進階搜尋


   電子論文尚未授權公開,紙本請查館藏目錄
(※如查詢不到或館藏狀況顯示「閉架不公開」,表示該本論文不在書庫,無法取用。)
系統識別號 U0026-2608201014003800
論文名稱(中文) 介白素十九單株抗體於關節炎動物模式中之特性分析
論文名稱(英文) Characterization of anti-IL-19 monoclonal antibody in experimental arthritis animal model
校院名稱 成功大學
系所名稱(中) 生物化學暨分子生物學研究所
系所名稱(英) of Biochemistry and Molecular Biology
學年度 98
學期 2
出版年 99
研究生(中文) 謝佩蓓
研究生(英文) Pei-Pei Hsieh
電子信箱 peipei7582@hotmail.com
學號 s1697412
學位類別 碩士
語文別 中文
論文頁數 81頁
口試委員 指導教授-張明熙
口試委員-張敏政
口試委員-鄭宏祺
中文關鍵字 介白素十九  滑液纖維母細胞  第二型膠原蛋白誘導關節炎動物模式  抗原決定位 
英文關鍵字 Interleukin-19  synovial fibroblast  collagen-induced arthritis  epitope 
學科別分類
中文摘要 介白素19屬於介白素10家族的一員,其中介白素10家族成員包括:介白素10、19、20、22、24、26、28及介白素29。文獻指出介白素19、20及22皆參與類風濕性關節炎的致病過程,其中介白素19會藉由活化介白素6與降低滑液細胞自我凋亡,在RA疾病當中扮演促進發炎反應的角色。已知,IL-19與IL-20共用了IL-20RI及IL-20R2此受體複合物。在實驗室過去的研究當中,利用電穿孔方式送入表現游離的IL-20RI受體次單元之質體,可以有效降低CIA大鼠之關節炎嚴重程度。因此,我們想進一步利用抗人類IL-19單株抗體-1BB1處理CIA大鼠,藉此改變IL-19於動物體內的表現量來探討其是否也可用於治療並緩解關節炎的嚴重程度。首先,利用免疫組織化學染色法及反轉錄-聚合酶連鎖反應,在CIA大鼠之關節滑液纖維母細胞中,可偵測到高表現量的IL-19。此外,IL-19也會作用在CIA大鼠關節滑液纖維母細胞上,促使關節滑液纖維母細胞表現大量的TNF-α、IL-1β及RANKL等細胞激素。我們利用第二型膠原蛋白所建立的CIA大鼠關節炎動物模式,在處理抗人類IL-19單株抗體-1BB1的組別可以明顯看出其腳掌腫脹程度與骨密度流失都有減輕的現象,並且在其關節滑液膜當中,利用反轉錄-聚合酶連鎖反應可偵測到TNF-α、IL-1β以及RANKL等細胞激素表現量皆明顯下降。而在處理1BB1之關節炎大鼠,其血清中的IL-6表現量也較mIgG控制組來得低許多。由以上實驗結果推測,1BB1與IL-19的結合位置為一重要的motif,IL-19可能藉由此位點來傳遞下游的訊息,也可能阻止IL-19與receptor的結合而阻擋下游訊號的傳遞,因此我們想要進一步找出此epitope。首先,我們構築並表現各種不同的hIL-19截短型蛋白質,經由西方墨點法與點突變的方式證實1BB1所辨認之IL-19的位置,最後找出1BB1所辨識之IL-19 motif位於三個連續的胺基酸序列上。而我們進一步將此胺基酸序列做突變,則可以破壞IL-19與1BB1之間的結合。為了証實此IL-19 motif的重要性,我們計劃分析IL-19突變型蛋白與IL-19野生型蛋白其生物功能性的差異,藉此,不僅可更深入了解IL-19於RA的角色為何,同時也提供了未來於類風濕性關節炎開發新藥的契機。
英文摘要 Interleukin-19 (IL-19) belongs to the IL-10 family, which includes IL-10, IL-19, IL-20, IL-22, IL-24, IL-26, IL-28, and IL-29. Previous studies showed that IL-19, IL-20, and IL-22 are involved in the pathogenesis of rheumatoid arthritis(RA). Thus, IL-19 has been defined as an pro-inflammatory cytokine by increasing IL-6 production and reducing RASC apoptosis. IL-19 signals through IL-20R1 and IL-20R2 receptor complex that is also utilized by IL-20. In our previous study, electroporated sIL-20R1 plasmid DNA decreased the severity of arthritis in the rats with CIA. Therefore, we investigated whether anti-IL-19 monoclonal antibody (1BB1) treatment could modulate the severity of the disease in the collagen-induced arthritis (CIA) rat model. In this study, we demonstrated that IL-19 was upregulated in CIA rat synovial fibroblasts(SFs) and induced SFs to secrete TNF-α and IL-1β in vitro. In CIA animal model, anti-IL-19 antibody (1BB1) significantly reduced the severity of arthritis by decreasing hind-paw thickness and downregulating the expression of TNF-α, IL-1β and RANKL in synovial membrane. The expression of IL-6 was downregulated in CIA rat serum after treatment with 1BB1. These results suggested that the binding motif of IL-19 by 1BB1 might be important in the IL-19-mediated cellular responses. Therefore, we aimed to determine the vital motif of IL-19 recognized by 1BB1. We performed a serial deletion on IL-19 coding region and expressed the recombinant proteins of these deleted mutants. We mapped the epitope of IL-19 which was recognized by 1BB1 by using Western blot analysis of truncated IL-19 proteins binding with 1BB1. To confirm the importance of the 1BB1-binding motif, we will compare the biological function and intracellular signaling between IL-19-mutant and wild-type IL-19 (IL-19-WT). This study will provide important information in developing
drug for treating inflammatory diseases associated with IL-19.
論文目次 致謝 i
摘要 iii
Abstract iv
目錄 v
圖目錄 viii
附錄目錄 x
縮寫檢索表 xi

