現在位置首頁 > 博碩士論文 > 詳目
  • 同意授權
論文中文名稱:合成具有沙利竇邁的水溶性碳六十衍生物,並探討其抗發炎效果 [以論文名稱查詢館藏系統]
論文英文名稱:Synthesis and anti-inflammation evaluation of new water soluble fulleropyrrolidine bearing thalidomide [以論文名稱查詢館藏系統]
院校名稱:臺北科技大學
學院名稱:工程學院
系所名稱:化學工程研究所
出版年度:97
中文姓名:何家欣
英文姓名:Chia-Shin Ho
研究生學號:95738007
學位類別:碩士
語文別:中文
口試日期:2008-06-18
論文頁數:64
指導教授中文名:黃聲東
指導教授英文名:Sheng-Tung Huang
口試委員中文名:林俊茂;郭憲壽;吳瑞裕
中文關鍵詞:抗發炎碳六十沙利竇邁
英文關鍵詞:FullereneAnti-inflammationthalidomide
論文中文摘要:近期的研究中發現,碳六十衍生物於生物活性方面具有廣泛的應用,包括在抑制HIV蛋白酶、神經保護、光動力療法、選擇性核苷酸切割及吸收自由基(例如ROS和NO)等,但是由於碳六十難溶於水,所以碳六十衍生物在生物活性方面的探討仍運用有限,尤其是運用於抗發炎方面之研究,更為少見。而已知藥物沙利竇邁(Thalidomide)則是能有效抑制腫瘤壞死因子(TNF-α)的生成。在發炎過程中往往會釋放過多的NO、ROS及TNF-α,遂我們期望結合碳六十吸收自由基的特性再加上已知用藥Thalidomide抑制TNF-α的性質,進而設計出具有Thalidomide的水溶性碳六十衍生物,CLT,來同時抑制NO、TNF-α和ROS的生成,並進一步的探討其抗發炎的功效。
於是本論文主要分成四大部分,第一部份為設計與合成含Thalidomide的水溶性碳六十衍生物,CLT;第二部份為CLT對於已發炎細胞所釋出的發炎激素NO和TNF-α進行其抑制效果之探討;第三部份為測試CLT對於抑制已發炎細胞之過氧化物(ROS)生成的情況;第四部份為CLT對於已發炎細胞內iNOS和phospho-ERK蛋白質表現的影響。
我們藉由巨噬細胞RAW 264.7來測試其抗發炎的效果,由實驗結果發現CLT無細胞毒性。而當細胞經由免疫刺激因子(Lipopolysaccharide, LPS)刺激後,加入CLT (10μM)有助於大幅降低NO 46.5%與TNF-α 74.1%的產生,也明顯降低ROS的生成,之後我們再針對NO和TNF-α在已發炎細胞中的蛋白質表現進行測試,其結果顯示CLT可有效的抑制iNOS (inducible nitric oxide) 的蛋白質表現與ERK的磷酸化程度,因此本研究成果顯示所合出的全新碳六十衍生物CLT具有良好的抗發炎效果。
論文英文摘要:Research studies in the field of C60 fullerene derivatives have significantly increased due to the broad range of biological activities that were found for these compounds. We designed and prepared a new C60 fullerene hybrid bearing thalidomide as a potential double-action anti-inflammatory agent, capable of simultaneous inhibition of LPS-induced NO and TNF-alpha production. The C60 fulleropyrrolidine-thalidomide dyad, CLT, was an effective agent to suppress the release of NO and TNF-alpha by the LPS-stimulated macrophages RAW 264.7. Ten microM of CLT effectively inhibited LPS-induced NO and TNF-alpha production by 46.5 + 4.2 % and 70.2 + 4 % with respected to the control, respectively. Furthermore, preliminary biochemical investigation revealed that CLT was a potent agent to suppress both LPS-induced intracellular ROS production and iNOS expression, and CLT also inhibited the phosphorylation of ERK which is an important protein kinase involved in the activation of TNF-alpha synthesis in LPS-activated macrophages. We believed that the studies herein would hold promise for future development of a new generation of potent anti-inflammatory agents.
