家族性原發性皮膚類澱粉沉積症 (Familial primary cutaneous amyloidosis, FPCA) 一種好發於東南亞及南美的慢性皮膚病，近年來已發現FPCA是由於角質細胞的IL-31 receptor-IL-31RA或oncostatin M receptor beta (OSMRb) subunit發生點突變而造成。一般皮膚角質細胞在受到IL-31的刺激時會促使MCP-1產生並吸引單核球及巨噬細胞來清除皮膚的細胞碎片，但當IL-31 receptor發生突變後，MCP-1的產生量會下降而導致細胞碎片無法清除，最後累積而造成皮膚類澱粉沉積的現象發生。FPCA現有的治療方法以塗抹外用類固醇 (corticosteroids) 及服用抗組織胺藥物為主，但病症大多有復發的情形。
在本篇研究中建立了以人類皮膚角質細胞 (HaCaT) 轉殖mutant IL-31RA的細胞株作為一個篩藥平台，用以找出可提升IL-31RA突變細胞株中MCP-1的藥物，進而達到改善家族性原發性皮膚類澱粉沉積症的目的，並進一步確認藥物的作用機制。
在藥物篩選結果發現MEK1/2 kinase inhibitor可提升IL-31RA突變細胞MCP-1產量。此外，上市的MEK1/2 kinase inhibitor, Trametinib, 也能夠提升IL-31RA突變細胞MCP-1產量；除了MCP-1產量以外，MCP-1 mRNA表現量在加入藥物後同樣有看到提升的現象。而MCP-1 promoter的轉錄因子之一- phospho-STAT1 (Tyr 701) 在加入藥物後，發現表現量有顯著增加的情形。
根據這些結果可推測藉由抑制MEK1/2的活性會增加phospho-STAT1 (Tyr701) 的表現，phospho-STAT1 (Tyr701) 作為transcription activator結合到MCP-1 promoter增加MCP-1 mRNA的表現量，進而提升IL-31RA突變細胞的MCP-1產量，顯示MEK1/2在調控IL-31RA突變細胞的MCP-1產量扮演著相當重要的角色，未來期待能更進一步研究MEK1/2與家族性原發型皮膚類澱粉沉積症的關聯性，也期待與現有療法搭配能更有助於疾病的改善與減少復發的情形

Primary cutaneous amyloidosis (PCA) is a chronic skin disease that most frequently occurs in Southeast Asia and South America. Based on the etiology, PCA can be classified into sporadic and familial types. Recently, mutations on IL-31RA or oncostatin M receptor β (OSMRβ) gene have been found to correlate with familial primary cutaneous amyloidosis (FPCA). MCP-1, upon induction by IL-31 can recruit monocytes/macrophages to clean the cell debris on the skin. However, when certain mutations occur in IL-31 receptor, the levels of MCP-1 would decrease, leading to the accumulation of cell debris on the surface of skin.
In this study, a cell-based drug screening system was established in which human keratinocyte cells (HaCaT) were transfected with mutant IL-31RA plasmid. In lieu of this system, several potential activators of MCP-1 in IL-31RA mutant cells were discovered. Subsequently, the underlying mechanism was explored.
Results from this study showed that the levels of MCP-1 were increased by MEK1/2 kinase inhibitors, PD 198306 or Trametinib. The expression levels of MCP-1 mRNA increased when the cells were treated with PD 198306. The expression levels phospho-STAT1 (Tyr701), one transcription factor of MCP-1 promoter, were also significantly increased when the HaCaT cells harboring mutant IL-31RA were treated with either MEK1/2 kinase inhibitors.
Overall, these results might suggest that inhibition of MEK1/2 activity might increase the expression levels of phospho-STAT1 (Tyr701). The phospho-STAT1 (Tyr701) can act as a transcription activator and bind on MCP-1 promoter to enhance the transcription. Although the exact interplays between MEK1/2 and FPCA remains to be further investigated, this study indicate that MEK1/2 pathway may be a novel target for development of effective FPCA treatment.

