全身性紅斑狼瘡 (Systemic Lupus Erythematosus， SLE) 是屬於典
型的全身性自體免疫疾病，其特色是在 SLE 病人血清中產生針對抗核抗
體。 DNase 是一種核酸內切，其主要的功能在於能夠將去氧核糖核
酸 (DNA) 水解，在正常人類的血液及尿液中此都存在。 在我們的研
究中顯示當利用酵素免疫分析的方式檢測 anti-DNase antibodies大約
有 62.4% 的SLE 病人是呈現陽性反應﹔而在抗核抗體為陰性的對照組
血清中僅 8% 為陽性反應。 另外 SLE 病人血清中 anti-DNase
antibodies 與 anti-DNA antibodies 是呈現正相關。 我們也收集
SLE 病人的血清經由親和性管柱純化的方式純化 anti-DNase IgG，
此種抗體經由酵素免疫法測試可以結合至 DNase 及 DNA﹔若將合成的
DNase peptide 與牛胰臟 DNase I 進行競爭性試驗則可以抑制與
anti-DNase IgG 和牛的 DNase I 結合。 當我們利用親和性管柱所
純化的 anti-DNase IgG 做間接免疫螢光分析法時，發現以 Hep-2
細胞作為受質時會呈現斑點型及核仁型的分佈，但不會在鞭毛蟲
(Crithidia luciliae) 作為受質時不會和其 kinetoplast 結合。
進一步利用親和性管柱所純化的 anti-DNase IgG 比正常人血清所
純化的 IgG 更明顯抑制鏈球菌、牛、人的 DNase 活性。 值得注意
的是，若利用牛的 DNase I 去免疫 BALB/c 及 NZB/NZW 老鼠所產生
的 anti-DNase antibodies 及 anti-DNA antibodies 的效價都較正
常之未被免疫老鼠來的高。此外，取用牛的 DNase I 免疫 BALB/c老
鼠的腎臟做組織學切片，發現在老鼠腎管壁有纖維細胞的替代。
綜合以上結果我們認為，anti-DNase antibodies 普遍存在於 SLE
病人血清中，並且經由親和性管柱所純化的此種 anti-DNase
antibodies 可以抑制 DNase的活性。 因此推測在 SLE 病人血清中，
可能由於 anti-DNase antibodies 的存在而抑制 DNase 水解 DNA
的活性，進而刺激或促進 anti-DNA antibodies的產生。

英文摘要
Systemic lupus erythematosus (SLE) is a prototypic systemic
autoimmune disease characterized by the production of
autoantibodies against nuclear components . Dexoxyribonuclease I
(DNase I), an endonuclease capable of hydrolyzing of DNA, is
normally present in human serum and urine. In our study,
antibodies binding to bovine DNase I were investigated by ELISA
in SLE patients and anti-nuclear antibody (ANA) negative
normal sera. About 62.4% of SLE patients (63/101) were positive
for anti-DNase antibodies while only 8% of normal controls
(8/98) were positive. A positive correlation was found between
the concentrations of anti-DNase and anti-DNA antibodies in
SLE patients. Affinity-purified anti-DNase IgG from pooled SLE
patients serum and BALB/c mice immunized with bovine DNase
bound to DNase as well as DNA coated ELISA plates. A synthetic
peptide which cross-response to the catalytic site of DNase was
able to completely inhibit anti-DNase IgG bound to DNase but
not DNA coated plate. Indirect immunofluorescence of anti-DNase
IgG displayed speckled and nucleolar patterns of Hep-2 cells
but no reaction to the kinetoplast DNA in Crithidia luciliae.
In addition to bovine DNase, anti-DNase IgG also bound to DNase
in streptococcal supernatants and human urine and inhibited
their enzymatic activities . It was also noticed that BALB/c
and NZB/NZW mice immunized with bovine DNase I produced more
anti-DNase and anti-DNA antibodies than na鴳e mice. Besides,
immunized BALB/c mice showed fibroblast cells in renal tubule
walls under histological examination showed.Based on the
data above, we conclude that it is possible that the
inhibition of DNase I activity by the anti-DNase antibodies
may cause a defect in DNA degradation and stimulate or
enhance anti-DNA antibodies production.

