臺灣博碩士論文加值系統

肝纖維化發生在大多數的肝臟疾病中，是由於細胞外基質蛋白(extracellular matrix, ECM)，包括膠原蛋白(collagen)的過度積累所導致。在受到損傷的肝臟中，膠原蛋白主要是由肝臟形狀細胞 (hepatic stellate cell, HSC)、靜脈的纖維細胞 (portal fibroblast)以及纖維母細胞 (myofibroblast)所分泌。而這些細胞主要是受到纖維化細胞激素，特別是乙型轉化生長因子（transforming growth factor β,TGF-β）的刺激而活化。前胸腺素 (prothymosin α, ProT)，是一個12.4 kDa的小分子蛋白, 且存在於一般哺乳動物組織中的細胞核內。在過去的研究中已經證明ProT參與在一些重要的生理功能，但其確切所扮演的角色仍然有待進一步的研究。在本實驗室過去的研究中，發現ProT可以透過穩定smad7蛋白，進而抑制TGF-β的訊息傳遞。所以在本研究中我們假設ProT可透過抑制纖維細胞的活化進而阻止肝纖維化的發生。在動物實驗上，我們利用ProT基因轉殖鼠來探討ProT與肝纖維化的關係。在四氯化碳誘發小鼠肝纖維化動物模式中發現，ProT基因轉殖鼠的肝纖維化程度相較非基因轉殖鼠有明顯下降。而我們同時也發現在ProT基因轉殖鼠肝臟中，TGF-β傳遞訊息路徑下游smad2/3的磷酸化程度也明顯下降，而抑制型smad，smad7的表現量相較提高，導致TGF-β訊息傳遞路徑受到抑制。在細胞實驗中也發現，過量表現ProT將提高smad7的乙醯化，穩定性以及活性，進而抑制了TGF-β傳遞訊息路徑。另一方面，金奈米 (nanogold)已經廣泛利用於生物醫學材料的研究上，主要是因為其生物相容性和易與生物分子產生共軛。由於金奈米與硫醇和胺會產生鍵結，我們發現金奈米會與TGF-β產生金硫鍵進而抑制其傳遞訊息路徑。在小鼠肝纖維化動物模式中發現，在金奈米的治療下，無論是在臨床上，超音波影像以及組織學上都發現肝纖維化的程度都明顯下降。在細胞實驗上我們也證明了金奈米會與TGF-β結合進而抑制纖維細胞的移動以及細胞外基質蛋白的分泌。總和以上結論，ProT和金奈米可透過抑制TGF-β傳遞訊息路徑阻止纖維細胞的活化，進而抑制肝纖維化的發生。

Liver fibrosis occurs in most types of chronic liver diseases with the excessive accumulation of extracellular matrix proteins including collagen. Activated hepatic stellate cells, portal fibroblasts, and myofibroblasts have been identified as the major collagen-producing cells in the injured liver. These cells are activated by fibrogenic cytokines such as TGF-β. Prothymosin α (ProT) is a small protein with 12.4 kDa that can be detected in nuclei of normal cells in various mammalian tissues. ProT has been shown to exert several essential biological functions. However, its exact physiologic roles remain to be elucidated. In our previous study, suggested that ProT can inhibit TGF-β signaling through stabilizing smad7, an inhibitory smad of TGF-β signaling. We hypothesized that ProT might inhibit the activation of fibroblasts and result in the suppression of liver fibrosis. ProT transgene mice served as a model to study the effect of ProT on liver fibrosis. After chronic CCl4 treatment, liver fibrosis remarkably decreased in ProT-overexpressing mice compared to that in non-transgenic mice. The phosphorylated Smad2/3 levels were reduced in ProT-overexpressing mice and levels of the inhibitory Smad, Smad7 increased, resulting in down regulation of TGF-β signaling. The in vitro study revealed that overexpression of ProT increased the acetylation, stability, and activity of Smad7, which could antagonize TGF-β signalling. In addition, nanogold has been actively investigated in a wide variety of biomedical applications, due to its biocompatibility and ease of conjugation to biomolecules. Since gold binds strongly to thiols and amines, we found that nanogold inhibited TGF-β signaling by interacting with its sulfur/amines. The in vivo study demonstrated that nanogold ameliorated liver fibrosis in mice in terms of clinical, ultrasound imaging, and histologic examinations. We also showed in vitro that nanogold bound to TGF-β and thereby inhibited fibroblast migration and the production of extracellular matrix. Taken together, ProT and nanogold might inhibit fibroblast activation and subsequently interfere with TGF-β signaling to suppress liver fibrosis.

