臺灣博碩士論文加值系統

胰島素依賴型糖尿病（insulin-dependent diabetes mellitus, IDDM），又稱第一型糖尿病 (type I diabetes) 主要致病原因是體內的T淋巴細胞選擇性的攻擊胰臟蘭氏小島 （islets of Langerhans）中具有產生胰島素（insulin）能力的β細胞。非肥胖型糖尿病小鼠 （nonobese diabetic mouse, NOD mouse）可以自發性產生與人類胰島素依賴型糖尿病相似的糖尿病，為目前研究這類疾病最佳的動物模式。4-1BB屬於腫瘤壞死因子受體家族（tumor necrosis factor receptor family）中的一員，表現在活化的CD4+ 和CD8+ T細胞表面，為細胞提供共刺激信號，促進細胞增殖以及釋放細胞激素。研究報導指出，施打agonistic anti-4-1BB單株抗體進入具有實驗誘導自體免疫腦脊髓炎（experimental autoimmune encephalomyelitis, EAE）病症的實驗小鼠中，可以有效減輕病狀。本論文將測試anti-4-1BB對IDDM的影響，我們分兩個策略進行，第一：施打agonistic anti-4-1BB進入NOD小鼠中，觀察其IDDM的發病情形，實驗結果發現，治療後NOD小鼠IDDM發病率降低，具有部分保護的效果，其Th1細胞族群先增加後降低，胰島淋巴細胞浸潤的現象減輕。第二：建構出能在β細胞上表現single-chain anti-4-1BB Fv的基因轉殖NOD小鼠，利用胰島啟動子（insulin promoter）攜帶一段anti-4-1BB scFv，當自體免疫T細胞攻擊β細胞時期望能藉由anti-4-1BB scFv活化4-1BB，進而促進T細胞進行活化誘導的細胞凋亡，以保護β細胞不受到自體免疫的破壞。然而從實驗結果中卻發現表現anti-4-1BB scFv的基因轉殖NOD小鼠在第四、五週大時就有發病的現象，經由anti-4-1BB scFv的表現提早並且加重IDDM的病發。從組織切片圖可看出在基因轉殖小鼠的胰島中，淋巴細胞浸潤的現象很嚴重。因表現single-chain anti-4-1BB而增強了自體免疫淋巴細胞攻擊β細胞的反應。

Abstract
Insulin-dependent diabetes mellitus (IDDM), or type I diabetes, is caused by progressive autoimmune destruction of the insulin-producing b cells in the pancreatic islets of Langerhan. A widely used animal model for dissecting the immunopathological mechanisms in IDDM and for developing preventive or therapeutic strategies is the non-obese diabetic (NOD) mouse, an inbred strain that spontaneously develops autoimmune diabetes resembling human IDDM. Obviously, the amelioration of the autoreactive T-cell responses or the induction of autoantigen-specific tolerance will be the key to preventing or curing IDDM. 4-1BB, a member of the tumor necrosis factor receptor (TNFR) superfamily, is a costimulatory receptor primarily expressed on activated T cells. The administration of an agonistic anti-4-1BB antibody (2A) dramatically reduced the incidence and severity of experimental autoimmune encephalomyelitis (EAE). Treatment with the same antibody in Fas-deficient MRL/lpr mice blocked lymphadenopathy and lupus-like autoimmune processes. Therefore, we in this study are trying to establish a 4-1BB-based immunotherapy for IDDM. Paradoxically, transgenic NOD mice overexpressing membrane-bound agonistic single-chain anti-4-1BB Fv in pancreatic b cells developed more severe diabetes than their non-transgenic littermates, with earlier onset, faster diabetic processes, and higher mortality. Forty percent of transgenic mice developed diabetes by four weeks of age, compared with their control littermates, which first exhibited diabetes at 14 weeks. The frequency of diabetes in female transgenics reached 70% by eight weeks of age. Most female transgenic mice died around 12 weeks. Consistent with this, transgenic mice developed earlier and more severe insulitis, compared with age-matched control littermates. Our results indicate an adverse effect of transgenic anti-4-1BB scFv in NOD mice and suggest a potential risk of this anti-4-1BB-based immunotherapy for autoimmune diseases. However, on the other hand, in vivo anti-4-1BB mAb injection caused partial protection in NOD mice and altered lymphocyte number and subpopulation in peripheral lymphoid organs. Our results suggest that further studies extending to additional systems must be undertaken, and a better understanding must be sought of the mechanisms by which 4-1BB is engaged, using monoclonal antibodies, natural ligands, or scFv.

