C. Immunization Schedule

免疫程序

根據(jù)小鼠品系和實驗?zāi)康牡牟煌?，誘導(dǎo)高發(fā)病率和嚴(yán)重關(guān)節(jié)炎的方法也各有不同。

1.通過單次免疫（無需加強(qiáng)免疫）誘發(fā)關(guān)節(jié)炎（易感品系）

注射膠原和完全弗式佐劑混合而成的含2mg/ml結(jié)合桿菌乳劑。對于易感品系小鼠DBA/1（H-2q）和B10.RIII(H-2r)注射乳劑后28-35天會引發(fā)關(guān)節(jié)炎。42-56天發(fā)病率達(dá)90-100%。關(guān)節(jié)炎嚴(yán)重程度高，臨床評分達(dá)10-12分 （最高16分）。

注：由于高濃度的結(jié)核桿菌會引起免疫部位嚴(yán)重的炎癥反應(yīng)，因此根據(jù)各研究機(jī)構(gòu)對動物實驗的要求，可選用方案2或方案4。

2.通過加強(qiáng)免疫誘發(fā)關(guān)節(jié)炎（易感品系）

注射膠原和完全佐劑混合而成的含0.5mg/ml結(jié)合桿菌乳.在初次注射后第21天，加強(qiáng)注射膠原和不完全佐劑（不含結(jié)核桿菌）混合而成的乳劑。尾部皮下注射0.1ml乳劑， 避開初次免疫部位。一般在初次免疫注射后28-35天引發(fā)關(guān)節(jié)炎。在第42-56天，發(fā)病率為80-100%，臨床評分可達(dá)8-12分（最高16分）。

3.用高劑量的結(jié)核桿菌完全佐劑加強(qiáng)免疫誘發(fā)關(guān)節(jié)炎（CIA抗性小鼠）

對于一些低反應(yīng)的小鼠，例如：B10 (H-2b), C57BL/6(H-2b), and C57BL/6x129/Sv (H-2b) ，采用以下方案可誘發(fā)關(guān)節(jié)炎。初次免疫，尾部皮下注射0.1ml膠原和完全佐劑混合而成的含2.5mg/ml結(jié)核桿菌的乳劑。在第21天加強(qiáng)免疫，再次注射0.1ml膠原和完全佐劑混合而成的含2.5mg/ml結(jié)核桿菌的乳劑。一般在初次免疫注射后28-35天引發(fā)關(guān)節(jié)炎。在第42-56天，發(fā)病率為50-70% （10）。

注：該方案引起的炎癥反應(yīng)比較嚴(yán)重，根據(jù)各研究機(jī)構(gòu)對動物實驗的要求，可選用膠原抗體（CAIA）誘發(fā)的小鼠關(guān)節(jié)炎模型。Chondrex公司的單克隆抗體合劑（Arthrogen-CIA）和LPS能在CIA抗性的小鼠中誘發(fā)關(guān)節(jié)炎。相關(guān)信息請參考www.chondrex.com或咨詢我們代理商欣博盛生物。

4. 通過LPS加強(qiáng)免疫

LPS和體內(nèi)二型膠原自身抗體水平對于誘發(fā)關(guān)節(jié)炎具有協(xié)同效應(yīng)(13)。此外，在經(jīng)典的CIA動物模型中 ，應(yīng)用LPS(B細(xì)胞分裂素)或MAM(支原體產(chǎn)生的T細(xì)胞分裂素)或SEB(金黃色葡萄球菌產(chǎn)生的T 細(xì)胞分裂素)都會加強(qiáng)關(guān)節(jié)炎發(fā)病率和嚴(yán)重程度（14-16）。因此這些毒素不僅用于引發(fā)和加強(qiáng)關(guān)節(jié)炎水平，而且用于指定關(guān)節(jié)炎發(fā)生時間。

這個方案，需要注射0.1ml二型膠原和含0.5mg/ml的CFA混合乳劑（參考方案2）。在第25-28天或在在關(guān)節(jié)炎發(fā)生前3-5天腹腔注射LPS(25-50μg，溶解在生理鹽水)。關(guān)節(jié)炎將在24-48小時發(fā)生，發(fā)病率90-100%。