目錄
中文摘要
英文摘要
誌謝
縮寫
圖目錄
前言
實驗設計
材料與方法
一、材料與方法
I. 人類血清
II. 實驗動物
III. Peptides的合成
IV. 酵素免疫
V. 親和性管柱層析法
VI. 測定抗體濃度
VII. 間接免疫螢光染色法
VIII. 硫酸十二酯鈉-聚丙烯醯氨膠電泳法
IX. 西方點墨法
X. 酵素活性膠體試驗
XI. 抗體抑制 DNase 活性試驗
XII. 免疫小鼠
XIII腎臟組織學切片
二、試藥與儀器
結果
I. 檢測 SLE 病人血清中的 anti-DNase antibodies
II. Anti-DNase IgG的特性
III. Anti-DNase antibodies對 NZB/NZW F1及
BABL/c老鼠的影響
討論
圖
參考文獻
自述
著作權聲明

參考文獻
1. Kater, L. and Baart, H.de la Faille, ed. Multi-systemic
autoimmune Derksen, H.W.N., and C. J. M. 1995. Stephens.
Lupus erythematosus. In: disease. 2 th ed. Elsevier Science
B.V., 85-128.
2. Liang, M. H., Socher, S. Larsen, A. M. G. and Schur, P. H. 1989. Reliability and validity of 6 systems for the clinical assessment of disease activity in SLE. Arthritis Rheum. 32: 1107-1118.
3. Bombardier, C. D. D., Gladman, M. B., Urowitz, D., C and C. H. Chang. 1992. Derivation of the SLEDAI: a disease activity index for lupus patients. The committee on prognosis studies in SLE. Arthritis Rheum. 35: 630-640.
4. Dubois E L. 1978. Antimalarials in the management of discoid and systemic lupus erythematosus. Semin. Arthritis Rheum. 8: 33-51.
5. Bermstein, K. A., Valerio, R. D. and Lefkowith, J. B. 1995. Glomerular binding activity in MRL/lpr serum consists of antibodies that bind to a DNA/histion/type/ IV collagen complex. J. Immunol. 154: 2424-2433.
6. Brian, L. K. 1996. Systemic lupus erythematosus. Cell 85: 303-306.
7. Brinkman, K., Termaat R., Berden, J. H. M., and Smeenk, R. J. T. 1990. Anti-DNA antibodies and lupus nephritis: the complexity of crossreactivity. Immunol. Today 11: 232-234.
8. Suzuki, N., Shoji, M. and Tsuyoshi, S. 1997. Development of pathogenic anti-DNA antibodies in patients with systemic lupus erythematosus. FASEB. 11:1033-1038.
9. Ebling, F., and Hahn, B. H. 1980. Restricted subpopulations of DNA antibodies in kidneys of mice with systemic lupus: comparison of antibodies in serum and renal eluates. Arthritis Rheum.23: 392-403.
10. Bruijn, J. A., Bergijk, E. C. DeHerr, E., Fleurence, G. J. and Hoedemaeke,r P. J. 1992 Induction and progression of experimental lupus nephritis: Exploration of a pathogenic pathway. Kidney Int. 41: 5-13
11. Frank, M. M., Hamburger, Lawley, M. I., T. J. Kimberly, R. P., and Plotz, P. H. 1979. Defective reticuloendothelial system Fc-recptor function in systemic lupus erythematosus. N. Engl. J. Med. 300: 518-523.
12. Wilson, J. G. and Fearon, D. T. 1984. Altered expression of complement receptors as a pathogenic factor in systemic lupus erythematosus. Arthritis Rheum. 27: 1321-1328.
13. Theofilopoulos, A.N. and Dixon, F.J. 1985. Murine models for systemic lupus erthematosus. Adv. Immunol. 37: 269-390.
14. Drake, C.G., Roozo, S. J., Vyse,T. J., Palmer, E. P., and Kotin, B. U. 1995. Genetic contributions to lupus-like disease in (NZB/NZW) F1 mice. Immunol. Rev. 144: 51-74.
