人Ⅱ型胶原蛋白的制备、鉴定及其应用的研究
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摘要
Ⅱ型胶原蛋白(type Ⅱ collagen，CⅡ)是与RA发病密切相关的自身抗原(autoantigen)之一。为了探讨牛CⅡ(bovine typeⅡcollagen，BCⅡ)与人CⅡ(human type Ⅱ collagen，HCⅡ)在类风湿性关节炎(rheumatoidarthritis，RA)患者中引起的特异性免疫反应是否相同，为口服耐受治疗RA的研究提供实验依据。本研究采用了细胞培养，特异性T细胞活化，流式细胞术(now cytometry，FCM)，western blot印迹法及ELISA技术，制备、鉴定鉴定HCⅡ，并初步研究其免疫原性和建立检测RA患者血清中抗CⅡ自身抗体ELISA系统。主要研究内容如下：
采用胃酶消化及盐析法，自引产胎儿的软骨中提取人CⅡ，经DEAE(DE-52)离子交换层析纯化，SDS聚丙烯酰胺凝胶电泳(SDS-polyacrylaminegel elecyrophoresis，SDS—PAGE)分析，western blot印迹法鉴定，证实所提取纯化的蛋白为人CⅡ，其分子量为130KD。用人CⅡ免疫小鼠制备抗血清，ELISA检测滴度为1：128的抗血清。证明所提纯人CⅡ具有免疫原性(immunogenicity)和抗原性(antigenicity)。
用HCⅡ、BCⅡ作为包被抗原，建立ELISA检测RA患者外周血抗CⅡ抗体
第四军医大学硕士学位论文
系统。结果显示：（l）RA患者外周血中抗则抗体与HCll和BCll分别反应的
阳性率均显著大于对照组，与 HC 11和 BC 11反应双阳性血清都来自 RA早期患
者；（2）RA患者外周血中抗 C 11抗体分别与 HC 11和 BCll反应的阳性率无显著
差异；③ RA患者抗 C 11抗体阳性组血沉平均水平及 RF阳性率均大于 RA患
者抗 C 11抗体阴性组。提示：＊）抗 C 11抗体在 RA的发病中，尤其在发病初
期，可能起着重要作用；（2）在检测 RA患者抗 C 11抗体可考虑以牛 C 11代替
人Cll作为包被抗原：m抗Cll抗体阳性预示队病情活动性和进展性。
为了探讨 BC 11与 HC 11在 RA患者体外诱导 C 11特异性 T细胞活化中所起的
作用，我们分离了 RA患者外周血及滑液单个核细胞，分别加 HC 11或 BC 11体
外培养，运用流式细胞术，检测表达 CD3“CD69细胞百分率，以此表示 C 11反
应性 T细胞的阳性率。可以观察到：（l）RA患者外周血 C 11反应性 T细胞的
阳性率大于对照组，RA患者滑液 C 11反应性 T细胞的阳性率大于外周血 C 11
反应性 T细胞的阳性率；①加 HC 11与 BC 11分别培养，RA患者外周血 C 11反
应性T细胞的阳性率无显著差别。提示：山 Cll反应性T细胞可能在RA的
发病中起重要作用，尤其在炎性关节局部：（2）SC 11在体外研究m的T细胞
特异性活化反应上可以代替 HC 11。
本研究结果表明：本方法成功地制备鉴定了人 C 11，且该蛋白具有抗原
性；抗 C 11抗体及 C 11反应性 T细胞在 RA的发病中都起着重要的作用；牛 C
11在该研究中可以代替人 C 11。这对于口服 C 11治疗 RA提供了理论基础、实
验依据和实验材料，同时也为从体液免疫及B细胞作用方面研究RA提供了
思路。
Rheumatoid arthritis (RA) is a chronic autoimmune disease that is characterized by destructive polyarthritis. The patients with RA will be changed to be disable if they can't been diagnosed and cured in early phase of RA, however the etiologic agent of RA remains unclear now. There is some evidences that type II collagen (CII) might act as an autoantigen of correlating with RA. So, we had extracted and purified human CII,detected the positive frequencies of CII -reactive T cells and the positive frequencies of antibodies to CII in RA.
CII was extracted from human cartilage by pepsin digestion and purified, then identified by SDS-PAGE analysis. It was found that the bands of CII samples could be seen as same as the bands of the standard CII. Then its antiserum was prepared with the extracted human CII by immunizing mice, next
the level of antiserum was verified by ELISA method. The result was that anti-C II antibody could be detected in the third immunization .The results suggest that the extracted human C II has immunogenicity. The extracted protein was identified with human CII by western blot method.
In order to explore the significance of serum antibodies against human and bovine type II collagen in the patients with rheumatoid arthritis, HCII and BCII were used as coating antigen respectively, to detect the anti-C II antibodies by indirect ELISA in 30 RA patients, 30 patients with other rheumatic diseases and 34 normal individuals. The results were that: the positive rates of serum anti-C II antibodies reaction with HC II or BC II from the RA patients were 30% and 33% respectively, all higher than that of controls. The difference between RA patients and control groups was very notable. In anti-C II antibody positive RA patients positive rate of RF was more than that in anti-C II antibody negative RA patients.The serums reaction with HC II and BC II were all from the early RA patients. The results suggest that detection of anti-C II antibody may be used as a means for judging RA patients' conditions, the replacement of HCllby BCII can be used to study the immunological role of B cells in the pathogenesis of RA.