注：CFA免疫注射小鼠會在2-4周發(fā)生嚴(yán)重的免疫抑制。所以一些小鼠對LPS（50μg）注射非常敏感。

Chondrex公司建議進(jìn)行正式實驗之前先對來自不同供應(yīng)商的動物進(jìn)行測試。

D. Onset of Arthritis

關(guān)節(jié)炎的發(fā)作

有效的免疫注射后，臨床上明顯的關(guān)節(jié)腫脹癥狀會在3-5周出現(xiàn)。關(guān)節(jié)炎的發(fā)作和發(fā)病率取決于小鼠品系和實驗方案。

EVALUATING ARTHRITIS

關(guān)節(jié)炎評估

A.Scoring

A.臨床評分

關(guān)節(jié)炎可通過臨床評分來定性評估或用測厚儀來測量爪子厚度來評估。這些方法適用于 所有的關(guān)節(jié)炎模型比如經(jīng)典的CIA，佐劑誘導(dǎo)的關(guān)節(jié)炎，單克隆抗體合劑和其他炎癥模型。Chondrex公司提供一下系統(tǒng)（表二）

注：由于小鼠的爪子太小，因此無法像測量大鼠爪子體積那樣通過浸沒法測定小鼠爪子體積。

表二 關(guān)節(jié)炎炎癥程度的臨床評分

B. Serum Analysis

B. 血清分析

高濃度自身二型膠原蛋白IgG抗體水平的產(chǎn)生是誘發(fā)關(guān)節(jié)炎的關(guān)鍵（10, 17）。二型膠原蛋白IgG2a 和 IgG2b抗體亞型對激活補(bǔ)體及隨后的關(guān)節(jié)炎的發(fā)生至關(guān)重要。

Chondrex公司提供小鼠膠原IgG及其各亞型抗體ELISA測試盒（具體信息參考www.chondrex.com或咨詢代理商欣博盛生物）。

REFERENCES

參考文獻(xiàn)

1. D. Trentham, A. Townes, A. Kang, Autoimmunity to Type II Collagen an Experimental Model of Arthritis. J Exp Med 146,857-68 (1977).

2. J. Courtenay, M. Dallman, A. Dayan, A. Martin, B. Mosedale,Immunisation Against Heterologous Type II Collagen Induces Arthritis in Mice. Nature 283, 666-8 (1980).

3. E. Cathcart, K. Hayes, W. Gonnerman, A. Lazzari, C.Franzblau, Experimental Arthritis in a Nonhuman Primate. I.Induction by Bovine Type II Collagen. Lab Invest 54, 26-31(1986).

4. Ivanov, K. Atarashi, N. Manel, E. Brodie, T. Shima, et al.,Induction of Intestinal Th17 Cells by Segmented Filamentous Bacteria. Cell 139, 485-98 (2009).

5. P. Wooley, Collagen-induced arthritis in the mouse.Methods Enzymol 162:361-373, 1988.

6. P. Wooley, H. Luthra, M. Griffiths, J. Stuart, A. Huse, C.David, et al., Type II Collagen-Induced Arthritis in Mice. IV.Variations in Immunogenetic Regulation Provide Evidence for Multiple Arthritogenic Epitopes on the Collagen Molecule.J Immunol 135, 2443-51 (1985).

7. R. Holmdahl, L. Jansson, E. Larsson, K. Rubin, L.Klareskog, Homologous Type II Collagen Induces Chronic

and Progressive Arthritis in Mice. Arthritis Rheum 29, 106-13 (1986).

8. R. Ortmann, E. Shevach, Susceptibility to Collagen-Induced Arthritis: Cytokine-Mediated Regulation. Clin Immunol 98,109-18 (2001).

9. W. Watson, A. Townes, Genetic Susceptibility to Murine Collagen II Autoimmune Arthritis. Proposed Relationship to the IgG2 Autoantibody Subclass Response, Complement C5, Major Histocompatibility Complex (MHC) and non-MHC Loci. J Exp Med 162, 1878-91 (1985).

