臺灣博碩士論文加值系統

肝纖維化的產生是肝臟受到持續性的慢性損傷，肝組織不斷進行傷口修復的結果。在纖維化的過程中，大量的胞外基質堆積形成疤痕組織。纖維化的形成主因是肝臟星狀細胞 (hepatic stellate cells, HSC) 的增生與活化，因此抑制肝星狀細胞的增生或促進其凋亡是目前被認為有效抑制肝纖維化的方法之一。過量的氧化壓力會促使肝星狀細胞活化，在過程中轉型生長因子 (transforming growth factor-β1, TGF-β1) 已被證實可誘導肝星狀細胞活化並導致胞外基質堆積，進而產生肝纖維化的現象。咖啡酸苯乙酯 (caffeic acid phenethyl ester, CAPE) 是蜂膠成分之一，被證實具有抗肝毒性、抗氧化、抗腫瘤及抗發炎等作用。本研究欲探討 CAPE 是否具有抗小鼠肝纖維化的作用。利用腹腔注射硫代乙醯氨 (thioacetamide, TAA) 誘導 C57BL/6Jnarl 小鼠產生肝纖維化，並以前處理 (pretreatment) 或合併處理 (cotreatment) CAPE。結果顯示：(1) 經由 Sirius red 組織染色及 Ki67 免疫組織染色發現 CAPE 可降低第一型膠原蛋白 (type I collagen) 生成量與細胞增生現象。(2) RT-PCR 結果顯示 CAPE 可減緩 �� SMA、��1(I) procollagen mRNA 的表現。(3) 以 RT-PCR 分析 TGF��1 和 TNF���n表現，發現各組動物體內沒有下降之情形，而且發炎反應並沒有因 CAPE 之加入而有所降低。(4) 經前處理 CAPE 一週後合併處理 TAA/CAPE 雖然能降低肝纖維化程度，但卻伴隨著高死亡率的危機。簡言之， CAPE 可用於治療肝纖維化，但使用上須十分謹慎。

In general, liver fibrosis can be regarded as a chronic wound healing process, characterized by increased deposition of connective tissue. Proliferation and activation of hepatic stellate cells (HSCs) are critical steps in the development of liver fibrosis. Previous studies have shown that excess oxidative stress causes activation of HSCs. During this process, transforming growth factor-b1 (TGF-b1) is known to be the most potent factor to induce HSCs activation and accumulation of exrtacellular matrix (ECM). Therefore, decrease of oxidative stress and suppression of growth or induction of apoptosis of HSCs are recognized as effective therapeutic targets for prevention of hepatic fibrosis. Caffeic acid phenethyl ester (CAPE), one of the major components of honeybee propolis, has been shown to have anti-oxidative, anti-hepatotoxic, anti-inflammatory and anti-tumor effects.
Our study is aimed to investigate the effect of CAPE against thioacetamide (TAA)-induced liver injury. We established a fibrotic C57BL/6 mouse model by TAA injections, and pretreated or co-treated with CAPE to test the anti-fibrotic effect of CAPE. Our data showed that (1) CAPE significantly abrogated hepatic fibrogenesis and reduced matrix density by immunostaining for type I collagen and Ki 67. (2) Mice treated with TAA and CAPE showed a significant reduction in aSMA、a1(I) procollagen mRNA expression. (3) RT-PCR results revealed CAPE could not down-regulate TGFb1 and TNFa mRNA expressions indicating the presence of CAPE could not reduce inflammatory reaction in liver after TAA-treatment. (4) CAPE pretreatment for one week and then combination with TAA/CAPE could attenuate hepatic fibrosis, but have associated with an intolerable high mortality rate. In conclusion, CAPE might be an effective therapeutic reagent for liver fibrosis, but be used cautiously.

