臺灣博碩士論文加值系統

摘要
關鍵字：肝癌(hepatocellular carcinoma, HCC); 肝癌衍生生長因子(hepatoma
derived growth factor; HDGF); 酵素免疫連結偵測(enzyme-linked
immunosorbent assay, ELISA); 中和作用 (neutralization); 單株抗體
(monoclonal antibodies)

肝癌在台灣為最常見之惡性腫瘤，全世界每年有超過壹佰萬人罹患肝癌。
目前已發現生長因子在肝細胞癌化過程中扮演著重要角色， 肝癌衍生生長
因子(hepatoma-derived growth factor; HDGF) 是由肝癌細胞株Huh-7 培養基
中分離出的新型生長因子，具有刺激纖維母細胞，內皮細胞和肝癌細胞生
長的能力。而且HDGF 過度表現與人類肝癌，肺癌和黑色素癌之癌化進行
也有密切關聯。除此之外HDGF 也會強烈影響肝癌患者手術後的存活情
況。在本論文中製造超過90% 純度(SDS-PAGE) 重組HDGF 蛋白質，然後
用這些重組HDGF 蛋白質，提升兔子血液中HDGF 多株抗體的濃度和生產
三株小鼠的HDGF 單株抗體。檢驗這些抗體特性沒有問題後，以多株抗體
作為捕捉抗體(capture antibodies) ，單株抗體作為偵測抗體(detection
antibodies) ，開發HDGF 酵素免疫連結偵測的檢驗方法(enzyme linked
immunosorbent assay; ELISA) 。在以重組HDGF 做為標準濃度的測試中，
這個ELISA 系統可以準確偵測到SK-Hep-1 細胞經病毒HDGF 基因傳送
後，培養液中HDGF 含量的變化。除此之外，本論文中也評估HDGF 抗體
對於肝癌細胞生長的影響，結果顯示，相較於用HDGF 免疫之前的兔子血
液，不論是HDGF 多株抗體或單株抗體皆具有抑制SK-Hep-1 肝癌細胞增
殖作用(p<0.05)，而且有劑量的效應。總而言之，在本研究中製造HDGF
單株抗體來發展偵測HDGF 的ELISA 偵測方法，同時也探討HDGF 單株
抗體應用在抑制肝癌細胞生長的能力。未來應再加強HDGF ELISA 偵測的
靈敏度，和評估HDGF 單株抗體治療肝癌潛力。

Hepatocellular carcinoma (HCC) is the most common malignant tumor in Taiwan with more than one million new cases annually in the world. Growth factors play important roles in liver carcinogenesis. Hepatoma-derived growth factor (HDGF), originally isolated from the cultured media of human hepatoma HuH-7 cells, stimulate the growth of fibroblast cells, endothelial cells, and hepatoma cells. Overexpression of HDGF is related to the transformation of human hepatoma, lung cancer and melanoma. Besides, HDGF also exerts strong influence on the prognosis of patients with hepatoma. In this study, recombinant HDGF was expressed and purified with higher purity than 90%. The recombinant protein was used to raise polyclonal HDGF antibodies in rabbits and to generate three lines of HDGF monoclonal antibodies in mice. After antibodies characterization, an in-house, sandwich HDGF ELISA system was established using the purified polyclonal anti-HDGF IgG as the capture antibodies and the monoclonal anti-HDGF IgG as the detection antibodies. By using recombinant HDGF as standard, this ELISA system accurately evaluate the changes of HDGF release in SK-Hep-1 cells after gene delivery. In addition, we also evaluated the effect of anti-HDGF on the growth of hepatoma cells. Application of either polyclonal or monoclonal HDGF antibodies, but not preimmune antibodies, inhibited the proliferation of SK-Hep-1 hepatoma cells in a dose-dependent manner. In summary, the present study generated HDGF monoclonal antibodies for development of HDGF ELISA and application on suppressing HCC progression. Future studies should be carried out to enhance the sensitivity of HDGF ELISA and to evaluate the therapeutic potential of HDGF antibodies for treatment of HCC.

