臺灣博碩士論文加值系統

在人體的免疫反應是由高度複雜的控制因子系統所調節。在這些調節控制因子之中，其中較顯著重要的是抗發炎的細胞素( anti-inflammatory cytokine ) 和特定細胞素的抑制者 ( specific cytokine inhibitor )。免疫系統中，發炎前期細胞素 ( proinflammatory cytokine ) 和抗發炎細胞素 ( anti-inflammatory cytokine ) 存在著一種動態平衡。濃度不足的抗發炎細胞素會導致過度發炎反應，而過多的抗發炎細胞素則會破壞宿主清除外來致病原的機制。
由於人體基因體計劃的進行，興起新穎基因的探索研究。因此，相似IL-10的新穎基因已漸被鑑定出來被稱為IL-10家族 ( IL-10 family )。IL-10家族包括IL-10，IL-19，IL-20，IL-22，AK155和MDA-7。Interleukin-10 ( IL-10 ) 是一種多功能性細胞素，在免疫反應中扮演相當重要的角色。由於IL-10經由降低抗原呈獻 ( antigen presentation )和巨噬細胞 ( macrophage )對刺激的反應來達到抗發炎反應的作用，所以早期IL-10被稱為細胞素合成抑制因子 ( cytokine synthesis inhibitory factor )。
我們自人類腎臟cDNA library分離出與IL-10相似的新穎細胞素-1 ( Novel cytokine-1；NC-1 )。分析人類NC-1基因體結構，發現NC-1基因具5個exon和4個intron。在NC-1 5’端未轉譯區域 ( untranslation region )，exon與intron排列頗多變化。
為了研究此新穎細胞素在人體中所扮演的角色，以及與IL-10功能相關性。我們構築人類NC-1的表現質體，分別在哺乳動物細胞、酵母菌和大腸桿菌表現系統中表達蛋白質，再進行蛋白質純化。將來自酵母菌和大腸桿菌表現系統中純化好的蛋白質處理人類單核球，發現會刺激單核球產生interleukin-6與tumor necrosis factor-a。
另一方面，為了能夠更進一步研究NC-1的生物功能，我們實驗室擬採老鼠作為研究模式。首先，我們在老鼠基因庫 ( database )中找尋到一段與人類NC-1相似的DNA序列。我們再經由5’RACE和3’RACE以找到全長的老鼠NC-1。分析老鼠NC-1的基因體結構 ( genomic structure )，發現老鼠NC-1的intron位置與人類相同。構築老鼠NC-1的表現質體，將老鼠NC-1在大腸桿菌系統中表達蛋白質，進行蛋白質再摺疊 (refolding )與純化。將純化好的老鼠NC-1處理單核球，以ELISA觀察對單核球之影響。發現老鼠NC-1會刺激老鼠單核球產生interleukin-6與tumor necrosis factor-a。此結果與人類NC-1相同。

The human immune response is regulated by a highly complex and intricate network of control elements. Prominent among these regulatory components are the anti-inflammatory cytokines and specific cytokine inhibitors. A dynamic balance exists between proinflammatory cytokines d anti-inflammatory components in human immune system. Inadequate concentrations of anti-inflammatory cytokines result in excess inflammation, yet excess anti-inflammatory cytokine concentrations disrupt clearance mechanisms of microbial pathogens in the host.
The overall sequencing of the human genome gave rise to the discovery of many novel genes. Thus, novel molecules which are homology to the cytokine interleukin-10 ( IL-10 ) have been identified. They were recently combined and referred to as IL-10 family. This family now comprises IL-10, IL-19, IL-20, IL-22, AK155 and melanoma differentiation associated gene 7 ( MDA-7 ). IL-10 has multiple biological activities and plays an import role in the immune system. IL-10 was known to be able to inhibit the inflammatory responses by reducing the antigen presenting capacity of monocytes and the response of macrophage to the stimuli. Therefore, interleukin-10 was originally described as cytokine synthesis inhibitory factor.
We isolated NC-1 ( novel cytokine-1 ) which was homologous to interleukin-10 from human kidney cDNA library. After the analysis of human NC-1 genomic structure, there are five exons and four introns found in the 5’end untranslation region. A lot of variable sorts were existed among these exons and introns
To understand the function of this novel cytokine and the relationship to interleukin-10, we constructed the expression clone of human NC-1 in mammalia cell, Pichia pastoris and E. coli expression system. We purified NC-1 protein from Pichia pastoris and E. coli expression system. Then we treated human monocytes with NC-1 recombinant protein. NC-1 can stimulate human monocytes to produce interleukin-6 and tumor necrosis factor-a.