論文目次:中文摘要.................................................i
英文摘要................................................iii
誌謝......................................................v
目錄.....................................................vi
圖目錄.................................................viii
第一章 前言..............................................1
1.1 碳六十簡介............................................1
1.1.1 碳六十的發現........................................1
1.1.2 碳六十的特性........................................1
1.1.3 碳六十在生物活性上的應用............................2
1.2 已知抗發炎藥物Thalidomide介紹.........................8
1.3 免疫系統與發炎反應介紹...............................10
1.3.1 巨噬細胞在發炎時所扮演的角色.......................11
1.3.2 TNF-α在生物系統所造成的效應........................12
1.3.3 自由基在生物系統所造成的效應.......................13
1.3.4 Nitric oxide與iNOS的關聯...........................15
1.3.5 ERK................................................16
1.4 研究動機與目的.......................................18
第二章 材料與方法.......................................21
2.1儀器與試藥............................................21
2.1.1 實驗儀器..........................................21
2.1.2 實驗試藥..........................................22
2.2實驗方法與流程........................................24
2.2.1 (Benzyloxycarbonyl- {2- [2- (2- {2-[3-(1,3- dioxo-1,3-dihydro- isoindol-2-yl)-2,6-dioxo-piperidin-1-yl]-acetylamino}-ethoxy)-ethoxy]-ethyl}-amino)-acetic acid benzyl ester 6 的合成方法................................24
2.2.2 {2-[2-(2-{2-[3-(1,3-Dioxo-1,3-dihydro-isoindol-2-yl)-2,6-dioxo- piperidin-1-yl]-acetylamino}-ethoxy)-ethoxy]-ethylamino}-acetic acid 7的合成方法......................25
2.2.3 Fulleropyrrolidine CLT的合成方法..................26
2.2.4 UV-visible spectra 測試...........................27
2.2.5 DLS 測試..........................................27
2.2.6 RAW 264.7細胞培養.................................28
2.2.7 細胞毒性測試(MTT assay)...........................28
2.2.8 一氧化氮的生成測量(NO assay)......................28
2.2.9 腫瘤壞死因子(TNF-α)的生成測量.....................29
2.2.10 Confocal Microscope..............................30
2.2.11 Flow cytomertry..................................30
2.2.12 蛋白質電泳分析-西方點墨法........................31
第三章 結果與討論.......................................34
3.1 具有Thalidomide的碳六十衍生物CLT產物的誕生...........34
3.2 碳六十衍生物CLT的水溶性與粒徑分佈測試................36
3.3 碳六十衍生物CLT的細胞毒性、NO與TNF-α測試.............39
3.4 碳六十衍生物CLT的ROS抑制活性測試.....................44
3.5碳六十衍生物CLT的iNOS和ERK蛋白質活性表現測試..........46
第四章 結論..............................................47
參考文獻.................................................48
附錄.....................................................54
論文參考文獻:[1] Kroto, H. W. ; Heath, J. R. ; O’brien, S. C. ; Curl, R. F. ; Smalley R. E. , C60: Buckminsterfullerene. Nature 1985, 318,162.
[2] Becker, L. ; Bada J. L.; Winans, R. E.; Hunt, J. E.; Bunch, T. E.; French, B. M. , Fullerene in the 1.85-billion-year-old Sudbury impact structure. Science 1994, 265, 642-645.
[3] Haddon, R. C. ; Brus, L. E. ; Raghavachari, K. , Rehybridization and π-orbital alignment: the key to the existence of spheroidal carbon clusters. Chem. Phys. lett. , 1986, 131, 3, 165.
[4] Kratschmer, W. ; Lamb, L. D. , Fostropoulos, K. ; Huffman, D. R. , Solid C60: a new form of carbon. Nature, 1990, 347, 354.
[5] Friedman, S. H. ; Decamp, D. L. ; Sijbesma, R. P. ; Srdanov, G. ; Wudl, F. ; Kenyon, G. L. J. , Inhibition of the HIV-1 protease by fullerene derivatives: model building studies and experimental verification. J. Am. Chem. Soc., 1993,115, 6506-6509.
[6] Sijbesma, R. ; Srdanov, G. ; Wudl, F. ; Castoro, J. A. ; Wilkens, C. ; Friedman, S. H. ; Decamp, D. L. ; Kenyon, G. L. , Synthesis of a fullerene derivative for the inhibition of HIV enzymes. J. Am. Chem. Soc. , 1993, 115, 6510-6514.