15. Nygard, N. R. Deborah M. M. Joel, S. and Benjamin, D. S. 1993. Mixed haplotypes and autoimmunity. Immunology Today 14: 53-56.
16. Klinman, D. M. and Steinberg, A. D. 1987. Systemic autoimmune diseasearise from polyclonal B cell activition. J Exp. Med. 165: 1775-1760.
17. Izui, S., McConahey, P. J., and Dixon, F. J. 1978. Increased spontaneous polyclonal activatein of B lymphocytes in mice with spontaneous autoimmune disease. J. Immunol. 121: 2213-2219.
18. Kilnman, D. M. 1990. Polyclonal B cells activation in lupus-prone mice precedes and predicts the development of autoimmune disease. J. Clin. Invest. 86: 1249-1254.
19. Reininger, L. Radaskiewicz, T. Kosco M. Melchers, F. and Rolink, A.G. 1992. Development of autoimmune disease in SCID mice populated with long-term "in-vitro" proliferating (NZB/NZW) F1 pre-B cells. J. Exp. Med. 174: 1023-1029.
20. Cyster, J. G., Hartley, S. B., and Goodnow, C. C. 1994. Competition for follicular niches excludes self-reactive cells from the recirculating B-cell repertoire. Nature 371: 389-395.
21. Chen, C., Nagy, Z., Radic, M.Z., Hardy, R. R., Huszar, D., Camper S.A. and Weigert M. 1995. The site and stage of anti-DNA B-cell deletion. Nature 373: 252-255.
22. Goodnow, C. C., Cyster, J. G. Hartley, S. B., Bell, S. E., Cooke M. P., Healey, J. I., Akkaraju, S., Rathmell, J. C., Pogue, S. L. and Shokat, K. P. 1995. Self-tolerance checkpoints in B lymphocyte development. Adv. Immunol. 59: 279-378.
23. Watangabe-Fukunage, R., Brannan, C. I., Copland, N. G., Jenkins, N. A. and Nagata, S. 1992. Lymphorproliferative disorder in mice explained by defects in Fas antigen that mediate apoptosis. Nature 356: 314-317.
24. Strasser, A., Whittingham, S., Vaux , D. L., Adams, J. M., Cory, S. and Harrism A. W. 1988. Enforced Bcl-2 expression in B lymphoid cells prolongs antibody responses and exhibits autoimmune disease. Proc. Natl. Acad. Sci. USA 88: 8661-8665.
25. Nisitanim, S., Tsubata, T., Murakami, M., Okamoto, M., and Honjo, T. 1993. The bcl-2 gene product inhibits colonal deletion of self-reactive B lymphocytes in the periphery but not in the bone-mar row. J. Exp. Med. 178: 1247-1254.
26. Romagnami, S. 1992. Induction of Th1 and Th2 response: a key role for the "nature" immune response? Immunol. Today 13: 379-381.
27. Spronk , P. E., Ter, Borg, E. J., Limburg P. C., and Kallenberg C. G. M.1992. Plasma concentration of interleukin-6 in systemic lupus erythematosus: An indictor of disease activity? Clin. Exp. Immunol. 90: 106-110.
28. Nagafuchi, H., Suzuki, N., Mizushima, Y., and Sakane, T., Constitutive expression of IL-6 receptors and their role in the excessive B cell function in patients with systemic lupus erythematosus. J. Immunol. 151: 6525-6534.
29. Llorente, L., Richaud-Patin, Y., Fior, R., Alcocer-Varela, J., Wijdene,s J., Fourrier, B. M., Galanaud, P., and Emilie, D.1994. In vivo production of interleukin-10 by non-T cells in rheumatoid arthritis, Sjogren* syndrome, and systemic lupus erythematosus. Arthritis Rheum. 37: 1647-1655.