On the other hand, to study the biological function of NC-1 much more, we processed some experiments using the mice model. First, we isolated a DNA fragment homology to human NC-1 from mouse database. We found the full-length cDNA of mouse NC-1 by 5’ end rapid amplification cDNA end and 3’ end rapid amplification cDNA end. After the analysis of mouse NC-1 genomic structure, we found the location of intron was identical to human NC-1. We constructed the expression clone of mouse NC-1 and expressed mouse NC-1 protein in E. coli expression system. After the refolding and purification of protein, we treated mouse monocytes with NC-1 protein to study the function of mouse NC-1. We also found that mouse NC-1 stimulated monocytes to produce interleukin-6 and tumor necrosis factor-a.

中文摘要 I
英文摘要 III
誌謝 V
目錄 VI
圖目錄 X
表目錄 XII
附錄目錄 XIII
縮寫檢索表 XIV
儀器 XV
第1 章 緒論 1
1-1 免疫反應與細胞素 1
1-2　interleukin-10 ( IL-10 )之介紹 2
1-3 研究動機及內容之簡介 4
第2 章 材料與方法 6
2-1 實驗菌株、質體與培養基配方 6
2-1-1 Host strains and genotypes 6
2-1-2 Vector 6
2-1-3 Growth medium 7
2-2　NC-1基因之來源 10
2-3　NC-1重組基因之構築 10
2-3-1 聚合脢連鎖反應 10
2-3-2 電泳法回收DNA片段 11
2-3-3　TA cloning 11
2-3-4　E. coli Transformation 13
2-3-5　Colony PCR 14
2-3-6 限制脢處理 15
2-3-7　PCR產物純化法　 15
2-3-8 接合反應 16
2-3-9 小量質體的抽取 17
2-4　Ligation independent cloning 17
2-5　Pichia pastoris EasyComp transformation 18
2-6 人類NC-1_pCEP4重組蛋白質的製備 19
2-6-1 蛋白質之表達 19
2-6-2 偵測蛋白質表達情形 21
2-7 人類NC-1_pPICZaA重組蛋白質的製備 24
2-8 人類 NC-1_pET 30 EK/LIC重組蛋白質的製備 26
2-9 人類 NC-1重組蛋白質之功能分析 27
2-9-1 人類單核球 ( monocyte ) 之純化 27
2-9-2 處理單核球 ( monocyte ) NC-1重組蛋白質 28
2-9-3　Enzyme linked immunoabsorbent assay ( ELISA ) 28
2-10 5’ Rapid amplification of cDNA ends ( 5’RACE ) 29
2-11 3’ Rapid amplification of cDNA ends ( 3’RACE ) 30
2-12 北方轉漬法 31
2-13 老鼠NC-1重組蛋白質的製備 31
2-14 老鼠NC-1重組蛋白質之功能分析 33
2-14-1老鼠單核球 ( monocyte ) 之純化 33
2-14-2處理單核球 ( monocyte ) NC-1重組蛋白質 33
2-14-3 Enzyme linked immunoabsorbent assay ( ELISA ) 33
第3章 35
3-1 人類NC-1與interleukin-10之胺基酸序列比對 35
3-2 人類NC-1之基因體結構 35
3-3人類NC-1之5’端未轉譯區域 35
3-4人類NC-1重組基因的製備 36
3-5人類NC-1重組蛋白質的製備 37
3-6人類NC-1重組蛋白質之功能分析 39
3-7全長( full-length )老鼠NC-1 cDNA 40
3-8北方轉漬之結果 42
3-9老鼠NC-1與人類NC-1之胺基酸序列比對 42
3-10老鼠NC-1之基因體結構 43
3-11老鼠NC-1之重組基因的製備 43
3-12老鼠NC-1重組蛋白質的製備 43
3-13老鼠NC-1重組蛋白質之功能分析 44
第4章 討論 45
參考文獻 50
圖 56
表 81
附錄 83
自述

Anne-Mari O’Farrell, Ying Liu, Kevin W.Moore and Alice L.-F.Mui. IL-10 inhibits macrophage activation ad proliferation by distinct signaling mechanisms: evidence for Stat3-dependent and —independent pathways. EMBO J. 17: 1006-1018, 1998.
Anthony R. Mire-Sluis , and Robin thorpe (1998) Cytokine, Academic Press Inc.
Benjamin, D., Knobloch, T.J. and Dayton, M.A. Human B-cell interleukin-10: B-cell lines derived from patients with acquired immunodeficiency syndrome and Burkitt’s lymphoma constitutively secrete large quantities of interleukin-10. Blood 80: 1289-1298, 1992.
Burdin, N., Peronne, C., Banchereau, J. and Rousser, F. Epstein-Barr virus transformation induces B lymphocytes to produce human interleukin 10. J. Exp. Med. 177: 295-304, 1993.