[7] Marcorin, G. ; Da Ros, T. ; Castellano, S. ; Stefancich, G. ; Borin, I. ; Miertus, S. ; Prato, M. , Design and Synthesis of Novel [60]Fullerene Derivatives as Potential HIV Aspartic Protease Inhibitors. Org. Lett. , 2000, 2, 3955-3958.
[8] Schinazi, R. F. ; Sijbesma, R. ; Srdanov, G. ; Hill, C. L. ; Wudl, F. , Synthesis and virucidal activity of a water soluble, configurationally stable, derivated C60 fullerene. Antimicrob Agents Chemother, 1993, 37, 1707-1710
[9] Lu, L. H. ; Lee, Y. T. ; Chen, H. W. ; Chiang, L. Y. ; Huang, H. C. , The possible mechanisms of the antiproliferative effect of fullerenol, polyhydroxylated C60, on vascular smooth muscle cells. Brit. J. Pharmacol. , 1998, 123, 1097-1102.
[10] Satoh, M. ; Matsuo, K. ; Kiriya, H. ; Hirobe, T. M. ; Takayanagi. I. , Inhibitory effect of a fullerene derivative, monomalonic acid C60, on nitric oxide- dependent relaxation of aortic smooth muscle. Gen. Pharmacol. , 1997, 29, 345-351.
[11] Dugan, L. L. ; Gabrielsen, J. K. ; Yu, S. P. ; Choi, D. W. , Buckminsterfullerenol Free Radical Scavengers Reduce Excitotoxic and Apoptotic Death of Cultured Cortical Neurons. Neurobiol. Dis. , 1996, 3, 129- 135.
[12] Cusan, C. ; Da Ros, T. ; Spalluto, G. ; Foley, S.; Janot, J. M. ; Seta, P. ; Larroque, C. ; Tomasini, M. C. ; Antonelli, T. ; Ferraro, L. ; Prato, M. , A New Multi-Charged C60 Derivative: Synthesis and Biological Properties. Eur. J. Org. Chem. 2002, 17, 2928–2934.
[13] Tzeng, S. F. ; Lee, J. L. ; Kuo, J. S. ; Yang, C. S. ; Murugan, P. ; Tai, L. A. ; Hwang, K. C. , Effects of malonate C60 derivatives on activated microglia. Brain Research, 2002, 940,61-68.
[14] Huang, Y. L. ; Shen, C. K. ; Luh, T. Y. ; Yang, H. C. ; Hwang, K. C. ; Chou, C. K. , Blockage of apoptotic signaling of transforming growth factor-beta in human hepatoma cells by carboxyfullerene. Eur. J. Biochem. , 1998, 254, 38-43.
[15] Tokuyama, H. ; Yamago, S. ; Nakamura, E. ; Shiraki, T. ; Sugiura, Y. , Photo-induced Biological Activity of Fullerene Carboxylic Acid. J. Am. Chem. Soc. , 1993, 115, 7918.
[16] Boutorine, A. S. ; Tokuyama, H. ; Takasugi, M. ; Isobe, H. ; Nakamura, E. ; Hélène, C. , Fullerene-oligonucleotide conjugates: photo-induced sequence- specific DNA cleavage. Angew. Chem. Int. Ed. Engl. , 1994, 33, 2462–2465.
[17] Rice-Evans, C. A. ; Miller, N. J. ; Paganga,G. , Sturcture-antioxidant activity relationships of flavonoids and phenolic acid. Free Radical Biol. Med. , 1996, 20, 933-956.
[18] Gonzalez, K. A. ; Wilson, L. J. ; Wu, W. ; Nancollas, G. H. , Synthesis and In Vitro Characterization of a Tissue-Selective Fullerene: Vectoring C60(OH)16 AMBP to Mineralized Bone. Bioorg. Med. Chem. , 2002, 10, 1991-1997.
[19] Chen, B. X. ; Wilson, S. R. ; Das, M. ; Coughlin, D. J. ; Erlanger, B. F. , Antigenicity of fullerenes: antibodies specific for fullerenes and their characteristics. Proc. Natl. Acad. Sci. , 1998, 95, 10809-10813.
[20] Cagle, D. W. ; Kennel, S. J. ; Mirzadeh, S. ; Alford, J. M. ; Wilson, L. J. , In vivo studies of fullerene-based materials using endohedral metallofullerene radiotracers. Proc. Natl. Acad. Sci. , 1999, 96, 5182-5187.
