC involvement. Carcinogenesis. 22, 1036-1042.

Kirchhofer, D., Yao, X., Peek, M., Eigenbrot, C., Lipari, M. T., Billeci, K. L., Maun, H. R., Moran, P., Santell, L., Wiesmann, C. and Lazarus, L. A. (2004). Structural and functional basis of the serine protease-like hepatocyte growth factor β-chain in Met binding and signaling. J. Biol. Chem. 279, 39915-39924.

Kuba, K., Matsumoto, K., Date, K., Shimura, H., Tanaka, M. and Nakamura, T. (2000). HGF/NK4, a four-kringle antagonist of heaptocyte growth factor, is an angiogenesis inhibitor that suppresses tumor growth and metastasis in mice. Cancer res. 60, 6737-6743.

Lokker N. A. and Godowski, P. J. (1993). Generation and characterization of a competitive antagonist of human hepatocyte growth factor, HGF/NK1. J. Biol. Chem. 268, 17145-17150.

Lokker, N. A., Mark, M. R., Luis, E. A., Bennett, G. L., Robbins, K. A., Baker, J. B. Godowski, P. J. (1992). Structure-function analysis of hepatocyte growth factor: identification of variants that lack mitogenic activity yet retain high affinity receptor binding. EMBO J. 11, 2503-2510.

Lokker N. A., Presta, L. G. and Godowski, P. J. (1994). Mutational analysis and molecular modeling of the N-terminal kringle-containing domain of hepatocyte growth factor identifies amino acid side chains important for interaction with the c-Met receptor. Protein Eng. 7, 895-903.

Ma, H., Calderon, T. M., Kessel, T., Ashton, A. W. and Berman, J. W. (2003). Mechanisms of hepatocyte growth factor-mediated vascular smooth muscle cell migration. Circ. Res. 93, 1066-1073.

Martin, T. A., Parr, C., Davies, G., Watkins, G., Lane, J., Matsumoto, K., Nakamura, T., Mansel, R. E. and Jiang, W. G. (2003). Growth and angiogenesis of human breast cancer in a nude mouse tumour model is reduced by NK4, a HGF/SF antagonist. Carcinogenesis. 24, 1317-1323.

Matsumoto, K., Takehara, T., Inoue, H., Hagiya, M., Shimizu, S. and Nakamura, T. (1991). Deletion of kringle domains or the N-terminal hairpin structure in hepatocyte growth factor results in marked decreases in related biological activities. Biochem. Biophy. Res. Commun. 181, 691-699.

Merry, C. L. R., Bullock, S. L., Swan, D. C., Backen, M. L., Beddingtonm R. S. P., Wilson, V. A. and Gallagher, J. T. (2001). The molecular phenotype of heparan sulfate in the Hs2st-/- mutant mouse. J. Biol. Chem. 276, 35429-35434.

Miyazawa, K., Shimomura, T., Kitamura, A., Kondo, J., Morimoto, Y. and Kitamura, N. (1993). Molecular cloning and sequence analysis of the cDNA for a human serine protease responsible for activation of hepatocyte growth factor. J. Biol. Chem. 268, 10024-10028.

Mizuno, K., Inoue, H., Hagiya, M., Shimizu, S., Nose, T., Shimohigashi, Y. and Nakamura, T. (1994). Hairpin loop and second kringle domain are essential sites for heparin binding and biological activity of hepatocyte growth factor. J. Biol. Chem. 269, 1131-1136.

Naka, D., Ishii, T., Yoshiyama, Y., Miyazawa, K., Hara, H., Hishida, T. and Kitamura, N.(1989). Activation of hepatocyte growth factor by proteolytic conversion of a single chain form to a heterodimer. J. Biol. Chem. 267, 20114-20119.

Nakamura, T., Nawa, K., Ichihara, A., Kaise, N. and Nishino, T. (1987). Purification and subunit structure of hepatocyte growth factor from rat platelets. FEBS Lett. 224, 311-316.

Nakamura, T., Nishizawa, T., Hagiya, M., Seki, T., Shimonishi, M., Sugimura, A., Tashiro, K. and Shimizu, S. (1989). Molecular cloning and expression of human hepatocyte growth factor. Nature. 342, 440-443.

Nakamura, T., Teramoto, H. and Ichihara, A. (1986). Purification and characterization of a growth factor from rat platelets for mature parenchymal hepatocytes in primary cultures. Proc. Natl. Acad. Sci. 83, 6489-6493.

Nicola, N. A. (1994). Guidebook to cytokines and their receptors. Oxford University Press.

