臺灣博碩士論文加值系統

慢性病毒性肝炎是一個全球性的健康問題，一般並無明顯症狀或只是輕微的症狀表現，卻可能持續性的使肝臟發炎和發展成肝臟纖維化，約有20-30% 的慢性肝病患者會導致肝硬化或肝細胞癌。肝硬化所引起之併發症常是肝病患者之主要死因，因此，肝硬化的早期診斷是非常重要的。由肝臟星狀細胞所分泌的細胞外間質在肝臟纖維化的過程中扮演重要的角色，特別是間質金屬性蛋白酶（matrix metalloproteinase, MMPs）及金屬性蛋白酶之組織抑制因子（tissue inhibitor of metalloproteinase, TIMPs）。我們收集了30個因肝細胞癌或轉移性肝癌而做過腫瘤切除手術之tumorus and non-tumorus的肝臟組織。我們將GAPDH、MMP-1, -2, -9 和TIMP-1, -2之mRNA以即時聚合酶連鎖反應（real-time polymerase chain reaction）分別定量。根據METAVIR 分類系統將non-tumorus之肝臟組織依纖維化的程度分為F0-F4，同時偵測肝組織內hydroxylproline的分佈，評估collagen 在肝組織中的含量。MMP-9之mRNA表現在肝臟纖維化後期（F3 - F4）明顯高於肝纖維化前期 （F0 - F2）（p < 0.03），然而MMP-1, -2和TIMP-1, -2之mRNA表現在肝臟纖維化的分期中並無差異。肝臟組織中Hydroxylproline的分佈，在肝臟纖維化後期亦明顯高於肝纖維化早期（p < 0.05），並和TIMP-1（r=0.39, p=0.03），TIMP-2（r=0.39, p=0.03）呈現正相關。我們的實驗結果顯示，MMP-9 mRNA的表現在早期肝臟纖維化的診斷上扮演著重要的角色。

Chronic viral hepatitis is a global health problem. It is usually clinically silent but may undergo persistent hepatic inflammation and progressive fibrosis, which lead to liver cirrhosis and hepatocellular carcinoma (HCC) in about 20-30 % of patients. Because patients with advanced cirrhosis carry a grave long-term prognosis, early diagnosis of liver fibrosis and early cirrhosis is of paramount importance. Hepatic extracellular matrix (ECM), which is secreted by hepatic stellate cells (HSC), plays an important role in the hepatic fibrogenesis. We concomitantly collected tumorous and non-tumorous liver tissues from 30 patients who underwent surgical resections for hepatocellular carcinoma or metastatic liver cancers. Real-time polymerase chain reaction (RT-PCR) was performed to detect and quantitatively measure mRNA for GAPDH, MMP-1, -2, -9 and TIMP-1, -2 in these tissue samples. In the non-tumorous liver tissues, hepatic fibrosis was staged by METAVIR system (F0 - F4), and the collagen contents were determined by the hydroxyproline levels. The expression of MMP-9 mRNA was significantly higher in advanced fibrosis (F3 - F4) than that in early fibrosis or no fibrosis (F0 - F2). (p< 0.03), while the expressions of MMP-1, -2, -3 and TIMP-1, -2 mRNA were not statistically significant. Hydroxyproline levels were also significantly higher in advanced fibrosis than that in early fibrosis (p < 0.05) and positively correlated with TIMP-1 (r=0.39, p=0.03) and TIMP-2 (r=0.39, p=0.03). Our results showed that the expression of MMP-9 mRNA was valuable in the detection of early hepatic fibrosis.