[21] Yamago, S. ; Tokuyama, H. ; Nakamura, E. ; Kikuchi, K. ; Kananishi, S. ; Sueki, K. ; Nakahara, H. ; Enomoto, S. ; Ambe, F. , In vivo biological behavior of a water-miscible fullerene: 14C labeling, absorption, distribution, excretion and acute toxicity. Chem. & Biol. , 1995, 2, 385-389.
[22] Bullard-Dillard, R. ; Creek, K. E. ; Scrivens, W. A. ; Tour, J. M. , Tissue sites of uptake of 14C-labeled C60. Bioorg. Chem. , 1996, 24, 376-385
[23] Capitosti, S. M. ; Hansen, T. P. ; Brown, M. L. , Facile synthesis of an azido-labeled thalidomide analogue. Organic Letters, 2003, 5, 2865-2867
[24] Melchert, M. ; List, A. , The thalidomide saga. The International Journal of Biochemistry & Cell Biology, 2007, 39, 1489–1499
[25] Palladino, M. A. ; Bahjat, F. R. ; Theodorakis, E. A. ; Moldawer, L. L. , Anti-TNF-alpha therapies: the next generation. Nature reviews Drug discovery, 2003, 2, 736-746
[26] 黎煥耀譯,免疫系統,台北,偉明圖書有限公司,2002,第1-18頁。
[27] 謝文欽譯,細胞與分子免疫學,台北,合記圖書出版社,2004,第1-15頁。
[28] 王政光、林玄原、洪小芳、楊舒如、張弘志、張瑞宏、張芸潔、蕭欣杰、賴志河譯,免疫學,台北,九州圖書文物有限公司,2004,第1-16頁。
[29] 王聖予、陳建和譯,免疫學,台北,藝軒圖書出版社,2002,第1-12頁。
[30] 黎煥耀譯,免疫系統,台北,偉明圖書有限公司,2002,第209-220頁。
[31] 謝文欽譯,細胞與分子免疫學,台北,合記圖書出版社,2004,第25-27頁。
[32] 王聖予、陳建和譯,免疫學,台北,藝軒圖書出版社,2002,第132-133頁。
[33] 王政光、林玄原、洪小芳、楊舒如、張弘志、張瑞宏、張芸潔、蕭欣杰、賴志河譯,免疫學,台北,九州圖書文物有限公司,2004,第39-44頁。
[34] Hu, W. H. ; Johnson, H. ; Shu, H. B. , Tumor necrosis factor-related apoptosis- inducing ligand receptors signal NF-κB and JNK activation and apoptosis through distinct pathway. J. Biol. Chem. , 1999, 274, 30603-30610.
[35] May, M. J. ; Ghosh, S. , Signal transduction through NF-κB. Immunol. Today, 1998, 19, 80-88.
[36] Dyke, M.; Montana, S. , Update on the Therapeutic Potential of PDE4 Inhibitors. Expert Opin. Investig. Drugs. , 2002, 11, 1-13.
[37] Meda, L. ; Cassatella, M. A. ; Szendrei, G. I. ; Otvos, L. Jr. ; Baron, P. ; Villalba, M. ; Ferrari, D. ; Rossi, F. , Activation of Microglial Cells by β-Amyloid Protein and Interferon-γ. Nature, 1995, 374, 647-650.
[38] 謝文欽譯,細胞與分子免疫學,台北,合記圖書出版社,2004,第252-253頁。
[39] Palmer, R. M. ; Rees, D. D. ; Ashton; D. S. ; Moncada S. , L-arginine is the physiological precursor for the formation of nitric oxide in endothelium- dependent relaxation. Biochem. Biophys. Res. Commun. , 1988, 153, 1251-1256
[40] Marletta, M.A., Nitric oxide synthase: aspects concerning structure and catalysis. Cell, 1994, 78, 927-930.