Blumberg, H., Conklin, D., Xu, W. F., Grossmann, A., Brender, T., Carollo, S., Eagan, M., Foster, D., Haldeman, B. A., Hammond, A., Haugen, H., Jelinek, L., Kelly, J. D., Madden, K., Maurer, M. F., Parrish-Novak, J., Prunkard, D., Sexson, S., Sprecher, C., Waggie, K., West, J., Whitmore, T. E., Yao, L., Kuechle, M. K., Dale, B. A., and Chandrasekher, Y. A. Interleukin 20 discovery, receptor identification, and role in epidermal function. Cell 104, 9-19, 2001
Chen, Q., Daniel, V., Maher, D.W. and Hersey, P. Production of IL-10 by melanoma cells: examination of its role in immunosuppression mediated by melanoma. Int J. Cancer 56: 755-760, 1994.
Di-Hwei, H., de Waal Malefyt, R., Fiorentino, D.F., minh-Ngoc Dang, Viera, P., de Vries, J., Spits, H., Mosmann, t.M.&Moore, K. M. Expression of interleukin-10 activity by Epstein-Barr virus protein BCRF1. Science 250: 830-832, 1990.
de Waal Malefyt RD, Abrams J, bennet B, Figdor GC, de Vries JE. Interleukin-10 inhibits cytokine synthesis by human monocytes: an autoregulatory role of IL-10 produced by monocytes. J. Exp Med 174: 1209-1220, 1991.
de Waal Malefyt R, Figdor CG, Huijbens R et al. Efects of IL-13 on phenotype, cytokine production, and cytotoxic function of human monocytes. Comparision with IL-4 and modulation by IFN-g or IL-10. J. Immunol 151: 6370-6381, 1993.
Daheshia, M., N. Kuklin, S. Kanangat, E. Manickan, and B.T. Rouse. Suppression of ongoing ocular inflammatory disease by topical administration of plasmid DNA IL-10. J. Immunol. 159: 1945-1952, 1997.
Dinarello CA. Interleukin-1, interleukin-1 receptors and interleukin-1 receptor antagonist. Int. Rev. Immunol. 16: 457-499, 1998.
Ding Y, Qin L, Kotenko SV, Pestka S, Bromberg JS. A single amino acid determines the immunostimulatory activity of interleukin 10. J. Exp Med 191: 213-224, 2000.
Dumoutier, L., J. Louahed, and J.-C. renauld. Cloning and characterizationof IL-10-related T cell derived inducible factor ( IL-TIF ), a novel cytokine structurally related to IL-10 and inducible by IL-9. J. Immunol. 164: 1814, 2000a.
Dumoutier, L., E. Van Roost, D. Colau, and J.-C. Renauld. Human IL-TIF: molecular cloning and functional characterization as an hepatocyte stimulating factor. Proc. Natl. Acad. Sci. USA 97: 10144, 2000b.
Dumoutier, L., Leemans, C., Lejeune, D., Kotenko, S. V., and Renauld, J. C. Cutting edge: STAT activation by IL-19, IL-20 and mda-7 through IL-20 receptor complexes of two types. J.Immunol. 167: 3545-3549, 2001.
Fei, N.F., B.E. Castle, R. Barrett, R. Kastelein, W. Dang, T.R. mosmann, K. W. Moore, and M. Howard. Interleukin-10, a novel B cell stimulatory factor: unresponsiveness of X chromosome-linked immunodeficiency B cell. J. Exp. Med. 172: 1625-1631, 1990.
Gastl, G.A., Abrams, J.S., Nanus, D.M., et al. Interleukin-10 production by humancarcinoma cell lies and its relationship to interleukin-6 expression. Int. J. Cancer 55: 96-101, 1993.
Gesser, B., Leffers, H., Jinquan, T., Vestergaard, C., Kirstein, N., Sindet-Pedersen, S., Jensen, S. L.,Thestrup-Pedersen, K., and Larsen, C. G. Identification of functional domains on human interleukin 10. Proc. Natl. Acad. Sci. USA 94: 14620-14625, 1997.
Gallagher, G., Dickensheets, H., Eskdale, J., Izotova, L. S., Mirochnitchenko, O. V., Peat, J. D., Vazquez, N., Pestka, S., Donnelly, R. P., and Kotenko, S. V. Cloning, expression and initial characterization of interleukin-19 (IL-19), a novel homologue of human interleukin-10 (IL-10). Genes Immun. 1: 442-450, 2000.
Gruenberg B.H., Schoenemeyer A., Weiss B., Toschi L., Kunz S., Wolk K., Asadullah K., Sabat R. A novel, soluble homologue of the human IL-10 receptor with preferential expression in placenta. Genes and Immunity 2: 329-334, 2001.