第一章 緒論 1
1-1 自體免疫疾病 (Autoimmune disease) 1
1-2 類風濕性關節炎 (Rheumatoid arthritis) 1
1-3 細胞激素 (Cytokines) 2
1-4 細胞激素之拮抗劑 (Antagonist of cytokines) 3
1-5 介白素10 (Interleukin-10) 4
1-6 介白素19 (Interleukin-19) 5
1-6-1 介白素19與類風濕性關節炎 6
1-7 第二型膠原蛋白誘導之關節炎 (CIA)動物模式 6
1-8 抗人類介白素19單株抗體1BB1融合瘤細胞來源 7

第二章 研究目的 8

第三章 材料與方法 9
3-1 實驗材料 9
3-1-1 實驗動物 9
3-1-2 細胞來源 9
3-1-3 實驗之菌株、質體與培養基 9
3-1-4 實驗溶劑 12
3-1-5 限制酵素 15
3-2 實驗方法 15
3-2-1 CIA大鼠關節滑液母細胞之分離與培養 15
3-2-2 免疫細胞化學染色法 16
3-2-3 反轉錄-聚合酶連鎖反應 16
3-2-4 融合瘤細胞之繼代與培養 17
3-2-5 生產與純化抗人類介白素19單株抗體 17
3-2-6 第二型膠原蛋白大鼠關節炎 18
(A) 大鼠關節言之誘導 18
(B) 大鼠關節炎臨床症狀評估 18
(C) 單株抗體的給予 19
(D) 利用測徑器量測大鼠腳掌厚度 19
(E) 利用超高解析度活體動物斷層掃描儀分析骨質密度 19
(F) 偵測滑液組織中發炎反應之媒介 19
3-2-7 酵素連結免疫吸附分析法 20
3-2-8 構築人類截短型 IL-19重組蛋白於 pMAL-c2X載體 20
(A) 聚合酶連鎖反應 21
(B) Insert及 vector的製備 21
(C) 限制酶處理 22
(D) 接合反應 23
(E) 製備E. coli的勝任細胞 23
(F) 形質轉移 23
(G) 單一菌落PCR 24
(H) 抽取質體 24
3-2-9 構築人類截短型 IL-19重組蛋白於 pMAL-c2X載體 24
3-2-10 由大腸桿菌系統表現人類截短型(突變型) IL-19重組蛋白 25
3-2-11 西方轉漬法 25
3-2-12 在 pSecTag2/Hygro A載體構築突變型 IL-19重組蛋白 26
3-2-13 由哺乳類細胞表現人類突變型 IL-19重組蛋白 26
3-2-14 純化人類突變型 IL-19重組蛋白 26
3-2-15 細胞內訊號傳遞分析 27