Abstrat in Chinese------------------------------------1
Abstrat in English------------------------------------2
Introduction------------------------------------------3
Material and Methods----------------------------------7
Results-----------------------------------------------12
Discussion--------------------------------------------15
Feature perspective-----------------------------------16
Figures and Legend------------------------------------17
References--------------------------------------------25
Appendix----------------------------------------------27

REFERENCES
Aaronson, S. A. (1991). Growth factors and cancer. Science 254, 1146-1153.
Beasley, R. P. (1988). Hepatitis B virus. The major etiology of hepatocellular carcinoma. Cancer 61, 1942-1956.
Boyer., Z. Hepatology, A textbook of Liver Disease 3rd ed 1, 1: 42.
Bustin, M., Lehn, D. A., and Landsman, D. (1990). Structural features of the HMG chromosomal proteins and their genes. Biochim Biophys Acta 1049, 231-243.
Cross, M., and Dexter, T. M. (1991). Growth factors in development, transformation, and tumorigenesis. Cell 64, 271-280.
Einck, L., and Bustin, M. (1985). The intracellular distribution and function of the high mobility group chromosomal proteins. Exp Cell Res 156, 295-310.
Everett, A. D., and Bushweller, J. (2003). Hepatoma derived growth factor is a nuclear targeted mitogen. Curr Drug Targets 4, 367-371.
Everett, A. D., Stoops, T., and McNamara, C. A. (2001). Nuclear targeting is required for hepatoma-derived growth factor-stimulated mitogenesis in vascular smooth muscle cells. J Biol Chem 276, 37564-37568.
Folkman, J. (1992). The role of angiogenesis in tumor growth. Semin Cancer Biol 3, 65-71.
Graham, F. L., and Prevec, L. (1995). Methods for construction of adenovirus vectors. Mol Biotechnol 3, 207-220.
Hu, T. H., Huang, C. C., Liu, L. F., Lin, P. R., Liu, S. Y., Chang, H. W., Changchien, C. S., Lee, C. M., Chuang, J. H., and Tai, M. H. (2003). Expression of hepatoma-derived growth factor in hepatocellular carcinoma. Cancer 98, 1444-1456.
Izumoto, Y., Kuroda, T., Harada, H., Kishimoto, T., and Nakamura, H. (1997). Hepatoma-derived growth factor belongs to a gene family in mice showing significant homology in the amino terminus. Biochem Biophys Res Commun 238, 26-32.
Javaherian, K., Sadeghi, M., and Liu, L. F. (1979). Nonhistone proteins HMG1 and HMG2 unwind DNA double helix. Nucleic Acids Res 6, 3569-3580.
Kishima, Y., Yamamoto, H., Izumoto, Y., Yoshida, K., Enomoto, H., Yamamoto, M., Kuroda, T., Ito, H., Yoshizaki, K., and Nakamura, H. (2002). Hepatoma-derived growth factor stimulates cell growth after translocation to the nucleus by nuclear localization signals. J Biol Chem 277, 10315-10322.
Kuehl, L., Rechsteiner, M., and Wu, L. (1985). Relationship between the structure of chromosomal protein HMG1 and its accumulation in the cell nucleus. J Biol Chem 260, 10361-10368.
Mosevitsky, M. I., Novitskaya, V. A., Iogannsen, M. G., and Zabezhinsky, M. A. (1989). Tissue specificity of nucleo-cytoplasmic distribution of HMG1 and HMG2 proteins and their probable functions. Eur J Biochem 185, 303-310.
Nakamura, H., Izumoto, Y., Kambe, H., Kuroda, T., Mori, T., Kawamura, K., Yamamoto, H., and Kishimoto, T. (1994). Molecular cloning of complementary DNA for a novel human hepatoma-derived growth factor. Its homology with high mobility group-1 protein. J Biol Chem 269, 25143-25149.
Nakamura, H., Kambe, H., Egawa, T., Kimura, Y., Ito, H., Hayashi, E., Yamamoto, H., Sato, J., and Kishimoto, S. (1989). Partial purification and characterization of human hepatoma-derived growth factor. Clin Chim Acta 183, 273-284.
Reeck, G. R., Isackson, P. J., and Teller, D. C. (1982). Domain structure in high molecular weight high mobility group nonhistone chromatin proteins. Nature 300, 76-78.
Thanos, D., and Maniatis, T. (1992). The high mobility group protein HMG I(Y) is required for NF-kappa B-dependent virus induction of the human IFN-beta gene. Cell 71, 777-789.
Tsukuma, H., Hiyama, T., Tanaka, S., Nakao, M., Yabuuchi, T., Kitamura, T., Nakanishi, K., Fujimoto, I., Inoue, A., Yamazaki, H., and et al. (1993). Risk factors for hepatocellular carcinoma among patients with chronic liver disease. N Engl J Med 328, 1797-1801.
Nakamura, H., Yoshida, K., Ikegame, K., Kishima, Y., Uyama, H., Enomoto, H., (2002). Antibodies against hepatoma-derived growth factor and mucosal repair in ulcerative colitis. J Gastroenterology ;37;8-14