目　錄
項 目 頁數
目錄………………………………………………………. I
摘要……………………………………………………….. V
Abstract………………………………………………….. VI
第一章　導 論…………………………………………… 1
　　　壹、研究IDDM的動物模式─NOD老鼠………………. 2
　　　　一、NOD老鼠IDDM的發病過程……………………. 2
　　　　二、摧毀β細胞的免疫反應機轉……………………… 3
貳、遺傳因子與IDDM的關係…………………………… 3
參、環境因子與IDDM的關係………………………….. 5
肆、IDDM相關的自體抗原(autoantigen)…………….. 5
伍、免疫系統失調與IDDM的關係…………………….. 6
一、 中央耐受性（central tolerance）失敗和週邊耐受性（peripheral tolerance）失敗……………………… 6
二、Th1和Th2細胞不平衡對IDDM的影響…………… 6
陸、以anti-4-1BB治療NOD小鼠，並探討其保護機制…………………………………………. 7
一、4-1BB的基本架構………………………………… 7
二、4-1BB所調控的免疫反應………………………… 8
三、4-1BB 對自體免疫性疾病造成的影響…………… 10
四、利用4-1BB治療自體免疫性糖尿病所進行的策略 11
第二章　材料與方法…………………………………………. 12
壹、 實驗材料…………………………………………….. 12
一、試劑與溶液…………………………………….. 12
二、實驗老鼠品系…………………………………… 18
貳、實驗方法……………………………………………… 19
一、細胞培養………………………………………… 19
二、Anti-4-1BB單株抗體的製備………………….. 19
三、以活化T淋巴細胞來確認anti4-1BB單株抗體的功能…………………………………………. 20
四、NOD小鼠施打anti-4-1BB單株抗體………….. 20
五、以流式細胞儀分析施打anti-4-1BB NOD小鼠脾臟細胞族群比例的變化……………………. 21
六、實驗動物老鼠之胰島炎及尿糖檢測 …………. 21
七、勝任細胞 (competent cell) 製備法……….. 22
八、轉形作用 (transformation)………………… 22
九、細菌質體的小量製備………………………….. 22
十、細菌質體的大量製備………………………….. 23
十一、DNA 片段之切割、接合及分析…………….. 24
十二、pIns1-single-chain anti-4-1BB Fv和pIns2-single-chain anti-4-1BB Fv表現載體之構築……………………………………. 25
十三、轉染作用(transfection)………………….. 25
十四、細胞中核醣核酸（ribonucleic acid, RNA）之萃取……………………………………. 26
十五、反轉錄聚合酵素連續反應………………….. 27
十六、建立HIT/pIns2-single-chain anti-4-1BB Fv 穩定表達之細胞株……………………… 28
十七、免疫細胞化學染色………………………….. 29
十八、蛋白質之萃取……………………………….. 29
十九、蛋白質濃度之測定………………………….. 30
二十、以西方點墨法（Western blot）確認表現之蛋白質………………………………………. 30
二十一、建立pIns1- single-chain anti-4-1BB Fv非肥胖型糖尿病基因轉殖小鼠……………. 31
二十二、實驗動物鼠染色體之萃取………………… 32
二十三、利用聚合酵素連續反應系統篩檢pIns1-single-chain anti-4-1BB Fv非肥胖型糖尿病基因轉殖鼠…………………. 32
二十四、實驗老鼠組織中核醣核酸（RNA）的萃取…… 33
第三章　結 果…………………………………………………. 35
一、以活化T淋巴細胞來確認anti4-1BB 單株抗體的功能………………………………………………… 35
二、NOD母鼠以腹腔注射anti-4-1BB後觀察其糖尿病病發率及胰島炎…………………………………………. 35
三、NOD母鼠以腹腔注射anti-4-1BB後觀察其脾臟細胞數目及細胞族群比例變化……………………………. 36
四、觀察pIns2-single-chain anti-4-1BB Fv在HIT-15腫瘤細胞株上，RNA以及蛋白質的表現情形………… 37
五 pIns1-single-chain anti-4-1BB Fv非肥胖型糖尿病轉殖鼠確認實驗……………………………………. 37
六、pIns1-single-chain anti-4-1BB Fv非肥胖型糖尿病基因轉殖鼠不同器官single-chain anti-4-1BB Fv cDNA的表現……………………………………………. 38
七、pIns1-single-chain anti-4-1BB Fv非肥胖型糖尿病基因轉殖鼠的糖尿病病發情形及胰島炎程度……. 38
第四章　討 論…………………………………………… 40
圖表……………………………………………………….. 44
第五章　參考文獻……………………………………….. 62

第五章 參考文獻
1. Dlovitch, T. L.,Singh, B. 1997. The nonobese diabetic mouse as a model of autoimmune diabetes: immune dysregulation gets the NOD. Immunity 7(6):727-38
2. Makins, J. F., Holt, G., Macdonald, K. D. 1980. Co-synthesis of penicillin following treatment of mutants of Aspergillus nidulans impaired in antibiotic production with lytic enzymes. J Gen Microbiol 119:397-404