30. Ishida, H., Muchamuel, T., Sakaguchi, S., Andradre, S., Menon, S., and Howard, M. 1994. Continous administration of anti-interleukin 10 antibodies delays onset of autoimmunity in NZB/W F1 mice. J. Exp. Med. 179: 305-310.
31. Ishida, H., Hastings, R., Kearney, J., and Howard, M. 1992. Continuous anti-interleukin 10 antibody administration depletes mice of Ly-1 B cells but not conventional B cells. J. Exp. Med. 175: 1213-1220.
32. Radic, M. Z., Mackle, J., Erickson, J. Anderson, W. F. and Weigert M.1993. Residues that mediate DNA binding of autoimmune antibodies. J. Immunol. 150: 4966-4977.
33. Datta, S. K., Arunan, K. and Ami, DM. 1997. T cells of lupus and molecular targets for immunotherapy. J. Clinc. Immunol. 17: 11-20.
34. Fink, B.K., Linsley, P.S., and Wofsy, D. 1994. Treatment of murine lupus with CTLA4 Ig. Science 265: 1225-1227.
35. Mohan, C., Shi, Y., Laman, J. D., and Datta, S. K. 1995. Interaction between CD4 and its ligand gp39 in the development of murine lupus nephritis. I. Immunol. 154: 1470-1480.
36. Mohan, C., Adams, S., Stanik, V., and Datta, S. K., 1993. Nucleosome: a major immunogen for pathogenic autoantibody-inducing Tcells of lupus nephritis. J. Exp. Med. 177: 1367-1381.
37. Bockenstedt, L. K., Gee R. J. 1995. Self peptides in the initiation of lupus autoimmunity. J. Immunol. 154: 3516-3524.
38. Sercarz, E. E., Lehmann, P. V., Ametani, A., Benichou, G., Miller, A. and Moudgill, K. 1993. Dominance and crypticity of T cell antigenic determinants. Annu. Rev. Immunol. 11: 729-766.
39. Hochberg, MC. 1992. Epidemiology of systemic lupus erythematosus, In: Lahita RG (ED) systemic lupus erythematous, 2nd edn. New York, Churchil Livinggstone. 103-117.
40. Arnett, FC. 1993. The genetic basis of lupus erythematous. In: Wallace DJ, Hahn BH (Eds) Dubois lupus erythematosus, 4th edn. Philadelphia, Lea and Febiger. 13-36
41. Lehman, T. J. 1995. A practical guide tosystemic lupus erythematosus. Pediatric Clincs of North America 42: 1223-1236.
42. Arnett, FC. 1993. The genetics basis of lupus erythematosus .in : wallwce DJ. Hahn BH (Eds) Dudois* lupus erthematosus ,4th edn. Philadelphai, Lea and Febiger, 13-36.
43. Arnett, FC, Reveille, JD. 1992. Genetics of systemic lupus erythematosus. Rheum Dis Clin North Am. 18(4):865-892.
44. Cook, L. Agenello, V. 1992. Complement deficiency and systemic lupus erythematosus. In : Lahita RG (Ed) Systemic Lupus Erythematosus. 2nd edn. New York, Churchil Livingstone. 569-586.
45. Timothy, J. V., and John, A. T. 1996. Genetic analysis of autoimmune disease. Cell 85: 311-318.
46. Ahmed, S.A., Penhale, W.J., Talal, N. 1985. Sex hormones, immune response, and autoimmune disease: mechanisms of sex hormone action. Am J Pathol. 531-551.
47. Fernandes, G., and Talal, N. 1986. SLE: hormones and diet. Clin. Exp. Rheum. 4: 183-185.
48. Sthoeger, Z., Chiorazzi, N., Lathita, RG. 1988. Regulation of the immune response by sex steroids. J. Immunol. 141: 91-98.
49. Hess, E.V. and Farhey, Y. 1995. Etiology environmental relationships, epidemiology, and genetics of systemic lupus erythematosus. Curr. Opin. Rheumatol. 11:371-375.
50. Petri, M., and Allbritton, J. 1992. Antibodies allergy in systemic lupus erythematosus: a case control study. J. Rheumatol. 19: 265-269.