Qualified Certificate．．．．．．．．．．．．．．．．．．．．．． I
Chinese introduction．．．．．．．．．．．．．．．．．．．．．． II
English introduction．．．．．．．．．．．．．．．．．．．．．． IV
Acknowledgements．．．．．．．．．．．．．．．．．．．．．．VI
Contents．．．．．．．．．．．．．．．．．．．．．．．．．．． VII
Figure contents．．．．．．．．．．．．．．．．．．．．．．．． X
Abbreviation．．．．．．．．．．．．．．．．．．．．．．．．． XI
Introduction
Liver fibrosis．．．．．．．．．．．．．．．．．．．．．．．．1
Pathogenesis of Liver Fibrosis．．．．．．．．．．．．．．．．．1
Transforming growth factor beta (TGF-β) ．．．．．．．．．．．．2
TGF-β signaling pathway．．．．．．．．．．．．．．．．．．．2
ProThymosin α．．．．．．．．．．．．．．．．．．．．．．．3
Gold nanoparticle．．．．．．．．．．．．．．．．．．．．．．3
Specific aim．．．．．．．．．．．．．．．．．．．．．．．．．．5
Materials and methods
Preparation of CCl4 solution．．．．．．．．．．．．．．．．．．6
Preparation of gold nanoparticles．．．．．．．．．．．．．．．．6
Cell lines．．．．．．．．．．．．．．．．．．．．．．．．． 6
Animals．．．．．．．．．．．．．．．．．．．．．．．．．．7
Plasmids．．．．．．．．．．．．．．．．．．．．．．．．．． 7
Reagent．．．．．．．．．．．．．．．．．．．．．．．．．． 8
Competent cell．．．．．．．．．．．．．．．．．．．．．．． 10
Antibodies used in immunohistochemistry staining．．．．．．．． 10
Cell culture．．．．．．．．．．．．．．．．．．．．．．．． 11
Lentivirus gene delivery system．．．．．．．．．．．．．．．． 11
Wound healing．．．．．．．．．．．．．．．．．．．．．．．12
Animal study．．．．．．．．．．．．．．．．．．．．．．． 12
Statistical analysis．．．．．．．．．．．．．．．．．．．．．． 12
Results
1 Prothymosin α suppresses liver fibrosis through the inhibition of TGF-β signaling pathway
1.1 Anti-fibrogenesis effect of ProT．．．．．．．．．．．．．．13
1.2 Overexpression of ProT downregulates TGF-β-Smad signal transduction．．．．．．．．．．．．．．．．．．．．．． 13
1.3 Overexpression of ProT decreases cell migration．．．．．．．． 14
1.4 Therapeutic effect of ProT by adenovirus gene delivery．．．．． 14
2 Amelioration of CCl4-Induced Liver Fibrosis in Mice by Gold nanoparticle
2.1 AuNP inhibited the TGF-β-mediated cell migration．．．．．．． 15
2.2 AuNP inhibited CCl4 induced liver fibrosis．．．．．．．．．． 15
Discussion．．．．．．．．．．．．．．．．．．．．．．．．．． 17
Conclusion．．．．．．．．．．．．．．．．．．．．．．．．． 19
References．．．．．．．．．．．．．．．．．．．．．．．．．． 20
Figures．．．．．．．．．．．．．．．．．．．．．．．．．．． 25

Attisano, L., and Wrana, J.L. (2002). Signal transduction by the TGF-beta superfamily. Science 296, 1646-1647.