3. Yoon, J. W., Jun, H. S. 2001. Cellular and molecular pathogenic mechanisms of insulin-dependent diabetes mellitus. Ann N Y Acad Sci 928:200-11
4. Reizis, B., Eisenstein, M., Bockova, J., Konen-Waisman, S., Mor, F., Elias, D. and Cohen, I. R. 1997. Molecular characterization of the diabetes-associated mouse MHC class II protein, I-Ag7. Int Immunol 9:43-51
5. Todd, J. A., 1999. From genome to aetiology in a multifactorial disease, type 1 diabetes. Bioessays 21(2):164-74.
6. Barnett, A. H., Eff, C., Leslie, R. D., and Pyke, D. A. 1981. Diabetes in identical twins. A study of 200 pairs. Diabetologia 20:87-93
7. Forrest, J. M., Menser, M. A., Slinn, R. F., Nowak, M. J., Murphy, A. M., and Stout, M. 1971. Rubella vaccination trial. Med J Aust 2:470-3
8. Tian, J., Lehmann, P. V., and Kaufman, D. L. 1994. T cell cross-reactivity between coxsackievirus and glutmate decarboxylase isassociated with a murine diabetes susceptibility allele. J Exp Med 180:1979-84.
9. Atkinson, M. A., Bowman, M. A., Campbell, L., Darrow, B. L., Kaufman, D. L., Maclaren, N. K. 1994. Cellular immunity to a determinant common to glutamate decarboxylase and coxsackie virus in insulin-dependent diabetes. J Clin Invest 94:2125-9
10. Kaufman, D. L., Clare-Salzler M., Tian J., Forsthuber T., Ting G. S. P., Robinson P., Atkinson M. A., Sercarz E. E., Tobin A. J., and Lehmann P. V. 1993. Spontaneous loss of T-cell tolerance to glutamic acid decarboxylase in murine insulin-dependent diabetes. Nature 366:69 -72.
11. Haskins, K., and Wegmann. D., 1996. Diabetogenic T-Cell clones. Diabetes. 45(10): 1299-1305.
12. Fox, C. J., and Danska. J. S. 1997. IL-4 expression at the onset of islet inflammation predicts nondestructive insulitis in nonobese diabetic mice. Immunol. 158: 2414-2424.
13. Gombert J. M., Herbelin A., Tancrede-Bohin E., Dy M., Carnaud C., Bach J. F. Early quantitative and functional deficiency of NK1+-like thymocytes in the NOD mouse. Eur J Immunol. 26(12):2989-98.
14. Gombert J. M., Tancrede-Bohin E., Hameg A., Leite-de-Moraes M. C., Vicari A., Bach J. F., Herbelin A. IL-7 reverses NK1+ T cell-defective IL-4 production in the non-obese diabetic mouseInt. Immunol. 8(11):1751-8.
15. Kwon, B. S., and Weissman S. M.. 1989. cDNA Sequencesof two inducible T-cell genes. Proc. Natl. Acad. Sci. USA 86: 1963.
16. Dass S. V. and Byoung S. K. 1998. Role of 4-1BB in immune responses. seminars in IMMUNOLOGY 10: 481-489.
17. Kwon B., Moon C. H., Kang S., Seo S. K., Kwon B. S. 2000. 4-1BB: Still in the midst of darkness. Mol Cells 10:119-26.