中文摘要 .................................................1
Abstract .................................................2
第一章 緒論 ........................................3
壹、 前言 ........................................3
貳、 肝臟的構造與功能 ...............................3
參、 肝星狀細胞與肝纖維化 ......................4
肆、 硫代乙醯氨 (thioacetamide, TAA) .............7
伍、 咖啡酸苯乙酯 (caffeic acid phenethyl ester, CAPE) .................................................7
第二章 研究目的 ........................................9
第三章 材料與方法 ..............................10
壹、 實驗材料 .......................................10
一、 試劑與藥品......................................10
二、 實驗動物 .......................................12
三、 小鼠肝細胞分離與培養............................12
四、 細胞培養........................................13
五、 實驗藥品配製....................................14
貳、 實驗方法........................................14
一、 動物模式：肝纖維化的誘發 (Animal model: Liver fibrosis induction).......................................14
二、 組織RNA之萃取 (Total RNA extraction)............15
三、 反轉錄酶反應 (Reverse transcriptase-polymerase chain reaction)..........................................16
四、 聚合酶連鎖反應 (Polymerase chain reaction, PCR) ................................................16
五、 膠體電泳分析 (Agarose gel electrophoresis)......16
六、 組織染色 (Histological examination).............17
七、 細胞內自由基分析 (Analysis of intercellular ROS) ................................................18
八、 TNFa 酵素連結免疫吸附分析法 (TNFa ELISA assay) ................................................19
九、 RT-PCR 結果量化分析.............................19
十、 統計分析 .......................................20
第四章 結果............................................21
壹、 肝臟病理變化....................................21
貳、 纖維化之生化指標改變............................22
一、 ALT/AST 之改變......................................22
二、 纖維化相關基因表現..................................22
參、 初代培養肝細胞之型態及其基因表現................23
肆、 TAA 誘導肝細胞內 ROS 生成之能力.................24
伍、 CAPE 抑制小鼠巨噬細胞 (RAW264.7) 分泌 TNFa 之研究 ................................................24
第五章 討論............................................25
第六章 結論............................................29
參考文獻 ................................................30
附圖.....................................................36
附錄 (表) ................................................61

Adrian, J. E. (2006). Addressing liver fibrosis with lipid-based drug carriers targeted to hepatic stellate cells. University of Groningen.
Bachem, M. G., Melchior, R., and Gressner, A. M. (1989). The role of thrombocytes in liver fibrogenesis: effects of platelet lysate and thrombocyte-derived growth factors on the mitogenic activity and glycosaminoglycan synthesis of cultured rat liver fat storing cells. J Clin Chem Clin Biochem 27, 555-565.
Bataller, R., and Brenner, D. A. (2005). Liver fibrosis. J Clin Invest 115, 209-218.
Benyon, R. C., and Iredale, J. P. (2000). Is liver fibrosis reversible? Gut 46, 443-446.
Bilzer, M., Roggel, F., and Gerbes, A. L. (2006). Role of Kupffer cells in host defense and liver disease. Liver Int 26, 1175-1186.
Busser, M. T., and Lutz, W. K. (1987). Stimulation of DNA synthesis in rat and mouse liver by various tumor promoters. Carcinogenesis 8, 1433-1437.
Cheng, J., Imanishi, H., Liu, W., Iwasaki, A., Ueki, N., Nakamura, H., and Hada, T. (2002). Inhibition of the expression of alpha-smooth muscle actin in human hepatic stellate cell line, LI90, by a selective cyclooxygenase 2 inhibitor, NS-398. Biochem Biophys Res Commun 297, 1128-1134.
Chieli, E., and Malvaldi, G. (1984). Role of the microsomal FAD-containing monooxygenase in the liver toxicity of thioacetamide S-oxide. Toxicology 31, 41-52.
Chung, T. W., Moon, S. K., Chang, Y. C., Ko, J. H., Lee, Y. C., Cho, G., Kim, S. H., Kim, J. G., and Kim, C. H. (2004). Novel and therapeutic effect of caffeic acid and caffeic acid phenyl ester on hepatocarcinoma cells: complete regression of hepatoma growth and metastasis by dual mechanism. FASEB J 18, 1670-1681.
Dai, K., Qi, J. Y., and Tian, D. Y. (2005). Leptin administration exacerbates thioacetamide-induced liver fibrosis in mice. World J Gastroenterol 11, 4822-4826.