Bataller, R., and Brenner, D.A. (2005). Liver fibrosis. The Journal of clinical investigation 115, 209-218.

Canbay, A., Friedman, S., and Gores, G.J. (2004). Apoptosis: the nexus of liver injury and fibrosis. Hepatology 39, 273-278.

Casini, A., Ceni, E., Salzano, R., Biondi, P., Parola, M., Galli, A., Foschi, M., Caligiuri, A., Pinzani, M., and Surrenti, C. (1997). Neutrophil-derived superoxide anion induces lipid peroxidation and stimulates collagen synthesis in human hepatic stellate cells: role of nitric oxide. Hepatology 25, 361-367.

Clinton, M., Graeve, L., el-Dorry, H., Rodriguez-Boulan, E., and Horecker, B.L. (1991). Evidence for nuclear targeting of prothymosin and parathymosin synthesized in situ. Proceedings of the National Academy of Sciences of the United States of America 88, 6608-6612.

Cotter, M.A., 2nd, and Robertson, E.S. (2000). Modulation of histone acetyltransferase activity through interaction of epstein-barr nuclear antigen 3C with prothymosin alpha. Molecular and cellular biology 20, 5722-5735.

Derynck, R., Akhurst, R.J., and Balmain, A. (2001). TGF-beta signaling in tumor suppression and cancer progression. Nature genetics 29, 117-129.

Desmet, V.J., and Roskams, T. (2004). Cirrhosis reversal: a duel between dogma and myth. Journal of hepatology 40, 860-867.



Dixit, V., Van den Bossche, J., Sherman, D.M., Thompson, D.H., and Andres, R.P. (2006). Synthesis and grafting of thioctic acid-PEG-folate conjugates onto Au nanoparticles for selective targeting of folate receptor-positive tumor cells. Bioconjugate chemistry 17, 603-609.

Eschenfeldt, W.H., and Berger, S.L. (1986). The human prothymosin alpha gene is polymorphic and induced upon growth stimulation: evidence using a cloned cDNA. Proceedings of the National Academy of Sciences of the United States of America 83, 9403-9407.

Faulk, W.P., and Taylor, G.M. (1971). An immunocolloid method for the electron microscope. Immunochemistry 8, 1081-1083.

Ferguson, M.W., and O'Kane, S. (2004). Scar-free healing: from embryonic mechanisms to adult therapeutic intervention. Philosophical transactions of the Royal Society of London Series B, Biological sciences 359, 839-850.

Friedman, S.L. (2003). Liver fibrosis -- from bench to bedside. Journal of hepatology 38 Suppl 1, S38-53.

Gines, P., Cabrera, J., Guevara, M., Morillas, R., Ruiz del Arbol, L., Solae, R., and Soriano, G. (2004). Consensus document on the treatment of ascites, dilutional hyponatremia and hepatorenal syndrome in liver cirrhosis]. Gastroenterologia y hepatologia 27, 535-544.

Hainfeld, J.F., Slatkin, D.N., and Smilowitz, H.M. (2004). The use of gold nanoparticles to enhance radiotherapy in mice. Physics in medicine and biology 49, N309-315.

Haritos, A.A., Tsolas, O., and Horecker, B.L. (1984). Distribution of prothymosin alpha in rat tissues. Proceedings of the National Academy of Sciences of the United States of America 81, 1391-1393.

Heine, U., Munoz, E.F., Flanders, K.C., Ellingsworth, L.R., Lam, H.Y., Thompson, N.L., Roberts, A.B., and Sporn, M.B. (1987). Role of transforming growth factor-beta in the development of the mouse embryo. The Journal of cell biology 105, 2861-2876
Hernandez-Gea, V., Aracil, C., and Villanueva, C. (2010). Management of varices in cirrhosis. The New England journal of medicine 362, 2332-2333; author reply 2333-2334.