CONTENTS

口試委員會審定書
誌謝
中文摘要
英文摘要
CONTENTS
LIST OF FIGURES
LIST OF TABLES
Chapter1 文獻回顧
1.1 類澱粉沉積 (Amyloidosis)
1.2 原發性皮膚類澱粉沉積 (Primary Cutaneous Amyloidosis)
1.3 家族性原發型皮膚類澱粉沉積 (Familial primary cutaneous
amyloidosis)
1.4 老藥新用
1.5 尋找治療藥物的目的
Chapter2 材料與方法
2.1 材料與試劑
2.2 細胞株培養、繼代與特性分析
2.3 藥物篩選
2.3.1 細胞分盤與藥物處理
2.3.2 MCP-1蛋白定量-酵素聯結免疫吸附分析法 (ELISA)
2.3.3 MTS assay
2.3.2.1 MTS assay介紹
2.3.2.3 MTS assay步驟
2.3.4 z-factor 計算
2.4 機制探討
2.4.1 即時定量聚合酶連鎖反應 (Real time PCR)
2.4.2 細胞蛋白質樣本製備
2.4.3 西方墨點法 (Western blot)
Chapter3 結果
3.1 細胞特性
3.1.1 HaCaT型態與特性分析
3.1.2 HaCaT各細胞株MCP-1分泌量
3.2 藥物篩選
3.2.1 ELISA靈敏度測試
3.2.2 初步篩選
3.2.3 再現性測試
3.3 機制探討
3.3.1 即時定量聚合酶連鎖反應 (Real time PCR)
3.3.2 西方墨點法 (Western blot)
Chapter4 討論
Chapter5 未來展望
Chapter6 圖片與表格
參考文獻REFERENCE
附表一
附表二
附表三
附表四
縮寫對照表

[1] M. W. Lin, D. D. Lee, T. T. Liu, Y. F. Lin, S. Y. Chen, C. C. Huang, et al., "Novel IL31RA gene mutation and ancestral OSMR mutant allele in familial primary cutaneous amyloidosis," Eur J Hum Genet, vol. 18, pp. 26-32, Jan 2010.
[2] K. Arita, A. P. South, G. Hans-Filho, T. H. Sakuma, J. Lai-Cheong, S. Clements, et al., "Oncostatin M receptor-beta mutations underlie familial primary localized cutaneous amyloidosis," Am J Hum Genet, vol. 82, pp. 73-80, Jan 2008.
[3] C. H. Shiao YM, Chen CC, Chiang KN, Chang YT, Lee DD, Lin MW, Tsai SF, Matsuura I, "MCP-1 as an effector of IL-31 signaling in familial primary cutaneous amyloidosis.," J Invest Dermatol, 2013.
[4] T. Murakami, N. Ishiguro, and K. Higuchi, "Transmission of Systemic AA Amyloidosis in Animals," Vet Pathol, vol. 51, pp. 363-71, Mar 2014.
[5] J. D. Sipe, M. D. Benson, J. N. Buxbaum, S. Ikeda, G. Merlini, M. J. Saraiva, et al., "Amyloid fibril protein nomenclature: 2010 recommendations from the nomenclature committee of the International Society of Amyloidosis," Amyloid, vol. 17, pp. 101-4, Sep 2010.
[6] R. S. Mitchell, V. Kumar, A. K. Abbas, and N. Fausto, Robbins Basic Pathology, 8 ed., 2007.
[7] L. M. Blancas-Mejia and M. Ramirez-Alvarado, "Systemic amyloidoses," Annu Rev Biochem, vol. 82, pp. 745-74, 2013.
[8] R. A. Kyle and M. A. Gertz, "Primary systemic amyloidosis: clinical and laboratory features in 474 cases," Semin Hematol, vol. 32, pp. 45-59, Jan 1995.
[9] W. Ollague, J. Ollague, and H. Ferretti, "Epidemiology of primary cutaneous amyloidoses in South America," Clin Dermatol, vol. 8, pp. 25-9, Apr-Jun 1990.
[10] T. Tan, "Epidemiology of primary cutaneous amyloidoses in southeast Asia," Clin Dermatol, vol. 8, pp. 20-4, Apr-Jun 1990.
[11] W. Ollague, "Primary cutaneous amyloidosis," Int J Dermatol, vol. 26, p. 135, Mar 1987.
[12] B. S. Qiu, "[715 cases of primary cutaneous amyloidosis]," Zhonghua Yi Xue Za Zhi, vol. 64, pp. 554-7, Sep 1984.