Desmet, V. J., and Roskams, T. (2004). Cirrhosis reversal: a duel between dogma and myth. J Hepatol 40, 860-867.
Fadillioglu, E., Oztas, E., Erdogan, H., Yagmurca, M., Sogut, S., Ucar, M., and Irmak, M. K. (2004). Protective effects of caffeic acid phenethyl ester on doxorubicin-induced cardiotoxicity in rats. J Appl Toxicol 24, 47-52.
Friedman, S. L. (2000). Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury. J Biol Chem 275, 2247-2250.
Friedman, S. L. (2008). Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver. Physiol Rev 88, 125-172.
Friedman, S. L., Roll, F. J., Boyles, J., and Bissell, D. M. (1985). Hepatic lipocytes: the principal collagen-producing cells of normal rat liver. Proc Natl Acad Sci U S A 82, 8681-8685.
Gallois, C., Habib, A., Tao, J., Moulin, S., Maclouf, J., Mallat, A., and Lotersztajn, S. (1998). Role of NF-kappaB in the antiproliferative effect of endothelin-1 and tumor necrosis factor-alpha in human hepatic stellate cells. Involvement of cyclooxygenase-2. J Biol Chem 273, 23183-23190.
Hemmann, S., Graf, J., Roderfeld, M., and Roeb, E. (2007). Expression of MMPs and TIMPs in liver fibrosis - a systematic review with special emphasis on anti-fibrotic strategies. J Hepatol 46, 955-975.
Honda, H., Ikejima, K., Hirose, M., Yoshikawa, M., Lang, T., Enomoto, N., Kitamura, T., Takei, Y., and Sato, N. (2002). Leptin is required for fibrogenic responses induced by thioacetamide in the murine liver. Hepatology 36, 12-21.
Hung, K. S., Lee, T. H., Chou, W. Y., Wu, C. L., Cho, C. L., Lu, C. N., Jawan, B., and Wang, C. H. (2005). Interleukin-10 gene therapy reverses thioacetamide-induced liver fibrosis in mice. Biochem Biophys Res Commun 336, 324-331.
Hunter, A. L., Holscher, M. A., and Neal, R. A. (1977). Thioacetamide-induced hepatic necrosis. I. Involvement of the mixed-function oxidase enzyme system. J Pharmacol Exp Ther 200, 439-448.
Ikejima, K., Honda, H., Yoshikawa, M., Hirose, M., Kitamura, T., Takei, Y., and Sato, N. (2001). Leptin augments inflammatory and profibrogenic responses in the murine liver induced by hepatotoxic chemicals. Hepatology 34, 288-297.
Ince, H., Kandemir, E., Bagci, C., Gulec, M., and Akyol, O. (2006). The effect of caffeic acid phenethyl ester on short-term acute myocardial ischemia. Med Sci Monit 12, BR187-193.
Iredale, J. P. (1997). Tissue inhibitors of metalloproteinases in liver fibrosis. Int J Biochem Cell Biol 29, 43-54.
Iredale, J. P., Benyon, R. C., Arthur, M. J., Ferris, W. F., Alcolado, R., Winwood, P. J., Clark, N., and Murphy, G. (1996). Tissue inhibitor of metalloproteinase-1 messenger RNA expression is enhanced relative to interstitial collagenase messenger RNA in experimental liver injury and fibrosis. Hepatology 24, 176-184.
Iredale, J. P., Murphy, G., Hembry, R. M., Friedman, S. L., and Arthur, M. J. (1992). Human hepatic lipocytes synthesize tissue inhibitor of metalloproteinases-1. Implications for regulation of matrix degradation in liver. J Clin Invest 90, 282-287.
Issa, R., Zhou, X., Constandinou, C. M., Fallowfield, J., Millward-Sadler, H., Gaca, M. D., Sands, E., Suliman, I., Trim, N., Knorr, A., Arthur, M. J., Benyon, R. C., and Iredale, J. P. (2004). Spontaneous recovery from micronodular cirrhosis: evidence for incomplete resolution associated with matrix cross-linking. Gastroenterology 126, 1795-1808.