Parr, C., Watkins, G., Mansel, R. E. and Jiang, W. G. (2004). The hepatocyte growth factor regulatory factors in human breast cancer. Clin. Cancer Res. 10, 202-211.

Rahimi, N., Etchells, S. and Elliott, B. (1996). Hepatocyte growth factor (HGF) is a copper-binding protein: a facile probe for purification of HGF by immobilized Cu(Ⅱ)-affinity chromatography. Protein Expr. Purif. 7, 329-333.

Rainon, T. M., England, P., Ding, S., Demerens, C. and Tobelem, G. (2003). The N-termial domain of hepatocyte growth factor inhibits the angiogenic behavior of endothelial cells independently from binding to the c-met receptor. J. Biol. Chem. 278, 37400-37408.

Rosen, E. M., Kleinman, H. K., Goldberg, I. D., Bhargava, M. M., Nickoloff, B. J., Kinsella, J. L. and Polverimi, P. (1993). Scatter factor (hepatocyte growth factor) is a potent angiogenesis factor in vivo. Symp. Soc. Exp. Biol. 47, 227-234.

Russell, W. E., McGowan, J. A. and Bucher, N. L. (1984). Partial characterization of a hepatocyte growth factor from rat platelets. J. Cell Physiol. 119, 183-192.

Saccone, S., Narsimhan, R. P., Gaudino, G., Dalpra, L., Comoglio, P. M. and Della Valle, G. (1992). Regional mapping of the human hepatocyte growth factor (HGF)-scatter factor gene to chromosome 7q21.1. Genomics. 13, 912-914.

Schiering, N., Knapp, S., Marconi, M., Flocco, M. M., Cui, J., Perego, R., Rusconi, L. and Cristiani, C. (2003). Crystal structure of the tyrosine kinase domain of the hepatocyte growth factor receptor c-Met and its complex with the microbial alkaloid K-252a. Proc. Nat. Acad. Sci. 100, 12654-12659.

Stamos, J., Lazarus, R. A., Yao, X., Kirchhofer, D. and Wiesmann. (2004). Crystal structure of the HGF b-chain in complex with the Sema domain of the Met receptor. EMBO J. 23, 2325-2335.

Soker, S., Gollamudi-Payne, S., Fidder, H. and Charmahelli, H. (1997). Inhibition of vascular endothelial growth factor (VEGF)-induced endothelial cell proliferation by a peptide corresponding to the exon 7-encoded domain of VEGF165. J. Biol. Chem. 272, 31582-31588.

Tomikawa, M., Hashizume, M., Highashi, H.,Ohta, M. and Sugimachi, K. (1996). The role of the spleen, platelets, and plasma hepatocyte growth factor activity on hepatic regeneration in rats. J. Am. Coll. Surg. 182, 12-16.

Ultsch, M., Lokker N. A., Godowski, P. J. and Vos, A. M. (1998). Crystal structure of the NK1 fragment of human hepatocyte growth factor at 2.0 Å resolution. Structure. 6, 1383-1393.

Weidner, K. M., Arakaki, N., Hartmann, G., Vandekerckhove, J., Weingart, S., Rieder, H., Fonatsch, C., Tsubouchi, H., Hishida, T., Daikuharat, Y. and Birchmeier, W. (1991). Evidence for the identity of human scatter factor and human hepatocyte growth factor. Proc. Nat. Acad. Sci. 88, 7001-7005.

Weidner, K. M., Behrens, J., Vandekerckhove, J. and Birchmeier, W. (1990). Scatter factor: molecular characteristics and effect on the invasiveness of epithelial cells. J. Cell. Biol. 111, 2097-2108.

Wright, T. G., Tsai, J., Jia, Z. and Elliott, B. E. (2004). Inhibition by copper(Ⅱ) binding of hepatocyte growth factor (HGF) interaction with its receptor Met and blockade of HGF/Met function. J. Biol. Chem. 279, 32499-32506.

Yamagata, T., Muroya, K., Mukasa, T., Igarashi, H., Momoi, M., Tsukahara, T., Arahata, K., Kumagai, H and Momoi, T. (1995). Hepatocyte growth factor specifically expressed in microglia activated Ras in the neurons, similar to the action of neurotrophic factors. Biochem. Biophys. Res. Commun. 5, 231-237.

Zioncheck, T. F., Richardson, L., Liu, J., Chang, L., King, K. L., Bennett, G. L., Fügedi, P., Chamow, S. M., Schwall, R. H. and Stack R. J. (1995). Sulfated oligosaccharides promote hepatocyte growth factor association and govern its mitogenic activity. J. Biol. Chem. 270, 16871-16878.
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