10. I. Campbell, J. Hamilton, I. Wicks, Collagen-induced Arthritis in C57BL/6 (H-2b) Mice: New Insights Into an Important Disease Model of Rheumatoid Arthritis. Eur J Immunol 30,1568-75 (2000).

11. E. Micha?lsson, V. Malmstr?m, S. Reis, A. Engstr?m, H.Burkhardt, R. Holmdahl, et al., T Cell Recognition of Carbohydrates on Type II Collagen. J Exp Med 180, 745-9(1994).

12. M. Andersson, R. Holmdahl, Analysis of Type II CollagenReactive T Cells in the Mouse. I. Different Regulation of Autoreactive vs. Non-Autoreactive Anti-Type II Collagen T Cells in the DBA/1 Mouse. Eur J Immunol 20, 1061-6 (1990).

13. K. Terato, D. Harper, M. Griffiths, D. Hasty, X. Ye, et al.,Collagen-induced Arthritis in Mice: Synergistic Effect of E.Coli Lipopolysaccharide Bypasses Epitope Specificity in the Induction of Arthritis With Monoclonal Antibodies to Type II Collagen. Autoimmunity 22, 137-47 (1995).

14. S. Yoshino, E. Sasatomi, Y. Mori, M. Sagai, Oral Administration of Lipopolysaccharide Exacerbates

Collagen-Induced Arthritis in Mice. J Immunol 163, 3417-22(1999).

15. B. Cole, M. Griffiths, Triggering and Exacerbation of Autoimmune Arthritis by the Mycoplasma Arthritidis Superantigen MAM. Arthritis Rheum 36, 994-1002 (1993).

16. Y. Takaoka, H. Nagai, M. Tanahashi, K. Kawada,Cyclosporin A and FK-506 Inhibit Development of

Superantigen-Potentiated Collagen-Induced Arthritis in Mice. Gen Pharmacol 30, 777-82 (1998).

17. R. Reife, N. Loutis, W. Watson, K. Hasty, J. Stuart, SWR Mice Are Resistant to Collagen-Induced Arthritis but Produce Potentially Arthritogenic Antibodies. Arthritis Rheum 34, 776-81 (1991).

18. T. Kagari, H. Doi, T. Shimozato, The Importance of IL-1 Beta and TNF-alpha, and the Noninvolvement of IL-6, in the Development of Monoclonal Antibody-Induced Arthritis. J

Immunol 169, 1459-66 (2002).

19. J. Stuart, M. Cremer, A. Townes, A. Kang, Type II CollagenInduced Arthritis in Rats. Passive Transfer With Serum and Evidence That IgG Anticollagen Antibodies Can Cause Arthritis. J Exp Med 155, 1-16 (1982).

20. W. Watson, P. Brown, J. Pitcock, A. Townes, Passive Transfer Studies With Type II Collagen Antibody in

B10.D2/old and New Line and C57Bl/6 Normal and Beige(Chediak-Higashi) Strains: Evidence of Important Roles for C5 and Multiple Inflammatory Cell Types in the Development of Erosive Arthritis. Arthritis Rheum 30, 460-5(1987).

21. K. Terato, K. Hasty, R. Reife, M. Cremer, A. Kang, J. Stuart,et al., Induction of Arthritis With Monoclonal Antibodies to Collagen. J Immunol 148, 2103-8 (1992).

22. K. Terato, K. Hasty, M. Cremer, J. Stuart, A. Townes, A.Kang, et al., Collagen-induced Arthritis in Mice. Localization of an Arthritogenic Determinant to a Fragment of the Type II Collagen Molecule. J Exp Med. 162, 637-46 (1985).

23. L. Myers, H. Miyahara, K. Terato, J. Seyer, A. Kang,Collagen-induced arthritis in B10.RIII mice (H-2

r):identification of an arthritogenic T cell determinant. Immunol84, 509-513 (1995).

24. K. Terato, X. Ye, H. Miyahara, M. Cremer, M. Griffiths,Induction by Chronic Autoimmune Arthritis in DBA/1 Mice by Oral Administration of Type II Collagen and Escherichia Coli Lipopolysaccharide. Br J Rheumatol 35, 828-38 (1996).