Jinquan, T., Larsen, C. G., Gessser, b., Matsushima, K.&Thestrup-Pedersen, K. Human IL-10 is a chemoattractant for CD8+ T lymphocytes and an inhibitor of IL-8-induced CD4+ T lymphocyte migration. J. Immunol. 151: 4545-4551, 1993.
Katsikis, P.D., C.-Q. Chu, F.M. Brennan, R.N. Maini, and M. Feldmann. Immunoregulatory role of interleukin 10 in rheumatoid arthritis. J. Exp. Med. 179: 1517-1527, 1994.
Kasai T., Inada K., Takakuwa T., et al. Anti-inflammatory cytokine levels in patients with septic shock. Res Commun Mol Pathol Pharmacol 98: 34-42, 1997.
Knappe, A., s. Hor, S. Wittmann, and H. Fickenscher. Induction of a novel cellular homolog of interleukin-10, Ak155, by transformation of T lymphocytes with herpesvirus saimiri. J. Virol. 74: 3881-3887, 2000.
Kotenko, S. V., L. S. Izotova, O. V. Mirochnitchenko, E. Esterova, H. Dickensheets, R. P. Donnelly, and S. Pestka. Identificationof the functional interleukin-22 ( IL-22 ) receptor complex : the IL-10R2 chain ( IL-10Rb ) is a common chain of both the IL-10 and IL-22 ( IL-TIF ) receptor complexes. J. Biol. Chem. 276: 2725-2732, 2000.
Laure D., Caroline L., Diane L., Sergei V. K., and Jean-Christophe R. Cutting Edge : STAT activation by IL-19, IL-20 and mda-7 through IL-20 receptor complexes of two types. J. Immunol. 167 : 3545-3549, 2001.
Moore K.W., Vieira P., Fiorentino D. et al. Homology of cytokine synthesis inhibitory factor ( IL-10 ) to Epstein-Barr virus gene BCRF1. Science 248: 1230-1234, 1990.
Munoz C., Carlet J, Fitting C., et al. Dysregulation of in vitro cytokine production by monocytes during sepsis. J. Clin Invest 88: 1747-1754, 1991.
Moore KW, O’Garra A, de waal Malefyt R, Vieira P, Mosmann TR. Interleukin-10. Annu Rev Immunol 11: 165-190, 1993.
Mignon-Godefroy, K., O. Rott, m.-P. Brazillet, and J. Charreire. Curative and protective effects of IL-10 in experimental autoimmune thyroiditis ( EAT ). J. Immunol. 154: 6634-6643, 1995.
Ploegh HL. Viral strategies of immune evasion. Science 280: 248-253, 1998.
Rousser, F., E. Garcia, T. Defrance, C. Peronne, n. Vezzio, D.-H. Hsu, R. Kastelein, K.W. Moore, and J. Banchereau. Interleukin-10 is a potent growth and differentiation factor for activated human B lymphocytes. Proc. Natl. Acad. Sci. USA 89: 1890-1893, 1992.
Spriggs MK. One step ahead of the game : viral immunomodulatory molecules. Annu Rev Immunol 130: 101-130, 1996.
Steven M. Opal, Vera A. Depalo. Impact of basic research on tomorrow’s medicine : Anti-inflammatory cytokine. Chest 117: 1162-1172, 2000.
Thompson-Snipes, L.A., V. Dhar, M.W. Bond. T. R. Mosmann, K.W. Moore and D.M. Rennick. Interleukin-10: a novel stimulatory factor for mast cells and their progenitors. J. Exp. Med. 173: 507-510, 1991.
Vieira, P., de Waal Malefyt, R., Dang, W., et al. Isolation oand expressiono fhuman cytokine synthesis inhibitory factor ( CSIF ) cDNA clones: homology to Epstein Barr virus open reading frame BCRFI. Proc. Natl Acad. Sci. USA 88: 1172-1176, 1991.
Wanidworanun C, Strober W. Predominant role of tumour necrosis factor-a in human monocyte IL-10 synthesis. J. Immunol 151: 6853-6861, 1993.
Xie, M. H., S. Aggarwal, W.H. Ho, J. Foster, Z. Zhang, J. Stinson, w.J. Wood, A.D. Goddard, and A. L. Gurney. Interleukin (IL)-22, a novel human cytokine that signals through the interferon receptor related proteins CRF2-4 and IL-22R. J. Biol. Chem. 275: 31335-31339, 2000.
Yssel, H., De Waal Malefyt, R., Roncarolo, M.G., et al. IL-10 is produced by subsets of human CD4+ T cell clones and peripheral blood T cells. J. Immunol. 149: 2378-2384, 1992.
Zdanov A., Schalk-Hihi C., Wlodawer A. Crystal struture of human interleukin-10 at 1.6 A resoution and a model of a complex with is soluble receptor. Proterin Sci. 5 : 1955-1962, 1996.