18. Kwon B, Lee HW, Kwon BS. 2002. New insights into the role of 4-1BB in immune responses: Beyond CD8 + T cells. Trends Immunol 23:378-80.
19. Heinisch IVWM, Daigle I, Knopfli B, Simon HU. 2000. CD137 activation abrogates granulocyte-macrophage colony-stimulating factor-mediated anti-apoptosis in neutrophils. Eur Jimmunol 30:3441-6.
20. Futagawa T., Akiba H., Kodama T., Takeda K., Hosoda Y.,Yagita H., Okumura K. 2002. Expression and function of 4-1BB and4-1BB ligand on murine dendritic cells. Int Immunol 14:275-86.
21. DeBenedette M. A., Shahnian A., Mak T.W., Watts T. H. 1997 Co-stimulation of CD28-T lymphocytes by 4-1BB ligand. J Immunol 158:551-559.
22. Lee H. W., Park S. J., Choi B. K., Kim H. H., Nam K. O., Kwon B. S. 2002. 4-1BB promotes the survival of CD8 + T lymphocytes by increasing expression of Bcl-xL and Bfl-1. J Immunol 169:4822-8.
23. DeBenedette M. A., Wen T., Bachmann M. F., Ohashi P. S., Barber B. H., Stockling K. I., Peschon J. J., Watts T. H. 1999. 4-1BB ligand (4-1BBL)-deficient mice and of mice lacking both 4-1BBL andCD28 reveals a role for 4-1BB in skin allograft rejection andin the cytotoxic T cell response to influenza virus. J Immunol 163:4833-41
24. Melero I., Shuford W. W., Newby S. A., Aruffo A., Ledbetter J. A., Hellstrom K. E., Mittler R. S., Chen L. 1999. Monoclonal antibodies against the 4-1BB T-cell activation molecules eradicate established tumors. Nat Med 3:682-56330-7
25. Wilcox R. A., Tamada K., Strome S. E., Chen L. 2002. Signaling through NK cell-associated CD137 promotes both helper function for CD8+ cytolytic T cells and responsiveness to IL-2 but not cytolytic activity. J Immumol 169:4230-6.
26. Ye Z., Hellstrom I., Hayden-Ledbetter M., Dahlin A., Ledbetter J. A., Hellstrom K. E. 2002. Gene therapy for cancer using single-chainFv fragments specific for 4-1BB. Nat Med 8:343-8
27. Seko Y., Takahashi N., Oshima H., Shimozato O., Akiba H., Takeda K., Kobata T., Yagita H., Okumura K., Azuma M., Naga R. 2001. Expression of tumor necrosis factor (TNF) ligand superfamily costimulatory molecules CD30L, CD27L, OX40L, and 4-1BBL in murine hearts with acute myocarditis causedby coxakievirus B3. J Pathol 195:593-603.
28. Seo S. K., Park H. Y., Cho J. H., Kim W. Y., Jung H. W., Kwon B., Kwon B. S. 2003. Blocking 4-1BB/4-1BBL prevents herpetic stromal keratitis. J Immunol (In press)
29. Sun Y., Lin X., Chen H., Wu Q., Subudhi S. K., Chen L., Fu Y. X. 2002. Administration of agonistic anti-4-1BB monoclonal antibody leads to the amelioration of experimental autoimmune encephalomyelitis. J Immunol 168:1457-65.
30. Sun Y., Lin X., Chen L. M., Subudhi S. K., Chen J., Koda R., Chen L., Fu Y. X. 2002. Costimulatory molecule-targeted antibody therapyof a spontaneous autoimmune disease. Nat Med 8:1405-13
31. Hsieh, S. L., Chen N. J., Tarbell K., Liao N. S., Lai Y. G., Lee K.
H., Lee K. M., Wu S. C., Sytwu H. K., Han S. H., and McDevitt H. 2000.Transgenic mice expressing surface markers for IFNγ and IL-4 producing cells. Mol. Immunol. 37: 281-293.
32. Bosma G. C., Custer R. P., Bosma M. J. 1983. A severe combined immunodeficiency mutation in the mouse. Nature. 301:527-30.