25. P. Wallace, J. MacMaster, K. Rouleau, T. Brown, J. Loy, et al., Regulation of Inflammatory Responses by Oncostatin M.J Immunol 162, 5547-55 (1999).

26. A. de, Fougerolles Sprague, C. Nickerson-Nutter, G. ChiRosso, P. Rennert, et al., Regulation of Inflammation by Collagen-Binding Integrins alpha1beta1 and alpha2beta1 in Models of Hypersensitivity and Arthritis. J Clin Invest 105,721-9 (2000).

27. S Larox, J. Fuseler, D. Merril, L. Gray, R. Reife, K. Terato,et al. #301 A novel model of polyarthritis induced in mice using monoclonal antibodies to type II collagen.Characterization and effects of chemically modified tetracycline. Arthritis Rheum 42:s121 (supplement).

28. Y. Tsuchiya, M. Maeda, K. Hanada, et al. Mouse arthritis model induced by anti-type II collagen monoclonal antibody cocktail: Difference in distribution of diseased joints by strain and immunodeficient status. Proc Japanese Society Animal Models for Human Diseases. 19, 14-22 (2003).

29. K. Takagishi, N. Kaibara, T. Hotokebuchi, C. Arita, M.Morinaga, K. Arai, et al., Serum Transfer of Collagen Arthritis in Congenitally Athymic Nude Rats. J Immunol 134, 3864-7(1985).

30. H. Kato, K. Nishida, A. Yoshida, I. Takada, C. McCown, et al., Effect of NOS2 Gene Deficiency on the Development of Autoantibody Mediated Arthritis and Subsequent Articular Cartilage Degeneration. J Rheumatol 30, 247-55 (2003).

31. K. Yumoto, M. Ishijima, S. Rittling, K. Tsuji, Y. Tsuchiya, et al., Osteopontin Deficiency Protects Joints Against Destruction in Anti-Type II Collagen Antibody-Induced Arthritis in Mice. Proc Natl Acad Sci U S A 99, 4556-61(2002).

32. L. Myers, A. Kang, A. Postlethwaite, E. Rosloniec, S.Morham, et al., The Genetic Ablation of Cyclooxygenase 2 Prevents the Development of Autoimmune Arthritis. Arthritis Rheum 43, 2687-93 (2000).

33. T. Itoh, H. Matsuda, M. Tanioka, K. Kuwabara, S. Itohara, R.Suzuki, et al., The Role of Matrix metalloproteinase-2 and Matrix metalloproteinase-9 in Antibody-Induced Arthritis. J Immunol 169, 2643-7 (2002).

34. J. Labasi, N. Petrushova, C. Donovan, S. McCurdy, P. Lira,et al., Absence of the P2X7 Receptor Alters Leukocyte Function and Attenuates an Inflammatory Response. J Immunol 168, 6436-45 (2002).

35. Z. Han, L. Chang, Y. Yamanishi, M. Karin, G. Firestein, Joint Damage and Inflammation in c-Jun N-terminal Kinase 2 Knockout Mice With Passive Murine Collagen-Induced Arthritis. Arthritis Rheum 46, 818-23 (2002).

36. J. McCoy, J. Wicks, L. Audoly, The Role of Prostaglandin E2 Receptors in the Pathogenesis of Rheumatoid Arthritis. J Clin Invest 110, 651-8 (2002).

37. R. Newton, K. Solomon, M. Covington, C. Decicco, P. Haley,et al., Biology of TACE Inhibition. Ann Rheum Dis 60 Suppl3, iii25-32 (2001).

38. R. Holmdahl, L. Jansson, M. Andersson, E. Larsson,Immunogenetics of Type II Collagen Autoimmunity and

Susceptibility to Collagen Arthritis. Immunology 65, 305-10(1988).

39. S. Thornton, G. Boivin, K. Kim, F. Finkelman, R. Hirsch,Heterogeneous Effects of IL-2 on Collagen-Induced Arthritis.J Immunol 165, 1557-63 (2000).

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