51. Talan, N., Flescher, E., and Dang, H. 1992. Are endogenous retroviruses involved in human autoimmune disease? J. Autoimmun. 5: 61-66.
52. Ranki, A., Kurki, P., Riepponene, S., and Stephannsson, E. 1992. Antibodies to retroviral proteins in autoimmune connective tissues disease. Arthritis Rheum. 35:1483-1493.
53. Raz, E., Ben-Bassat, H., Davidi, T., Shlomai, Z. and Eila, D. 1993. Cross reaction of anti-DNA autoantibodies with cell surface proteins. Eur. J. Immunol. 23: 383-390.
54. Gladman, D. D. 1995. Prognosis and treatment of systemic lupus erythematous: clinical and laboratory correlation. Curr. Opin. Rheumatol. 7: 402-408.
55. Lacks, S.A., 1981. Deoxyribonuclease I in mammalian tissues. J. Bio. Chem. 256: 2644-2648.
56. Wannamaker, L. W. and Yasmineh. 1967. Streptococcal nuclease I further studies on the A, B and C enzymes. J. Exp. Med. 126: 475-496.
57. Price, P. A., Stein, WH., Moore, S. 1969. Effective of divalent cations on the reduction and reformation of the disulifide bonds of deoxyribonuclease. J. Bio. Chem. 244: 929-932.
58. Love, J. D. and Roger, R. H. 1979. The relationship between human serum and human pancreatic DNase I. J. Bio. Chem. 254: 12588-12594.
59. Drew, H. R. and Trvers, A. A. 1984. DNA structural variations in the E. coil tyr T promoter. 37: 491-502.
60. Oefner, C., and Dietrich, S. 1986. Crystallographic refinement and structure of DNase I at 2 A resolution. J. Mol. Bio. 192: 605-632.
61. Laskowski, M., Sr. 1961 in The Enzymes (Boyer, P. D., Lardy, H., and Myrback, K., eds) 2nd Ed.,Vol. 5,. Academic Press, New York 123-147.
62. Moore, S.1981 in The Enzymes (Boyer,P. D., ed) 3rd Ed., Academic Press, New York Vol. 14: 281-296,
63. Liao, T. H. 1981. Multiple forms of deoxyribonuclease I. Mol. Cell Biochem. 34: 15-22.
64. Liao, T. H. 1997. Deoxyribonuclease I and its clinical applications. J. Formos. Med. Assoc. 96: 481-487.
65. Kishi, K., Toshihiro, Y., Shuichi, A., and Kelko, M.1989. Gentic polymorphism of human urine deoxyribonuclease I. Hum Genet 81: 295-297.
66. Petisch, M.C., Polzar, B. Stephan, T., Crompton, H.R. and Tschopp, J. 1993. Characterization of the endogenous deoxyribonuclease in involvd in nuclear DNA fragmention during apoptosis. EMBO J. 12: 371-377.
67. Miyauchi, K., Ogaawa, M., Murata, A., Nakanon, I., Funakoshi, A., and Mori, T. 1989. Serum deoxyribonuclease I determined by a radioimmunoassay and an enzymatic assay in malignant disease. Clin. Chim. Acta. 184: 115-120.
68. Aitken, ML., Burke, W., McDonald, G., Shak, S., Montdomery, AB. and Smith, A. 1992. Recombinant human DNase inhalation in normal subjects and patients with cystic fibrosis. A phase 1 study . JAMA. 267: 1947-1951.
69. Miyauchi, K., Ogaawa, M., Murata, A., Nakanon, I., Funakoshi, A., and Mori, T. Ito, K. Minaniura, N. and Yamamoto. 1986. Development of a radioimmunoassay for human deoxyribonuclease I. Clin. Chim. Acta. 154: 115-123.
70. Scully, C., Spandios, DA., Ward, B. P., McGregor, IA., and Boyle, P. 1981. Serum alkaline deoxyribonuclease in oral cancer and permalignant lesions. Biomedicine 35: 179-180.