目錄
誌　 謝.……………………………………………………………..... I
中文摘要 ………………………………………………..…………..… II
英文摘要 …………………………………………….……….............. IV
目　　錄 ………………………………………………………………VI
圖表目次………………………………………………...................... VIII
第一章　緒論 ..…………… .…..…………………………………...… 1
第二章 實驗材料及方法
一、 Liver samples .……………………….………........................ 4
二、 Detection and Quantitation of the messenger RNA for GAPDH, MMP-1, -2, -9 and TIMP-1, -2 ……………………….…..… 4
三、 Demonstration of fibrous septa in non-tumorous liver tissue ..5
四、 Determination of hydroxyproline level in non-tumorous liver tissue …………………………………………………..…….. 5
五、 Statistics analysis ……...……………..……………………. 6
第三章 實驗結果與分析
一、 Fibrosis status of the studied liver tissue sample ………….... 7
二、 Hydroxyproline levels and fibrosis status in liver samples .…7
三、 Expression of MMPs and TIMPs mRNA in liver tissues ...… 7
四、 Correlation of mRNA expression of MMPs and TIMPs to hydroxyproline levels ……………………………………….. 8
五、 Relationship between mRNA expression of various MMP genes and various TIMP genes ……………………………… 8
第四章　討論 …….……..………………………………...………..… 9
第五章 參考文獻 ………………….………………………….…..... 15
第六章 附錄 ……………………………………………….……….. 43

1.Anselmi, K., et al., Accelerated reversal of carbon tetrachloride-induced cirrhosis in rats by the endothelin receptor antagonist TAK-044. J Gastroenterol Hepatol, 2002. 17(5): p. 589-97.
2.Fallowfield, J.A. and J.P. Iredale, Reversal of liver fibrosis and cirrhosis--an emerging reality. Scott Med J, 2004. 49(1): p. 3-6.
3.Abonyi, M.E. and P.L. Lakatos, Ribavirin in the treatment of hepatitis C. Anticancer Res, 2005. 25(2B): p. 1315-20.
4.Papatheodoridis, G.V., et al., Impact of interferon-alpha therapy on liver fibrosis progression in patients with HBeAg-negative chronic hepatitis B. J Viral Hepat, 2005. 12(2): p. 199-206.
5.Sauerbruch, T., [Serologic diagnosis of liver fibrosis]. Dtsch Med Wochenschr, 1999. 124(41): p. 1191.
6.Schuppan, D., C. Jax, and E.G. Hahn, [Serum markers of liver fibrosis]. Dtsch Med Wochenschr, 1999. 124(41): p. 1213-8.
7.Tao, J., et al., Influence factors of serum fibrosis markers in liver fibrosis. World J Gastroenterol, 2003. 9(11): p. 2497-500.
8.Murphy, F., et al., N-Cadherin cleavage during activated hepatic stellate cell apoptosis is inhibited by tissue inhibitor of metalloproteinase-1. Comp Hepatol, 2004. 3 Suppl 1: p. S8.
9.Gardi, C., et al., Effect of free iron on collagen synthesis, cell proliferation and MMP-2 expression in rat hepatic stellate cells. Biochem Pharmacol, 2002. 64(7): p. 1139-45.
10.Iredale, J.P., et al., Mechanisms of spontaneous resolution of rat liver fibrosis. Hepatic stellate cell apoptosis and reduced hepatic expression of metalloproteinase inhibitors. J Clin Invest, 1998. 102(3): p. 538-49.
11.Yang, C., et al., Liver fibrosis: insights into migration of hepatic stellate cells in response to extracellular matrix and growth factors. Gastroenterology, 2003. 124(1): p. 147-59.
12.Xu, G.F., et al., Dynamic changes in the expression of matrix metalloproteinases and their inhibitors, TIMPs, during hepatic fibrosis induced by alcohol in rats. World J Gastroenterol, 2004. 10(24): p. 3621-7.
13.Benyon, R.C. and M.J. Arthur, Extracellular matrix degradation and the role of hepatic stellate cells. Semin Liver Dis, 2001. 21(3): p. 373-84.
14.Siller-Lopez, F., et al., Treatment with human metalloproteinase-8 gene delivery ameliorates experimental rat liver cirrhosis. Gastroenterology, 2004. 126(4): p. 1122-33; discussion 949.
15.Siller-Lopez, F., et al., Truncated active matrix metalloproteinase-8 gene expression in HepG2 cells is active against native type I collagen. J Hepatol, 2000. 33(5): p. 758-63.
16.Lee, H.S., et al., Differential role of p38 in IL-1alpha induction of MMP-9 and MMP-13 in an established liver myofibroblast cell line. J Biomed Sci, 2003. 10(6 Pt 2): p. 757-65.
17.Lichtinghagen, R., et al., Expression of matrix metalloproteinase-2 and -9 and their inhibitors in peripheral blood cells of patients with chronic hepatitis C. Clin Chem, 2000. 46(2): p. 183-92.
18.Tichopad, A., A. Didier, and M.W. Pfaffl, Inhibition of real-time RT-PCR quantification due to tissue-specific contaminants. Mol Cell Probes, 2004. 18(1): p. 45-50.