第四章 結果 29
4-1 CIA大鼠動物模式 29
4-2 於CIA之關節滑液纖維母細胞中,有較高量IL-19的表達 29
4-3 以抗IL-19單株抗體1BB1處理CIA大鼠,可有效改善其關節炎之嚴重性 29
4-4 利用Micro-CT及放射線分析CIA大鼠脛骨骨密度與後腳腳踝軟骨及硬骨破壞程度 30
4-5 IL-19可誘導CIA大鼠關節滑液纖維母細胞 (SFs)產生大量TNF-α、IL-1β及RANKL表現 31
4-6 IL-19藉由活化p-ERK、p-JNK、p-c-Fos以及p-TRAF-6於CIA SFs傳遞下游訊息 31
4-7 處理抗IL-19單株抗體1BB1可抑制於CIA大鼠滑液膜表達TNF-α、IL-1β與RANKL 32
4-8 處理抗IL-19單株抗體1BB1,可降低CIA大鼠血清中IL-6的表現 33
4-9 界定IL-19與其單株抗體1BB1之抗原決定位 (epitope) 34
4-10 純化人類IL-19突變型蛋白 35

第五章 討論 37

參考文獻 41
圖 47
附錄 69
自述 81
參考文獻 1. Muller-Ladner, U., Pap, T., Gay, R.E., Neidhart, M., and Gay, S. (2005). Mechanisms of disease: the molecular and cellular basis of joint destruction in rheumatoid arthritis. Nat Clin Pract Rheumatol 1, 102-110.

2. FitzGerald O, Soden M, Yanni G, Robinson R, Bresnihan B. (1991). Morphometric analysis of blood vessels in synovial membranes obtained from clinically affected and unaffected knee joints of patients with rheumatoid arthritis. Ann Rheum Dis 50, 792-6.

3. Lee DM, Weinblatt ME. (2001). Rheumatoid arthritis. Lancet 358, 903-11.

4. Yamanishi Y, Firestein GS. (2001). Pathogenesis of rheumatoid arthritis: the role of synoviocytes. Rheum Dis Clin North Am 27, 355-71.

5. Feldmann M, Brennan FM, Maini RN. (1996). Role of cytokines in rheumatoid arthritis. Annu Rev Immunol 14, 397-440

6. Panayi GS, Lanchbury JS, Kingsley GH. (1992). The importance of the T cell in chronic synovitis of rheumatoid arthritis. Arthritis Rheum 35, 729-35.

7. Firestein GS, Zvaifler NJ. (2002). How important are T cells in chronic rheumatoid synovitis?:II. T cell-independent mechanisms from beginning to end. Arthritis Rheum 46, 298-308.

8. Wood NC, Dickens E, Symins JA, Duff GW. (1992). In situ hybridization of interleukin-1 in CD14-positive cells in rheumatoid arthritis. Clin Immunol Immunopathol 62, 295-300.

9. Chu CQ, Field M, Feldmann M Maini RN. (1991). Localization of tumor necrosis factor alpha in synovial tissues and at the cartilage-pannus junction in patients with rheumatoid arthritis. Arthritis Rheum 34, 1125-32.

10. Cope AP, Aderka D, Doherty M, Engelmann H, Gibbons D, Jones AC, et al. (1992). Increased levels of soluble tumor necrosis factor receptors in the sera and synovial fluid of patients with rheumatic disease. Arthritis Rheum 35, 1160-9.

11. Moore KW, deWaal Malefyt R, Coffman RL, and O’Garra A. (2001). Interleukin-10 and the Interleukin-10 receptor. Annu Rev Immuno 19, 683-765.

12. Moore KW, Vieira P, Fiorentino DF, Trounstine ML, Khan TA, Mosmann TR. (1990). Homology of cytokine synthesis inhibitory factor (IL-10) to the Epstein-Barr virus gene BCRFI. Science 248, 1230-4.

13. Thompson-Snipes L, Dhar V, Bond MW, Mosmann TR, Moore KW, Rennick DM. (1991). Interleukin 10: a novel stimulatory factor for mast cells and their progenitors. J Exp Med 173, 507-10.

14. Rousset F, Garcia E, Defrance T, Péronne C, Vezzio N, Hsu DH, Kastelein R, Moore KW, Banchereau J. (1992). Interleukin 10 is a potent growth and differentiation factor for activated human B lymphocytes. Proc Natl Acad Sci U S A. 89, 1890-3.

15. Go NF, Castle BE, Barrett R, Kastelein R, Dang W, Mosmann TR, Moore KW, Howard M. (1990). Interleukin 10, a novel B cell stimulatory factor: unresponsiveness of X chromosome-linked immunodeficiency B cells. J Exp Med. 172, 1625-31.