[41] Chi, D. S. ; Qui M. ; Krishnaswamy, G. ; Li, C. ; Stone, W. , Regulation of nitric oxide production from macrophages by lipopolysaccharide and catecholamines. Nitric oxide. 2003, 8, 127-132
[42] Nathan, C. ; Xie, Q. W. , Nitric oxide synthases: Role, Tolls, and Control. Cell,1994, 78, 915-918
[43] Bredt, D. S. ; Snyder, S. H., Isolation of nitric oxide synthetase, a calmodulin-requireing enzyme. Proc. Natl. Acad. Sci. ,1990, 87, 682-685
[44] Pollockm, J. S. ; Forsternann, U. ; Mitchell, J. A. ; Warner, T. D. ; Schmidt H.W. Nakane, M. ; Murad, F. , Purification and characterization of particulate endothelium-derived relaxing factor synthase from cultured and native bovine aortic endothelial cells. Proc. Natl. Acad. Sci. , 1991, 88, 10480-10484
[45] Nathan, C. ; Xie, Q. W. , Regulation of biosynthesis of nitric oxide. J. Biol. Chem. , 1994, 269, 13725-13728
[46] Knowles, R. G. ; Moncada, S. , Nitric oxide synthases in mammals. Biochem . J. , 1994, 298, 249-258
[47] Robbins, R. A. ; Hadeli, K. ; Nelson, D. ; Sato, E. ; Hoyt, J. C. , Nitric oxide, peroxynitrite, and low respiratory tract inflammation. Immunopharmacology. , 2000, 48, 217-221
[48] Larlux, F. S. ; Pavlick, K. P. ; Hines, I. N. ; Kawachi, S. ; Harada, H. ; Bharwani, S. ; Hoffman, J. M. ;Grisham, M. B. , Role of nitric oxide in inflammation. Acta. Physiol. Scand. , 2001, 173, 113-118
[49] Vural, C. ; Gungor, A. , Nitric oxide and the upper airways: recent discoveries. Journal of Ear, Nose&Throat: Kbb. , 2003, 10, 39-44
[50] Cobb, J. P. ; Danner, R. L. , Nitric oxide and septic shock. J Am Med Assoc. , 1996, 275, 1192-1196
[51] Radomski, M. W. ; Salas, E. , Nitric oxide-biological mediator, modulator and factor of injury: it is role in the pathogenesis of atherosclerosis. Atherosclerosis. ,1995, 118, S69-80
[52] Sporn, M. B. ; Robert, A. B. , Peptide growth factors and inflammation, tissue repair, and cancer. J. Clin. Invest. , 1986, 78, 329-332
[53] Graziewicz, M. ; Wink, D. A. ; Laval, F. , Nitric oxide inhibit DNA ligase activity: potential mechanisms for NO-mediated DNA damage. Carcinogenesis. , 1996, 17, 2501-2505
[54] Sautebin, L. , Prostaglandins and nitric oxide as molecular target for anti- inflammatory therapy. Fitoterapia. , 2000, 71, S48-S57
[55] Ottiz, J.; Harris, H. W.; Guitart, X. ; Terwilliger, R. Z., Haycock, J. W.; Nestler E. J., Extracellular Signal-Regulated Protein Kinases (ERKs) and ERK Kinase (MEK) in Brain: Regional Distribution and Regulation by Chronic Morphine. Journal of Neuroscience, 1995, 15, 1285-1297
[56] Prato, M. ; Maggini, M. ; Scorrano G. , Addition of Azomethine Ylides to CM: Synthesis, Characterization, and Functionalization of Fullerene Pyrrolidines. J. Am. Chem. Soc. , 1993, 115, 9798-9799
[57] Partha, R. ; Lackey, M. ; Hirsch, A. ; Casscells, S. W. ; Conyers, J. L. , Self assembly of amphiphilic C60 fullerene derivatives into nanoscale supramolecular structures. Journal of Nanobiotechnology, 2007, 5, 6
[58] Hu, Z. ; Guana, W. ; Wang, W. ; Huanga, L. ; Tang, X. ; Xu H. ; Zhu, Z. ; Xie. X. ; Xing, H. , Synthesis of amphiphilic amino acid C60 derivatives and their protective effect on hydrogen peroxide-induced apoptosis in rat pheochromocytoma cells. CARBON, 2008, 46, 99-109
[59] Qiao, R. ; Roberts, A. P. ; Mount, A. S. ; Klaine, S. J. ; Ke, P. C. , Translocation of C60 and Its Derivatives Across a Lipid Bilayer. Nano Lett. , 2007, 7, 614-619
[60] Barron, A. R. ; Yang, J. ; Driver, J. ; Wang, K. ; Yang, J. , The use of fullerene substituted phenylalanine amino acid as a passport for peptides through cell membranes. Org. Biomol. Chem. , 2007, 5, 260–266
論文全文使用權限:同意授權於2013-07-14起公開