19.Fontaine, H., et al., Hepatitis activity index is a key factor in determining the natural history of chronic hepatitis C. Hum Pathol, 2001. 32(9): p. 904-9.
20.Schlemmer, B.O., et al., Quantitative PCR--new diagnostic tool for quantifying specific mRNA and DNA molecules: HER2/neu DNA quantification with LightCycler real-time PCR in comparison with immunohistochemistry and fluorescence in situ hybridization. Scand J Clin Lab Invest, 2004. 64(5): p. 511-22.
21.Christoph, F., et al., Quantitative detection of cytokeratin 20 mRNA expression in bladder carcinoma by real-time reverse transcriptase-polymerase chain reaction. Urology, 2004. 64(1): p. 157-61.
22.Lee, H.S., et al., Hydroxyproline content of needle biopsies as an objective measure of liver fibrosis: Emphasis on sampling variability. J Gastroenterol Hepatol, 2005. 20(7): p. 1109-14.
23.Li, D. and S.L. Friedman, Liver fibrogenesis and the role of hepatic stellate cells: new insights and prospects for therapy. J Gastroenterol Hepatol, 1999. 14(7): p. 618-33.
24.Lichtinghagen, R., et al., Expression and coordinated regulation of matrix metalloproteinases in chronic hepatitis C and hepatitis C virus-induced liver cirrhosis. Clin Sci (Lond), 2003. 105(3): p. 373-82.
25.Reif, S., et al., Matrix metalloproteinases 2 and 9 are markers of inflammation but not of the degree of fibrosis in chronic hepatitis C. Digestion, 2005. 71(2): p. 124-30.
26.Zhou, X., et al., Expression of matrix metalloproteinase-2 and -14 persists during early resolution of experimental liver fibrosis and might contribute to fibrolysis. Liver Int, 2004. 24(5): p. 492-501.
27.Okazaki, I., et al., [Gene expression of MMPs and TIMPs in the process of hepatic fibrosis]. Nippon Rinsho, 1993. 51(2): p. 428-34.
28.Walsh, K.M., et al., Plasma levels of matrix metalloproteinase-2 (MMP-2) and tissue inhibitors of metalloproteinases -1 and -2 (TIMP-1 and TIMP-2) as noninvasive markers of liver disease in chronic hepatitis C: comparison using ROC analysis. Dig Dis Sci, 1999. 44(3): p. 624-30.
29.Murawaki, Y., et al., Serum matrix metalloproteinase-1 in patients with chronic viral hepatitis. J Gastroenterol Hepatol, 1999. 14(2): p. 138-45.
30.Schnur, J., et al., Thioacetamide-induced hepatic fibrosis in transforming growth factor beta-1 transgenic mice. Eur J Gastroenterol Hepatol, 2004. 16(2): p. 127-33.
31.Wang, X.B., et al., [The role of changes of MMP-2, 9 activity in the development of liver fibrosis in rats]. Zhonghua Gan Zang Bing Za Zhi, 2004. 12(5): p. 267-70.
32.Parsons, C.J., et al., Antifibrotic effects of a tissue inhibitor of metalloproteinase-1 antibody on established liver fibrosis in rats. Hepatology, 2004. 40(5): p. 1106-15.
33.Visse, R. and H. Nagase, Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry. Circ Res, 2003. 92(8): p. 827-39.
34.Okamoto, T., et al., Molecular mechanism for activation and regulation of matrix metalloproteinases during bacterial infections and respiratory inflammation. Biol Chem, 2004. 385(11): p. 997-1006.
35.Paquette, B., et al., Activation of matrix metalloproteinase-2 and -9 by 2- and 4-hydroxyestradiol. J Steroid Biochem Mol Biol, 2003. 87(1): p. 65-73.
36.Xu, L., et al., Human hepatic stellate cell lines, LX-1 and LX-2: new tools for analysis of hepatic fibrosis. Gut, 2005. 54(1): p. 142-51.
37.Reif, S., et al., Matrix Metalloproteinases 2 and 9 Are Markers of Inflammation but Not of the Degree of Fibrosis in Chronic Hepatitis C. Digestion, 2005. 71(2): p. 124-130.
38.Tambunting, F., et al., Increased lung matrix metalloproteinase-9 levels in extremely premature baboons with bronchopulmonary dysplasia. Pediatr Pulmonol, 2005. 39(1): p. 5-14.