16. Gallagher G, Dickensheets H, Eskdale J, Izotova LS, Mirochnitchenko OV, Peat JD, et al. (2000). Cloning, expression and initial characterization of interleukin-19 (IL-19), a novel homologue of human interleukin-10 (IL-10). Genes Immun. 1, 442-50.

17. Blumberg H, Conklin D, Xu WF, Grossmann A, Brender T, Carollo S, et al. (2001). Interleukin 20: discovery, receptor identification, and role in epidermal function. Cell 104, 9-19.

18. Dumoutier L, Louahed J, Renauld JC. (2000). Cloning and characterization of 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, 1814-9.

19. Jiang H, Lin JJ, Su ZZ, Goldstein NI, Fisher PB. (1995). Subtraction hybridization identifies a novel melanoma differentiation associated gene, mda-7, modulated during human melanoma differentiation, growth and progression. Oncogene 11, 2477-86.

20. Knappe A, Hör S, Wittmann S, Fickenscher H. (1995). Induction of a novel cellular homolog of interleukin-10, AK155, by transformation of T lymphocytes with herpesvirus saimiri. J Virol 74, 3881-7.

21. Wei CC, Chen WY, Wang YC, Chen PJ, Lee JY, Wong TW, Chen WC, Wu JC, Chen GY, Chang MS, Lin YC. (2005). Detection of IL-20 and its receptors on psoriatic skin. Clin Immunol 117, 65-72.

22. Hsu YH, Li HH, Hsieh MY, Liu MF, Huang KY, Chin LS, Chen PC, Cheng HH, Chang MS. (2006). Function of interleukin-20 as a proinflammatory molecule in rheumatoid and experimental arthritis. Arthritis Rheum 54, 2722-33.

23. Ikeuchi H, Kuroiwa T, Hiramatsu N, Kaneko Y, Hiromura K, Ueki K, Nojima Y. (2005). Expression of interleukin-22 in rheumatoid arthritis: potential role as a proinflammatory cytokine. Arthritis Rheum 52, 1037-46.

24. Hör S, Pirzer H, Dumoutier L, Bauer F, Wittmann S, Sticht H, et al. (2004). The T-cell lymphokine interleukin-26 targets epithelial cells through the interleukin-20 receptor 1 and interleukin-10 receptor 2 chains. J Biol Chem 279, 33343-51.

25. Sheikh F, Baurin VV, Lewis-Antes A, Shah NK, Smirnov SV, Anantha S, et al. (2004). Cutting edge: IL-26 signals through a novel receptor complex composed of IL-20 receptor 1 and IL-10 receptor 2. J Immunol 172, 2006-10.

26. al-Janadi M, al-Dalaan A, al-Balla S, al-Humaidi M, Raziuddin S. (1996). Interleukin-10 (IL-10) secretion in systemic lupus erythematosus and rheumatoid arthritis: IL-10-dependent CD4+CD45RO+ T cell-B cell antibody synthesis. J Clin Immunol 16, 198-207.

27. Walmsley M, Katsikis PD, Abney E, Parry S, Williams RO, Maini RN, Feldmann M. (1996). Interleukin-10 inhibition of the progression of established collagen-induced arthritis. Arthritis Rheum 39, 495-503.

28. Finnegan A, Kaplan CD, Cao Y, Eibel H, Glant TT, Zhang J. (2003). Collagen-induced arthritis is exacerbated in IL-10-deficient mice. Arthritis Res Ther. 5, R18-24.

29. Katsikis PD, Chu CQ, Brennan FM, Maini RN, Feldmann M. (1994). Immunoregulatory role of interleukin 10 in rheumatoid arthritis. J Exp Med 179, 1517-27.

30. Gallagher G, Dickensheets H, Eskdale J, Izotova LS, Mirochnitchenko OV, Peat JD, Vazquez N, Pestka S, Donnelly RP, Kotenko SV. (2000). Cloning, expression and initial characterization of interleukin-19 (IL-19), a novel homologue of human interleukin-10 (IL-10). Genes Immun 1, 442-450.

31. Foster D, Parrish-Novak J, Fox B, Xu W. (2004). Cytokine-receptor pairing: accelerating discovery of cytokine function. Nat Rev Drug Discov 3, 160-170.

32. Pestka S, Krause CD, Sarkar D, Walter MR, Shi Y, Fisher PB. (2004). Interleukin-10 and related cytokines and receptors. Annu Rev Immunol 22, 929-979.

33. Gallagher G, Eskdale J, Jordan W, Peat J, Campbell J, Boniotto M, Lennon GP, Dickensheets H, Donnelly RP. (2004). Human interleukin-19 and its receptor: a potential role in the induction of Th2 responses. Int Immunopharmacol 4, 615-626.

