臺灣博碩士論文加值系統

肝癌在台灣為最常見的惡性腫瘤之一。近年來，許多生長因子與其接受體被鑑定出來，與肝癌發展有關的分子病變都曾被探討，他們的作用在肝癌生成的不同階段可能為正調節或負調節。肝癌衍生生長因子(hepatoma derived growth factor; HDGF)一種新的生長因子，於肝癌細胞株中被發現，表現出具有刺激纖維母細胞和肝癌細胞生長的能力。HDGF結構上類似於HMG蛋白的氨基酸序列和功能。HDGF被認為在細胞週期和腫瘤增殖上扮演某些角色。最近文獻更發現HDGF在腎臟胚胎發育中扮演重要角色，並認為此因子為血管內皮增生因子。此外HDGF 也和細胞間的相互作用(cell-to-cell interaction)及細胞移動(cell movement) 有關，HDGF為一生長因子，在血管的發育及疾病時可刺激平滑肌細胞 (SMCs) 增殖，HDGF帶有兩個nuclear localization sequence (NLS) 序列具有nuclear targeting 功能，可刺激細胞生長。在本研究當中，首先利用transfection與藥物選殖出表現HDGF之細胞株3T3-HDGF。實驗數據顯示，表現HDGF使細胞株之形態發生改變且生長速率變快約2倍，並增加細胞在血清缺乏情況下之生長。在動物實驗方面，注射3T3-HDGF 細胞之裸鼠會導致產生腫瘤，可能和HDGF在細胞週期和腫瘤增殖上扮演的角色有關。cellular stress 效應上包括H2O2、dexamethasome、CoCl2之作用並無明顯的差異。而腫瘤治療藥物taxol、cisplatin 對 3T3-HDGF 細胞較有效果， 3T3-HDGF 細胞則對KCN具有保護作用。利用不同強度UV照射與不同照射時間之研究發現HDGF表現會增加細胞株對UV 照射之敏感性並會造成細胞apoptosis。希望本研究之結果能對於肝癌衍生生長因子 (HDGF) 之細胞功能和可能影響血管內皮細胞和肝癌細胞生長之效應有進一步瞭解，並評估HDGF用於早期診斷肝癌、治療方法選擇及作為預後指標的可能性。

Hepatocellular carcinoma (HCC) is the most common and devastating malignant tumor in Taiwan. The major factors involved in the molecular pathogenesis for the development of HCC have been explored in recent years. An extensive array of growth factors and their receptors have been identified and may act as positive and negative modulators in different stages of hepatocarcinogenesis. HDGF (hepatoma-derived growth factor) is a novel growth factor, identified from conditioned medium of hepatoma cell line. HDGF has growth stimulating activity for fibroblast and some hepatoma cells. The high homology of protein sequence to HMG (high mobility group) protein but with distinct structure indicate it is a novel growth factor with mitogenic effect. Recently, elevated HDGF expression was found in developing kidneys but little in adult kidney. Besides, HDGF expression was found to be correlated with angiogenic status of tissues. Thus, it is speculated that HDGF plays a role during embryonic development and angiogenesis. Besides, HDGF also plays a role in cell-to-cell interaction and cell movement. HDGF is a growth factor that is involved in stimulating vascular smooth muscle cells (SMCs) proliferation during development and in disease. HDGF contains a true bipartite nuclear localization sequence necessary for nuclear targeting. It is required for HDGF stimulation of DNA replication and cell proliferation in vascular smooth muscle cell. In present studies, have transfected HDGF cDNA to 3T3 cell and the HDGF expression was verified by Western blot analysis. HDGF expression altered the morphologies and growth rate of 3T3 cells by 2-fold. Besides, HDGF-expressing cells were more resistant to serum-starvation. Injection of 3T3-HDGF cells, but not 3T3 cells, resulted in tumor formation in nude mice, suggesting that the angiogenic and mitogenic functions of HDGF might contribute to carcinogenesis. By using various reagents including H2O2, dexamethasome, taxol, cisplatin, CoCl2 and KCN, the cellular stress studies revealed differential responses between in 3T3 and 3T3-HDGFH. analyzed dose and time-dependent effects of UV irradiation and found that 3T3-HDGF cells are more sensitive to UV irradiation than 3T3 cells and susceptible to apoptosis. hope these experiments will bring further insights into the cellular function of HDGF, particularly during angiogenic process, thereby enable to evaluate its role during HCC progression and its potential as clinical marker and therapeutic target for HCC.