[13] D. B. Vasily, S. G. Bhatia, and S. R. Uhlin, "Familial primary cutaneous amyloidosis. Clinical, genetic, and immunofluorescent studies," Arch Dermatol, vol. 114, pp. 1173-6, Aug 1978.
[14] D. D. Lee, C. K. Huang, P. C. Ko, Y. T. Chang, W. Z. Sun, and Y. J. Oyang, "Association of primary cutaneous amyloidosis with atopic dermatitis: a nationwide population-based study in Taiwan," Br J Dermatol, vol. 164, pp. 148-53, Jan 2011.
[15] J. Borowicz, M. Gillespie, and R. Miller, "Cutaneous amyloidosis," Skinmed, vol. 9, pp. 96-100; quiz 101, Mar-Apr 2011.
[16] W. D. James, T. Berger, and D. M. Elston, Andrews' Diseases of the Skin, 11 ed., 2006.
[17] C. K. Wong, "Lichen amyloidosus. A relatively common skin disorder in Taiwan," Arch Dermatol, vol. 110, pp. 438-40, Sep 1974.
[18] C. Errol, "Lichen amyloidosis of the auricular concha: report of two cases and review of the literature," Dermatol Online J, vol. 12, p. 1, 2006.
[19] V. Eswaramoorthy, I. Kaur, A. Das, and B. Kumar, "Macular amyloidosis: etiological factors," J Dermatol, vol. 26, pp. 305-10, May 1999.
[20] S. A. Ritchie, T. Beachkofsky, S. Schreml, A. Gaspari, and C. M. Hivnor, "Primary localized cutaneous nodular amyloidosis of the feet: a case report and review of the literature," Cutis, vol. 93, pp. 89-94, Feb 2014.
[21] C. K. Wong and C. S. Lin, "Friction amyloidosis," Int J Dermatol, vol. 27, pp. 302-7, Jun 1988.
[22] Y. T. Chang, C. K. Wong, K. C. Chow, and C. H. Tsai, "Apoptosis in primary cutaneous amyloidosis," Br J Dermatol, vol. 140, pp. 210-5, Feb 1999.
[23] Y. T. Chang, H. N. Liu, C. K. Wong, K. C. Chow, and K. Y. Chen, "Detection of Epstein-Barr virus in primary cutaneous amyloidosis," Br J Dermatol, vol. 136, pp. 823-6, Jun 1997.
[24] H. Furumoto, Y. Hashimoto, M. Muto, T. Shimizu, and K. Nakamura, "Apolipoprotein E4 is associated with primary localized cutaneous amyloidosis," J Invest Dermatol, vol. 119, pp. 532-3, Aug 2002.
[25] Y. T. Chang, S. F. Tsai, W. J. Wang, C. J. Hong, C. Y. Huang, and C. K. Wong, "A study of apolipoproteins E and A-I in cutaneous amyloids," Br J Dermatol, vol. 145, pp. 422-7, Sep 2001.
[26] P. F. Cheung, C. K. Wong, A. W. Ho, S. Hu, D. P. Chen, and C. W. Lam, "Activation of human eosinophils and epidermal keratinocytes by Th2 cytokine IL-31: implication for the immunopathogenesis of atopic dermatitis," Int Immunol, vol. 22, pp. 453-67, Jun 2010.
[27] Q. Zhang, P. Putheti, Q. Zhou, Q. Liu, and W. Gao, "Structures and biological functions of IL-31 and IL-31 receptors," Cytokine Growth Factor Rev, vol. 19, pp. 347-56, Oct-Dec 2008.
[28] S. Kasraie, M. Niebuhr, K. Baumert, and T. Werfel, "Functional effects of interleukin 31 in human primary keratinocytes," Allergy, vol. 66, pp. 845-52, Jul 2011.
[29] M. Shen, S. Siu, S. Byrd, K. H. Edelmann, N. Patel, R. R. Ketchem, et al., "Diverse functions of reactive cysteines facilitate unique biosynthetic processes of aggregate-prone interleukin-31," Exp Cell Res, vol. 317, pp. 976-93, Apr 15 2011.
[30] C. Cornelissen, J. Luscher-Firzlaff, J. M. Baron, and B. Luscher, "Signaling by IL-31 and functional consequences," Eur J Cell Biol, vol. 91, pp. 552-66, Jun-Jul 2012.