34. Romer J, Hasselager E, Norby PL, Steiniche T, Thorn Clausen J, Kragballe K. (2003). Epidermal overexpression of interleukin-19 and -20 mRNA in psoriatic skin disappears after short-term treatment with cyclosporine a or calcipotriol. J Invest Dermatol 121, 1306-1311.

35. Wolk K, Kunz S, Asadullah K, Sabat R. (2002). Cutting edge: immune cells as sources and targets of the IL-10 family members? J Immunol 168, 5397-5402.

36. Liao YC, Liang WG, Chen FW, Hsu JH, Yang JJ, Chang MS. (2002). IL-19 induces production of IL-6 and TNF-alpha and results in cell apoptosis through TNF-alpha. J Immunol 169, 4288-97.

37. Jordan WJ, Eskdale J, Boniotto M, Lennon GP, Peat J, Campbell JD, Gallagher G.(2005). Human IL-19 regulates immunity through auto-induction of IL-19 and production of IL-10. Eur J Immunol 35, 1576-1582.

38. Liao SC, Cheng YC, Wang YC, Wang CW, Yang SM, Yu CK, et al. (2004). IL-19 induced Th2 cytokines and was up-regulated in asthma patients. J Immunol 173, 6712-8.

39. Oral HB, Kotenko SV, Yilmaz M, Mani O, Zumkehr J, Blaser K, Akdis CA, Akdis M. (2006). Regulation of T cells and cytokines by the interleukin-10 (IL-10)-family cytokines IL-19, IL-20, IL-22, IL-24 and IL-26. Eur J Immunol 36, 380-8.

40. Sakurai N, Kuroiwa T, Ikeuchi H, Hiramatsu N, Maeshima A, Kaneko Y, Hiromura K, Nojima Y. (2008). Expression of IL-19 and its receptors in RA: potential role for synovial hyperplasia formation. Rheumatology (Oxford) 47, 815-20.

41. Alanärä T, Karstila K, Moilanen T, Silvennoinen O, Isomäki P. (2009). Expression of IL-10 family cytokines in rheumatoid arthritis: elevated levels of IL-19 in the joints. Scand J Rheumatol 39, 118-26.

42. Trentham DE, Townes AS, Kang AH. (1977). Autoimmunity to type II collagen an experimental model of arthritis. J Exp Med 146, 857-868.

43. Durie FH, Fava RA, Noelle RJ. (1994). Collagen-induced arthritis as a model of rheumatoid arthritis. Clin Immunol Immunopathol 73, 11-8.

44. Klareskog L, Holmdahl R, Larsson E, Wigzell H. (1983). Role of T lymphocytes in collagen II induced arthritis in rats. Clin Exp Immunol 51, 117-25.

45. Hsu YH, Chang MS. (2010). Interleukin-20 antibody is a potential therapeutic for rheumatoid arthritis. Arthritis Rheum (Impress)

46. Stephanou A, Latchman DS. (2005). Opposing actions of STAT-1 and STAT-3. Growth factors. 23, 177-82.

47. Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C. (1995). A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J Immunol Methods. 184, 39-51.

48. Cohen GM. (1997). Caspases: the executioners of apoptosis. Biochem J. 326:1-16.

49. Firestein GS. (2003). Evolving concepts of rheumatoid arthritis. Nature. 423:356-61.

50. Abramson SB, Amin A. (2002). Blocking the effects of IL-1 in rheumatoid arthritis protects bone and cartilage. Rheumatology(Oxford). 41:972-80.

51. Hsing CH, Chiu CJ, Chang LY, Hsu CC, Chang MS. (2008). IL-19 is involved in the pathogenesis of endotoxic shock. Shock. 29:7-15.

52. Chang C, Magracheva E, Kozlov S, Fong S, Tobin G, Kotenko S, Wlodawer A, Zdanov A. (2002). Crystal Structure of Interleukin-19 Defines a New Subfamily of helical cytokines. 278:3308-13.

53. Trivella DB, Ferreira-Júnior JR, Dumoutier L, Renauld JC, Polikarpov I. (2010). Structure and function of interleukin-22 and other members of the interleukin-10 family. (Impress)
論文全文使用權限
  • 同意授權校內瀏覽/列印電子全文服務,於2020-12-31起公開。


  • 如您有疑問,請聯絡圖書館
    聯絡電話:(06)2757575#65773
    聯絡E-mail:etds@email.ncku.edu.tw