39.Iglesias, D., et al., Metalloproteinases and tissue inhibitors of metalloproteinases in exudative pleural effusions. Eur Respir J, 2005. 25(1): p. 104-9.
40.May, A.E., et al., Plasminogen and matrix metalloproteinase activation by enzymatically modified low density lipoproteins in monocytes and smooth muscle cells. Thromb Haemost, 2005. 93(4): p. 710-5.
41.Jiang, Z.Q., et al., [Relationship between expression of matrix metalloproteinase (MMP-9) and tumor angiogenesis, cancer cell proliferation, invasion, and metastasis in invasive carcinoma of cervix]. Ai Zheng, 2003. 22(2): p. 178-84.
42.Wang, L., et al., [Expression of MMP-9 and MMP-9 mRNA in gastric carcinoma and its correlation with angiogenesis]. Zhonghua Yi Xue Za Zhi, 2003. 83(9): p. 782-6.
43.Zhang, S., et al., Imbalance between expression of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 in invasiveness and metastasis of human gastric carcinoma. World J Gastroenterol, 2003. 9(5): p. 899-904.
44.Chen, J.J., et al., Tumor-associated macrophages: the double-edged sword in cancer progression. J Clin Oncol, 2005. 23(5): p. 953-64.
45.Gu, Z.D., et al., Clinical significance of matrix metalloproteinase-9 expression in esophageal squamous cell carcinoma. World J Gastroenterol, 2005. 11(6): p. 871-4.
46.de Vicente, J.C., et al., Expression and clinical significance of matrix metalloproteinase-2 and matrix metalloproteinase-9 in oral squamous cell carcinoma. Oral Oncol, 2005. 41(3): p. 283-93.
47.Morelli, C., et al., Activity of the matrix metalloproteinase-9 promoter in human normal and tumor cells. J Cell Physiol, 2004. 199(1): p. 126-33.
48.Masson, V., et al., Contribution of host MMP-2 and MMP-9 to promote tumor vascularization and invasion of malignant keratinocytes. Faseb J, 2005. 19(2): p. 234-6.
49.Fridman, R., et al., Cell surface association of matrix metalloproteinase-9 (gelatinase B). Cancer Metastasis Rev, 2003. 22(2-3): p. 153-66.
50.Takeuchi, T., et al., The membrane-anchored matrix metalloproteinase (MMP) regulator RECK in combination with MMP-9 serves as an informative prognostic indicator for colorectal cancer. Clin Cancer Res, 2004. 10(16): p. 5572-9.
51.Masui, T., et al., RECK expression in pancreatic cancer: its correlation with lower invasiveness and better prognosis. Clin Cancer Res, 2003. 9(5): p. 1779-84.
52.Span, P.N., et al., Matrix metalloproteinase inhibitor reversion-inducing cysteine-rich protein with Kazal motifs: a prognostic marker for good clinical outcome in human breast carcinoma. Cancer, 2003. 97(11): p. 2710-5.
53.Sasahara, R.M., et al., Transcriptional control of the RECK metastasis/angiogenesis suppressor gene. Cancer Detect Prev, 2002. 26(6): p. 435-43.
54.Oh, J., et al., The membrane-anchored MMP inhibitor RECK is a key regulator of extracellular matrix integrity and angiogenesis. Cell, 2001. 107(6): p. 789-800.
55.Ozaki, I., et al., Hepatocyte growth factor induces collagenase (matrix metalloproteinase-1) via the transcription factor Ets-1 in human hepatic stellate cell line. J Hepatol, 2002. 36(2): p. 169-78.
56.Schwabe, R.F., et al., CD40 activates NF-kappa B and c-Jun N-terminal kinase and enhances chemokine secretion on activated human hepatic stellate cells. J Immunol, 2001. 166(11): p. 6812-9.
57.Kousidou, O.C., et al., Expression of MMPs and TIMPs genes in human breast cancer epithelial cells depends on cell culture conditions and is associated with their invasive potential. Anticancer Res, 2004. 24(6): p. 4025-30.
58.Kasprzak, A., et al., Expression of cytokines (TNF-alpha, IL-1alpha, and IL-2) in chronic hepatitis C: comparative hybridocytochemical and immunocytochemical study in children and adult patients. J Histochem Cytochem, 2004. 52(1): p. 29-38.