Kang, J. S., Wanibuchi, H., Morimura, K., Wongpoomchai, R., Chusiri, Y., Gonzalez, F. J., and Fukushima, S. (2008). Role of CYP2E1 in thioacetamide-induced mouse hepatotoxicity. Toxicol Appl Pharmacol 228, 295-300.
Knittel, T., Mehde, M., Grundmann, A., Saile, B., Scharf, J. G., and Ramadori, G. (2000). Expression of matrix metalloproteinases and their inhibitors during hepatic tissue repair in the rat. Histochem Cell Biol 113, 443-453.
Kornek, M., Raskopf, E., Guetgemann, I., Ocker, M., Gerceker, S., Gonzalez-Carmona, M. A., Rabe, C., Sauerbruch, T., and Schmitz, V. (2006). Combination of systemic thioacetamide (TAA) injections and ethanol feeding accelerates hepatic fibrosis in C3H/He mice and is associated with intrahepatic up regulation of MMP-2, VEGF and ICAM-1. J Hepatol 45, 370-376.
Kus, I., Colakoglu, N., Pekmez, H., Seckin, D., Ogeturk, M., and Sarsilmaz, M. (2004). Protective effects of caffeic acid phenethyl ester (CAPE) on carbon tetrachloride-induced hepatotoxicity in rats. Acta Histochem 106, 289-297.
Li, D., and Friedman, S. L. (1999). Liver fibrogenesis and the role of hepatic stellate cells: new insights and prospects for therapy. J Gastroenterol Hepatol 14, 618-633.
Low, T. Y., Leow, C. K., Salto-Tellez, M., and Chung, M. C. (2004). A proteomic analysis of thioacetamide-induced hepatotoxicity and cirrhosis in rat livers. Proteomics 4, 3960-3974.
Lu, B., Wang, L., Medan, D., Toledo, D., Huang, C., Chen, F., Shi, X., and Rojanasakul, Y. (2002). Regulation of Fas (CD95)-induced apoptosis by nuclear factor-kappaB and tumor necrosis factor-alpha in macrophages. Am J Physiol Cell Physiol 283, C831-838.
Milani, S., Herbst, H., Schuppan, D., Grappone, C., Pellegrini, G., Pinzani, M., Casini, A., Calabro, A., Ciancio, G., Stefanini, F., and et al. (1994). Differential expression of matrix-metalloproteinase-1 and -2 genes in normal and fibrotic human liver. Am J Pathol 144, 528-537.
Montpied, P., de Bock, F., Rondouin, G., Niel, G., Briant, L., Courseau, A. S., Lerner-Natoli, M., and Bockaert, J. (2003). Caffeic acid phenethyl ester (CAPE) prevents inflammatory stress in organotypic hippocampal slice cultures. Brain Res Mol Brain Res 115, 111-120.
Morrison, S. J., White, P. M., Zock, C., and Anderson, D. J. (1999). Prospective identification, isolation by flow cytometry, and in vivo self-renewal of multipotent mammalian neural crest stem cells. Cell 96, 737-749.
Neal, R. A., and Halpert, J. (1982). Toxicology of thiono-sulfur compounds. Annu Rev Pharmacol Toxicol 22, 321-339.
Nomura, M., Kaji, A., Ma, W., Miyamoto, K., and Dong, Z. (2001). Suppression of cell transformation and induction of apoptosis by caffeic acid phenethyl ester. Mol Carcinog 31, 83-89.
Olaso, E., and Friedman, S. L. (1998). Molecular regulation of hepatic fibrogenesis. J Hepatol 29, 836-847.
Oliver, J. R., Jiang, S., and Cherian, M. G. (2006). Augmented hepatic injury followed by impaired regeneration in metallothionein-I/II knockout mice after treatment with thioacetamide. Toxicol Appl Pharmacol 210, 190-199.
Poli, G. (2000). Pathogenesis of liver fibrosis: role of oxidative stress. Mol Aspects Med 21, 49-98.
Porter, W. R., and Neal, R. A. (1978). Metabolism of thioacetamide and thioacetamide S-oxide by rat liver microsomes. Drug Metab Dispos 6, 379-388.