誌謝1
中文摘要3
英文摘要5
目錄7
前言11
實驗目的16
材料與方法17
細胞培養液 17
細胞培養 17
細胞數目測定18
Transfection 與選殖表現HDGF之細胞株 18
蛋白質濃度分析 19
蛋白質電泳分析 19
西方墨點法 (Western blotting) 20
細胞增殖分析(cell proliferation assay) 21
Hoechst 33258 DNA 染色 .21
體內( in vivo )試驗22
UV-irradiation22
細胞週期(cell cycle)之分析 23
結果24
Transfection並選殖表現HDGF之3T3 細胞株 24
探討表現HDGF細胞株之生長效應 24
探討表現HDGF細胞株之致癌效應 25
探討cellulra stress對於表現HDGF細胞株之效應26
研究化療藥物對於表現HDGF細胞株之效應 26
研究hypoxic agent(引發缺氧藥物)對於
表現HDGF細胞株之效應 26
研究經UV照射後表現HDGF細胞株之效應26
討論29
參考文獻36
圖表39
圖1. HDGF之構圖 39
圖2. HDGF在3T3 細胞表現與細胞功能
研究之流程圖 40
圖3. 3T3細胞經transfection後經Western blot確定41
圖4. 3T3與3T3-HDGF型態的改變 42
圖5. 3T3與3T3-HDGF細胞生長速率之比較 43
圖6. 3T3與3T3-HDGF細胞於
缺乏血清情況之生長速率之比較44
圖7. 注射3T3-HDGF細胞導致nude mice
產生腫瘤(相片) 45
圖8. 注射3T3-HDGF細胞導致nude mice
產生腫瘤46
圖9. 氧化劑與glucocorticoid對3T3和
3T3-HDGF細胞的生長效應圖47
圖10. chemotherapy drugs 對兩種細胞之作用48
圖11. 引發缺氧藥物對兩種細胞之作用49
圖12.3T3與3T3-HDGF細胞經UV照射後
型態改變之比較50
圖13. 3T3細胞經UV照射後不同時間
之細胞週期分析圖 51
圖14. 3T3-HDGF細胞經UV 照射後
不同時間之細胞週期分析52
圖15. 3T3與3T3-HDGF細胞經UV 照射後
不同時間之細胞週期分布之比較53
圖16 .不同UV照射強度對3T3與3T3-HDGF
細胞生長之影響54
圖17. 3T3 cell經不同強度UV照射後之細胞週期分析55
圖18. 3T3-HDGF細胞經不同強度UV照射後
之細胞週期分析56
圖19. 3T3與3T3-HDGF細胞經不同UV強度
照射後之細胞週期各期之比較57

1.Nakamura H.,Kambe H., and Egawa T, et al. (1989) Partial purification and characterization of human hepatoma-derived growth factor Clin.ChimActa. 183:273-284.
2.Nakamura H. , lzumoto Y., and Kambe H., et al. (1994) Molecular cloning of complementary DNA for a novel human hepatoma-derived growth factor. The Journa of Biological chemistry .269:25143-25149.
3.lzumoto Y., Kuroda T., and Harada H., et al. (1997) Hepatoma-Derived growth factor belongs to a gene family in mice showing significant homology in the amino terminus. Biochemical and biophysical research communications. 238:26-32.
4.Ikegame K., Yamamoto M.,and Kishima Y.., et al. (1999) A new member of a hepatoma-derived growth factor gene family can translocate to the nucleus. Biochem Biophys Res Commun 266(1):81-87.
5.Bustin M., Lehn DA., and Landsman D.,(1990) Biochim. Biophys. Acta 1049: 231-43.
6.Shirakaya H., Tsuda K., and Yoshida M. (1990) Biochemistry 29:4419-23.
7. Javaherian K., Liu LF., and Wang JC. (1978) Science 199: 1345-46.
8. Kuehl L., Rechsteiner M., and Wu L. (1985) J. Biol. Chem. 260: 10361-68.
9. Thanos D., and Maniatis T. (1992) Cell 71: 777-789.
10 Reeck GR.,Isackson PJ., and Teller DC. (1982) Nature 300: 76-78.
11.Einck L., and Bustin M. (1988) Exp. Cell Res. 156: 295-310.