71. Zollner, EJ., Muller, WE., and Zahn, RK. 1971. Moglichkiten zur gezitlten diagnose durch beastimmung der sauren deoxyribonuclease im urim gesunder kontrollpersonen und xeroderma pigmentosum-patienten.Klin Wochenscher 49: 1290-1294.
72. Funakoshi, A., Wakasugi, H. and Lbayashi, H. 1979. Clinical investiga.tion of serum deoxyribonuclease LL. Clinical studies of serum deoxyribonuclease activity in pancreatic disease. Gastroenterol Jpn. 14: 434-440.
73. Frost, PG., and Lachmann, PJ. 1968. The relationship of deoxyribonuclease inhibitor levels in human sera to the occurrence of antinuclear antibodies. Clin. Exp. Immunol. 3: 447-455.
74. Chitrabamrung, S., Rubin, RL. and Tan, EM. 1981. Serum deoxyribonuclease I and clinical activity in systemic lupus erythematosus. Rheumatol. Int. 106: 243-252.
75. Aitken, M. L., Wylie, B., Gwen, MD., Steven, S., A. Bruce, M., Arnold, S. 1992 . Recombinant Human DNase Inhalation in Normal Subject and Patients With Cystic Fibrosis. JAMA. 267: 1947-1951.
76. Rubin, R. L., Joslin, F. G. and Tan, E. M. 1983. An improved ELISA for anti-native DNA elimination of interference by antihistone antibodies. J. Immunol. Methods 63: 359-371.
77. Rosenthal, A., L., and Lacks, S. A. 1977. Nuclease detection in SDS-polyacrylamide gel electrophoresis. Analy. Biochem. 80: 76-90.
78. Ito, K., Aminamiura, N., and Yamamoto, T. 1984. Human urine DNase I, and enzymic and protecochemical properties of the enzyme. J. Biochem. 95: 1399-1406.
79. Arden, L., DEGroot, E. R., and Feltkamp, E. W. 1975. Immunology of DNA III. Crithidia lucilia, a simple substrate for the determination of anti-dsDNA with the immunoflourescent technique. Ann. N. Y. Acad. Sci. 254:505-525.
80. Pisetsky, D. S. 1996. The immunologic properties of DNA. J. Immunol. 156: 421-423.
81. Dharmesh, D. D., Meera, R. K. John T. S., and Tony, N. M. 1993. Antigen-specific induction of antibodies against native mammalin DNA in nonautoimmune mice. J. Immunol. 151: 1614-1626.
82. Tan, EM. Schur, PH. Carr, RI. and Kunkel, HG. 1973. The occurence of single-strand DNA in the serum of patients with systemic lupus erythematosus and other disease. J. Clin. Invest. 52:198-204.
83. Macanovic, M. D., Sinicropi, S. S., Baunghman, S. T. and Lachmann, P. J. 1996. The treatment of systemic lupus erythematosus (SLE) in NZB/NZW F1 hybrid mice; studies with recombinant murine DNase and with dexamethasone. Clin. J. Immunol. 106:243-252.
84. Carlos, A. Muhlen V. and Tan, E. M. 1995. Autoantibodies in the diagnosis of systemic rheumatic disease. 24: 323-358.
85. Andreas, P., Iris, Z., Annegret, F. and Josephine, W. H. 1996. Molecular characterization of a major serotype M49 Group A streptococcal DNase Gene (sdaD). Infect. Immun. 64: 5349-5356.
86. Lin, J. L., Wang, W. Y., Liao, T. H. 1994. Thermal inactivation of shrimp deoxyribonuclease with and without sodium dodecyl sulfate. Biochim. Biophys. Acta. 1209: 209-214.
87. Marchini, B. Puccetti, A., Dolcher, M. P., Madaio, M. P. and Minglorini, P. 1995. Induction of anti-DNA antibodies in non- autoimmune mice by immunization with a DNA-DNase I complex. Clin. Exp. Rheum. 13: 7-10.
88. Judith, A. J. and John, B. H. 1998. A module of peptide-induced autoimmune B cell epitope spreading is strain specific and is not H-2 restricted in mice. J. Immunol. 160: 502-508.