[31] S. L. Deshmane, S. Kremlev, S. Amini, and B. E. Sawaya, "Monocyte chemoattractant protein-1 (MCP-1): an overview," J Interferon Cytokine Res, vol. 29, pp. 313-26, Jun 2009.
[32] A. Yadav, V. Saini, and S. Arora, "MCP-1: chemoattractant with a role beyond immunity: a review," Clin Chim Acta, vol. 411, pp. 1570-9, Nov 11 2010.
[33] P. Sartipy and D. J. Loskutoff, "Monocyte chemoattractant protein 1 in obesity and insulin resistance," Proc Natl Acad Sci U S A, vol. 100, pp. 7265-70, Jun 10 2003.
[34] J. Panee, "Monocyte Chemoattractant Protein 1 (MCP-1) in obesity and diabetes," Cytokine, vol. 60, pp. 1-12, Oct 2012.
[35] J. H. Gong, L. G. Ratkay, J. D. Waterfield, and I. Clark-Lewis, "An antagonist of monocyte chemoattractant protein 1 (MCP-1) inhibits arthritis in the MRL-lpr mouse model," J Exp Med, vol. 186, pp. 131-7, Jul 7 1997.
[36] C. R. Chong and D. J. Sullivan, Jr., "New uses for old drugs," Nature, vol. 448, pp. 645-6, Aug 9 2007.
[37] N. D. Yeomans, "Aspirin: old drug, new uses and challenges," J Gastroenterol Hepatol, vol. 26, pp. 426-31, Mar 2011.
[38] J. I. Sirven, "New Uses for Older Drugs: The Tales of Aspirin, Thalidomide, and Gabapentin," Mayo Clinic Proceedings, vol. 85, pp. 508-511, 2010.
[39] R. J. D'Amato, M. S. Loughnan, E. Flynn, and J. Folkman, "Thalidomide is an inhibitor of angiogenesis," Proc Natl Acad Sci U S A, vol. 91, pp. 4082-5, Apr 26 1994.
[40] A. Palumbo, T. Facon, P. Sonneveld, J. Blade, M. Offidani, F. Gay, et al., "Thalidomide for treatment of multiple myeloma: 10 years later," Blood, vol. 111, pp. 3968-77, Apr 15 2008.
[41] Y. Z. Patt, M. M. Hassan, R. D. Lozano, A. K. Nooka, Schnirer, II, J. B. Zeldis, et al., "Thalidomide in the treatment of patients with hepatocellular carcinoma: a phase II trial," Cancer, vol. 103, pp. 749-55, Feb 15 2005.
[42] J. H. Zhang, T. D. Chung, and K. R. Oldenburg, "A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays," J Biomol Screen, vol. 4, pp. 67-73, 1999.
[43] C. J. Wright and P. L. McCormack, "Trametinib: first global approval," Drugs, vol. 73, pp. 1245-54, Jul 2013.
[44] A. K. Salama and K. B. Kim, "Trametinib (GSK1120212) in the treatment of melanoma," Expert Opin Pharmacother, vol. 14, pp. 619-27, Apr 2013.
[45] N. Alluri and A. Jimeno, "Trametinib for the treatment of melanoma," Drugs Today (Barc), vol. 49, pp. 491-8, Aug 2013.
[46] R. Roskoski, Jr., "ERK1/2 MAP kinases: structure, function, and regulation," Pharmacol Res, vol. 66, pp. 105-43, Aug 2012.
[47] K. Maki and K. Ikuta, "MEK1/2 induces STAT5-mediated germline transcription of the TCRgamma locus in response to IL-7R signaling," J Immunol, vol. 181, pp. 494-502, Jul 1 2008.
[48] Y. Zhang, Y. Y. Cho, B. L. Petersen, F. Zhu, and Z. Dong, "Evidence of STAT1 phosphorylation modulated by MAPKs, MEK1 and MSK1," Carcinogenesis, vol. 25, pp. 1165-75, Jul 2004.
[49] Z. J. Tian and W. An, "ERK1/2 contributes negative regulation to STAT3 activity in HSS-transfected HepG2 cells," Cell Res, vol. 14, pp. 141-7, Apr 2004.
[50] N. Jain, T. Zhang, S. L. Fong, C. P. Lim, and X. Cao, "Repression of Stat3 activity by activation of mitogen-activated protein kinase (MAPK)," Oncogene, vol. 17, pp. 3157-67, Dec 17 1998.