12.Mosevitsky M., Novitskaya VA., and logannsen MG., et al. (1989) Tissue specificity of nucleo-cytoplasmic distribution of HMG1 and HMG2 proteins and their probable functions Eur.J. Biochem. 185:303-310.
13.Juan A. Oliver and Qais AI-Awquti. (1998) An endothelial growth factor involved in rat renal development. J. Clin. Invest. 102(6): 1208-1219.
14.Allen D., Everett, David R.,and Lobe, (2000) Hepatoma-derived
growth factor stimulates smooth muscle cell growth and is expressed
in vascular development J. Clin. Invest. 105:567-575.
15.Allen D., Everett , Tamara Stoops, and Coleen A., McNamara, (2001) Nuclear Targeting Is Required for Hepatoma-derived Growth Factor stimulated Mitogenesis in Vascular Smooth Muscle Cells The J. of Biolo. Chem. 276(40):7564—37568.
16.Everett AD. (2001) Identification cloning and developmental expression of hepatoma-derived growth factor in the developing rat heart.Dev. Dyn. 222(3):450-458.
17.Yoshihiko K., Hiroyasu Y., Yoshitaka I. ( 2002) Hepatoma-derived Growth Factor Stimulates Cell Growth after Translocation to the Nucleus by Nuclear Localization Signals J. Biol. Chem 277:10315-10322.
18.Inoue H., Shibuta K.,and Tanaka Y. (2001) Hepatoma-derived growth factor is associated with reduced sensitivity to irradiation in esophageal cancer. Cancer Res. 61:5714-5717.
19.Folkman, J. (1993) Diagnostic and therapeutic applications of angiogenesis research. C R Acad Sci.316: 909-918.
20.Pepper M. S., Ferrara N., Orci L., and Montesano R. (1992) Potent synergism between vascular endothelial growth factor and basic fibroblast growth factor in the induction of angiogenesis in vitro. Biochem Biophys Res Commun 189: 824-831.
21.Folkman J., and Shing Y. (1992) Angiogenesis. J Biol Chem 267: 10931-10934.
22.Ernst S., Henle and Stuart Linn, (1997) Formation, Prevention, and
Repair of DNA Damage by Iron/Hydrogen Peroxide.The J. of Biolo. Chem. 272( 31):19095—19098.
23.Li-Fen Lee, Guoxuan Li., Dennis J.( 1998) Paclitaxel (Taxol)-induced Gene Expression and Cell Death Are Both Mediated by the Activation of c-Jun NH2 —terminal Kinase (JNK/SAPK). The J. of Biolo. Chem 273(43): 28253—28260.
24.Xiantao Wang, Jennifer L., Martindale, and Nikki J. Holbrook.(2000) Requirement for ERK Activation in Cisplatin-induced Apoptosis The J. of Biolo. Chem. 275(50): 39435—39443.
25.Lison D1., De Boeck M2., Verougstraete, (2001) Update on the genotoxicity and carcinogenicity of cobalt compounds. Occupational & Environmental Medicine.58(10): 619-625.
26.Sakagami .T, Satoh K., Ishihara M. (2000) Effect of cobalt ion on radical intensity and cytotoxic activity of antioxidants. Anticancer Res.20(5A): 3143-50.
27.Torsten R., Dunkern and Bernd Kaina,( 2002) Cell Proliferation and DNA Breaks Are Involved in Ultraviolet Light-induced Apoptosis in Nucleotide Excision Repair-deficient Chinese Hamster Cells.
Molecular Biology of the Cell.(13): 348—361.
28.Dunkern T.R.,, Fritz G., Kaina B. (2001) Ultraviolet light-induced DNA damage triggers apoptosis in nucleotide excision repair-deficient cells via Bcl-2 decline and caspase-3/-8 activation. Oncogene.(20): 6026-6038.
29.Shiyong Wu., Yuanyuan Hu. , Ju-Lin Wang. (2002) Ultraviolet light inhibits translation through activation of the unfolded protein response kinase PERK in the lumen of the endoplasmic reticulum JBC Papers in Press. Published on March 4, as Manuscript M110164200.
30.Pavey S., Russell T., Gabrielli B. (2001) G2 phase cell cycle arrest in human skin following UV irradiation. Oncogene. 20(43): 6103-6110.