Hernandez-Gea, V., and Friedman, S.L. (2011). Platelets arrive at the scene of fibrosis......studies. Journal of hepatology 54, 1063-1065.

Karetsou, Z., Sandaltzopoulos, R., Frangou-Lazaridis, M., Lai, C.Y., Tsolas, O., Becker, P.B., and Papamarcaki, T. (1998). Prothymosin alpha modulates the interaction of histone H1 with chromatin. Nucleic acids research 26, 3111-3118.

Kinnman, N., and Housset, C. (2002). Peribiliary myofibroblasts in biliary type liver fibrosis. Frontiers in bioscience : a journal and virtual library 7, d496-503.

Kuhn, C., and McDonald, J.A. (1991). The roles of the myofibroblast in idiopathic pulmonary fibrosis. Ultrastructural and immunohistochemical features of sites of active extracellular matrix synthesis. The American journal of pathology 138, 1257-1265.

Li, K.J., Shiau, A.L., Chiou, Y.Y., Yo, Y.T., and Wu, C.L. (2005). Transgenic overexpression of prothymosin alpha induces development of polycystic kidney disease. Kidney Int 67, 1710-1722.

Lin, J.K., and Chou, C.K. (1992). In vitro apoptosis in the human hepatoma cell line induced by transforming growth factor beta 1. Cancer research 52, 385-388.

Manrow, R.E., Sburlati, A.R., Hanover, J.A., and Berger, S.L. (1991). Nuclear targeting of prothymosin alpha. The Journal of biological chemistry 266, 3916-3924.

Meran, S., Thomas, D.W., Stephens, P., Enoch, S., Martin, J., Steadman, R., and Phillips, A.O. (2008). Hyaluronan facilitates transforming growth factor-beta1-mediated fibroblast proliferation. The Journal of biological chemistry 283, 6530-6545.

Oddy, W.H., and McMahon, R.J. (2011). Milk-derived or recombinant transforming growth factor-beta has effects on immunological outcomes: a review of evidence from animal experimental studies. Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology 41, 783-793.

Orre, R.S., Cotter, M.A., 2nd, Subramanian, C., and Robertson, E.S. (2001). Prothymosin alpha functions as a cellular oncoprotein by inducing transformation of rodent fibroblasts in vitro. J Biol Chem 276, 1794-1799.

Paciotti, G.F., Myer, L., Weinreich, D., Goia, D., Pavel, N., McLaughlin, R.E., and Tamarkin, L. (2004). Colloidal gold: a novel nanoparticle vector for tumor directed drug delivery. Drug delivery 11, 169-183.

Palvimo, J., and Linnala-Kankkunen, A. (1990). Identification of a low-Mr acidic nuclear protein as prothymosin alpha. FEBS letters 277, 257-260.

Popper, H., and Uenfriend, S. (1970). Hepatic fibrosis. Correlation of biochemical and morphologic investigations. The American journal of medicine 49, 707-721.

Sandstrom, P., and Akerman, B. (2004). Electrophoretic properties of DNA-modified colloidal gold nanoparticles. Langmuir : the ACS journal of surfaces and colloids 20, 4182-4186.

Schaffner, F., and Klion, F.M. (1968). Chronic hepatitis. Annual review of medicine 19, 25-38.

Segade, F., and Gomez-Marquez, J. (1999). Prothymosin alpha. The international journal of biochemistry ＆ cell biology 31, 1243-1248.

Sperling, R.A., Rivera Gil, P., Zhang, F., Zanella, M., and Parak, W.J. (2008). Biological applications of gold nanoparticles. Chemical Society reviews 37, 1896-1908.


Watts, J.D., Cary, P.D., and Crane-Robinson, C. (1989). Prothymosin alpha is a nuclear protein. FEBS letters 245, 17-20.