Reeves, H. L., and Friedman, S. L. (2002). Activation of hepatic stellate cells--a key issue in liver fibrosis. Front Biosci 7, d808-826.
Roberts, B. J., Song, B. J., Soh, Y., Park, S. S., and Shoaf, S. E. (1995). Ethanol induces CYP2E1 by protein stabilization. Role of ubiquitin conjugation in the rapid degradation of CYP2E1. J Biol Chem 270, 29632-29635.
Roderfeld, M., Weiskirchen, R., Wagner, S., Berres, M. L., Henkel, C., Grotzinger, J., Gressner, A. M., Matern, S., and Roeb, E. (2006). Inhibition of hepatic fibrogenesis by matrix metalloproteinase-9 mutants in mice. FASEB J 20, 444-454.
Russo, A., Longo, R., and Vanella, A. (2002). Antioxidant activity of propolis: role of caffeic acid phenethyl ester and galangin. Fitoterapia 73 Suppl 1, S21-29.
Song, Y. S., Park, E. H., Hur, G. M., Ryu, Y. S., Lee, Y. S., Lee, J. Y., Kim, Y. M., and Jin, C. (2002). Caffeic acid phenethyl ester inhibits nitric oxide synthase gene expression and enzyme activity. Cancer Lett 175, 53-61.
Sud'ina, G. F., Mirzoeva, O. K., Pushkareva, M. A., Korshunova, G. A., Sumbatyan, N. V., and Varfolomeev, S. D. (1993). Caffeic acid phenethyl ester as a lipoxygenase inhibitor with antioxidant properties. FEBS Lett 329, 21-24.
Torres, M. I., Fernandez, M. I., Gil, A., and Rios, A. (1998). Dietary nucleotides have cytoprotective properties in rat liver damaged by thioacetamide. Life Sci 62, 13-22.
Tsai, L. Y., Lee, K. T., Tsai, S. M., Lee, S. C., and Yu, H. S. (1993). Changes of lipid peroxide levels in blood and liver tissue of patients with obstructive jaundice. Clin Chim Acta 215, 41-50.
Wang, C. H., Lee, T. H., Lu, C. N., Chou, W. Y., Hung, K. S., Concejero, A. M., and Jawan, B. (2006). Electroporative alpha-MSH gene transfer attenuates thioacetamide-induced murine hepatic fibrosis by MMP and TIMP modulation. Gene Ther 13, 1000-1009.
Watanabe, T., Niioka, M., Hozawa, S., Kameyama, K., Hayashi, T., Arai, M., Ishikawa, A., Maruyama, K., and Okazaki, I. (2000). Gene expression of interstitial collagenase in both progressive and recovery phase of rat liver fibrosis induced by carbon tetrachloride. J Hepatol 33, 224-235.
Xu, G. F., Li, P. T., Wang, X. Y., Jia, X., Tian, D. L., Jiang, L. D., and Yang, J. X. (2004). Dynamic changes in the expression of matrix metalloproteinases and their inhibitors, TIMPs, during hepatic fibrosis induced by alcohol in rats. World J Gastroenterol 10, 3621-3627.
Yoshiji, H., Kuriyama, S., Miyamoto, Y., Thorgeirsson, U. P., Gomez, D. E., Kawata, M., Yoshii, J., Ikenaka, Y., Noguchi, R., Tsujinoue, H., Nakatani, T., Thorgeirsson, S. S., and Fukui, H. (2000). Tissue inhibitor of metalloproteinases-1 promotes liver fibrosis development in a transgenic mouse model. Hepatology 32, 1248-1254.
Zhao, W. X., Zhao, J., Liang, C. L., Zhao, B., Pang, R. Q., and Pan, X. H. (2003). Effect of caffeic acid phenethyl ester on proliferation and apoptosis of hepatic stellate cells in vitro. World J Gastroenterol 9, 1278-1281.
Zhou, X., Murphy, F. R., Gehdu, N., Zhang, J., Iredale, J. P., and Benyon, R. C. (2004). Engagement of alphavbeta3 integrin regulates proliferation and apoptosis of hepatic stellate cells. J Biol Chem 279, 23996-24006.