59.Giannelli, G., et al., Clinical role of MMP-2/TIMP-2 imbalance in hepatocellular carcinoma. Int J Cancer, 2002. 97(4): p. 425-31.
60.Oakley, F., et al., Inhibition of inhibitor of kappaB kinases stimulates hepatic stellate cell apoptosis and accelerated recovery from rat liver fibrosis. Gastroenterology, 2005. 128(1): p. 108-20.
61.Frankenberger, M., et al., All trans-retinoic acid selectively down-regulates matrix metalloproteinase-9 (MMP-9) and up-regulates tissue inhibitor of metalloproteinase-1 (TIMP-1) in human bronchoalveolar lavage cells. Mol Med, 2001. 7(4): p. 263-70.
62.Iredale, J.P., Tissue inhibitors of metalloproteinases in liver fibrosis. Int J Biochem Cell Biol, 1997. 29(1): p. 43-54.
63.Siddique, K., et al., Expression of matrix metalloproteinases, their inhibitors, and urokinase plasminogen activator in human meningiomas. Int J Oncol, 2003. 22(2): p. 289-94.
64.Fassina, G., et al., Tissue inhibitors of metalloproteases: regulation and biological activities. Clin Exp Metastasis, 2000. 18(2): p. 111-20.
65.Gakiopoulou, H., et al., Tissue inhibitor of metalloproteinase-2 as a multifunctional molecule of which the expression is associated with adverse prognosis of patients with urothelial bladder carcinomas. Clin Cancer Res, 2003. 9(15): p. 5573-81.
66.Jiang, Y., I.D. Goldberg, and Y.E. Shi, Complex roles of tissue inhibitors of metalloproteinases in cancer. Oncogene, 2002. 21(14): p. 2245-52.
67.Joo, Y.E., et al., Expression of tissue inhibitors of metalloproteinases (TIMPs) in hepatocellular carcinoma. Korean J Intern Med, 2000. 15(3): p. 171-8.
68.Pares, A., et al., Serum hyaluronate reflects hepatic fibrogenesis in alcoholic liver disease and is useful as a marker of fibrosis. Hepatology, 1996. 24(6): p. 1399-403.
69.Roskams, T., et al., Heparan sulfate proteoglycan expression in chronic cholestatic human liver diseases. Hepatology, 1996. 24(3): p. 524-32.
70.Gallai, M., et al., Expression of extracellular matrix proteoglycans perlecan and decorin in carbon-tetrachloride-injured rat liver and in isolated liver cells. Am J Pathol, 1996. 148(5): p. 1463-71.
71.Mongiat, M., et al., Fibroblast growth factor-binding protein is a novel partner for perlecan protein core. J Biol Chem, 2001. 276(13): p. 10263-71.
72.Grisanti, S., et al., Decorin modulates wound healing in experimental glaucoma filtration surgery: a pilot study. Invest Ophthalmol Vis Sci, 2005. 46(1): p. 191-6.
73.Vij, N., et al., Lumican regulates corneal inflammatory responses by modulating Fas-Fas ligand signaling. Invest Ophthalmol Vis Sci, 2005. 46(1): p. 88-95.
74.Davies, J.E., et al., Decorin suppresses neurocan, brevican, phosphacan and NG2 expression and promotes axon growth across adult rat spinal cord injuries. Eur J Neurosci, 2004. 19(5): p. 1226-42.
75.Strazynski, M., et al., Interleukin (IL)-6 and IL-10 induce decorin mRNA in endothelial cells, but interaction with fibrillar collagen is essential for its translation. J Biol Chem, 2004. 279(20): p. 21266-70.
76.Hascall, V.C., et al., Intracellular hyaluronan: a new frontier for inflammation? Biochim Biophys Acta, 2004. 1673(1-2): p. 3-12.
77.Jakab, L., [Glycosaminoglycans, proteoglycans, atherosclerosis]. Orv Hetil, 2004. 145(15): p. 795-803.
78.McKee, C.M., et al., Hyaluronan (HA) fragments induce chemokine gene expression in alveolar macrophages. The role of HA size and CD44. J Clin Invest, 1996. 98(10): p. 2403-13.
79.Goldshmidt, O., et al., Heparanase expression during normal liver development and following partial hepatectomy. J Pathol, 2004. 203(1): p. 594-602.
80.Senoo, H., Structure and function of hepatic stellate cells. Med Electron Microsc, 2004. 37(1): p. 3-15.