[51] T. K. Sengupta, E. S. Talbot, P. A. Scherle, and L. B. Ivashkiv, "Rapid inhibition of interleukin-6 signaling and Stat3 activation mediated by mitogen-activated protein kinases," Proc Natl Acad Sci U S A, vol. 95, pp. 11107-12, Sep 15 1998.
[52] J. Chung, E. Uchida, T. C. Grammer, and J. Blenis, "STAT3 serine phosphorylation by ERK-dependent and -independent pathways negatively modulates its tyrosine phosphorylation," Mol Cell Biol, vol. 17, pp. 6508-16, Nov 1997.
[53] N. Li, J. E. McLaren, D. R. Michael, M. Clement, C. A. Fielding, and D. P. Ramji, "ERK is integral to the IFN-gamma-mediated activation of STAT1, the expression of key genes implicated in atherosclerosis, and the uptake of modified lipoproteins by human macrophages," J Immunol, vol. 185, pp. 3041-8, Sep 1 2010.
[54] M. E. Condren and M. D. Bradshaw, "Ivacaftor: a novel gene-based therapeutic approach for cystic fibrosis," J Pediatr Pharmacol Ther, vol. 18, pp. 8-13, Jan 2013.
[55] M. J. Harrison, D. M. Murphy, and B. J. Plant, "Ivacaftor in a G551D homozygote with cystic fibrosis," N Engl J Med, vol. 369, pp. 1280-2, Sep 26 2013.
[56] E. D. Deeks, "Ivacaftor: a review of its use in patients with cystic fibrosis," Drugs, vol. 73, pp. 1595-604, Sep 2013.
[57] J. R. Infante, L. A. Fecher, G. S. Falchook, S. Nallapareddy, M. S. Gordon, C. Becerra, et al., "Safety, pharmacokinetic, pharmacodynamic, and efficacy data for the oral MEK inhibitor trametinib: a phase 1 dose-escalation trial," Lancet Oncol, vol. 13, pp. 773-81, Aug 2012.
[58] Q. An, L. Zhang, S. Zheng, J. Lin, Y. Hong, H. D. Chen, et al., "Thalidomide improves clinical symptoms of primary cutaneous amyloidosis: report of familiar and sporadic cases," Dermatol Ther, vol. 26, pp. 263-6, May-Jun 2013.
[59] J. Das and R. K. Gogoi, "Treatment of primary localised cutaneous amyloidosis with cyclophosphamide," Indian J Dermatol Venereol Leprol, vol. 69, pp. 163-4, Mar-Apr 2003.
[60] E. Ozkaya-Bayazit, C. Baykal, and A. Kavak, "[Local DMSO treatment of macular and papular amyloidosis]," Hautarzt, vol. 48, pp. 31-7, Jan 1997.
[61] M. H. Lien, D. Railan, and B. R. Nelson, "The efficacy of dermabrasion in the treatment of nodular amyloidosis," J Am Acad Dermatol, vol. 36, pp. 315-6, Feb 1997.
[62] A. G. Jin, A. Por, L. K. Wee, C. K. Kai, and G. C. Leok, "Comparative study of phototherapy (UVB) vs. photochemotherapy (PUVA) vs. topical steroids in the treatment of primary cutaneous lichen amyloidosis," Photodermatol Photoimmunol Photomed, vol. 17, pp. 42-3, Feb 2001.
[63] A. Lesiak, A. Rakowski, A. Brzezinska, M. Rogowski-Tylman, P. Kolano, A. Sysa-Jedrzejowska, et al., "Effective treatment of nodular amyloidosis with carbon dioxide laser," J Cutan Med Surg, vol. 16, pp. 372-4, Sep-Oct 2012.
[64] I. Hamzavi and H. Lui, "Excess tissue friability during CO2 laser vaporization of nodular amyloidosis," Dermatol Surg, vol. 25, pp. 726-8, Sep 1999.
[65] A. P. Truhan, J. M. Garden, and H. H. Roenigk, Jr., "Nodular primary localized cutaneous amyloidosis: immunohistochemical evaluation and treatment with the carbon dioxide laser," J Am Acad Dermatol, vol. 14, pp. 1058-62, Jun 1986.