We isolated the peripheral blood mononuclear cells (PBMC) and synovial fluid mononuclear cells (SFMC) from RA patients by Lymphoprep density gradient centrifugation and cultured them with human CII and bovine C II respectively. Then a two-colour cytometric analysis was performed using a peridinin chorophyll protein(PerCP) conjugated anti-CD3 antibody in combination with phycoerythrin(PE) labeled anti-CD69 monoclonal antibody(mAb). Last,we investigated the expression of CD69 on CD3+ cells by flow cytometry (FCM) and believed the frequencies of CD3+ CD69+ T cells
6
as the frequencies of the CII -reactive T cells detected by FCM. The results of being stimulated with HCII were that: the frequency of the c II -reactive T cells in peripheral blood(PB) from 10 RA patients was (9.08 ?.42)%, significantly higher than controls', however that was (1.68?.47)%(P<0.05): the percent of the CII -reactive T cells in synovial fluid (SF) from 6 RA patients was (18.03 ?.62) %, higher than the percent of the CII -reactive T cells ((8.65 ?3.20 ) % ) (P <0. OS) in PB. The results of being stimulated with HC II ((9.08 ?4.42 ) % ) and stimulated with BCII ((10.83 ?. 37) %) had no difference. The results suggest that: CII -reactive T cells play an important role in the pathogenesis of RA,peripheral tolerance induction might be useful in the treatment of RA, BC II could take the place of HC II in the study of T cells correlation with the pathogenesis of RA.
The studies indicate that anti-C II antibody and CII -reactive T cells were all important in the pathogenesis of RA and bovine CII could take the place of human CII. The studies can provide the evidences for the treatment of RA by oral tolerance.
采用胃酶消化及盐析法，自引产胎儿的软骨中提取人CⅡ，经DEAE(DE-52)离子交换层析纯化，SDS聚丙烯酰胺凝胶电泳(SDS-polyacrylaminegel elecyrophoresis，SDS—PAGE)分析，western blot印迹法鉴定，证实所提取纯化的蛋白为人CⅡ，其分子量为130KD。用人CⅡ免疫小鼠制备抗血清，ELISA检测滴度为1：128的抗血清。证明所提纯人CⅡ具有免疫原性(immunogenicity)和抗原性(antigenicity)。
用HCⅡ、BCⅡ作为包被抗原，建立ELISA检测RA患者外周血抗CⅡ抗体
第四军医大学硕士学位论文
系统。结果显示：（l）RA患者外周血中抗则抗体与HCll和BCll分别反应的
阳性率均显著大于对照组，与 HC 11和 BC 11反应双阳性血清都来自 RA早期患
者；（2）RA患者外周血中抗 C 11抗体分别与 HC 11和 BCll反应的阳性率无显著
差异；③ RA患者抗 C 11抗体阳性组血沉平均水平及 RF阳性率均大于 RA患
者抗 C 11抗体阴性组。提示：＊）抗 C 11抗体在 RA的发病中，尤其在发病初
期，可能起着重要作用；（2）在检测 RA患者抗 C 11抗体可考虑以牛 C 11代替
人Cll作为包被抗原：m抗Cll抗体阳性预示队病情活动性和进展性。
为了探讨 BC 11与 HC 11在 RA患者体外诱导 C 11特异性 T细胞活化中所起的
作用，我们分离了 RA患者外周血及滑液单个核细胞，分别加 HC 11或 BC 11体
外培养，运用流式细胞术，检测表达 CD3“CD69细胞百分率，以此表示 C 11反
应性 T细胞的阳性率。可以观察到：（l）RA患者外周血 C 11反应性 T细胞的
阳性率大于对照组，RA患者滑液 C 11反应性 T细胞的阳性率大于外周血 C 11
反应性 T细胞的阳性率；①加 HC 11与 BC 11分别培养，RA患者外周血 C 11反
应性T细胞的阳性率无显著差别。提示：山 Cll反应性T细胞可能在RA的
发病中起重要作用，尤其在炎性关节局部：（2）SC 11在体外研究m的T细胞
特异性活化反应上可以代替 HC 11。
本研究结果表明：本方法成功地制备鉴定了人 C 11，且该蛋白具有抗原
性；抗 C 11抗体及 C 11反应性 T细胞在 RA的发病中都起着重要的作用；牛 C
11在该研究中可以代替人 C 11。这对于口服 C 11治疗 RA提供了理论基础、实
验依据和实验材料，同时也为从体液免疫及B细胞作用方面研究RA提供了
思路。
Rheumatoid arthritis (RA) is a chronic autoimmune disease that is characterized by destructive polyarthritis. The patients with RA will be changed to be disable if they can't been diagnosed and cured in early phase of RA, however the etiologic agent of RA remains unclear now. There is some evidences that type II collagen (CII) might act as an autoantigen of correlating with RA. So, we had extracted and purified human CII,detected the positive frequencies of CII -reactive T cells and the positive frequencies of antibodies to CII in RA.
CII was extracted from human cartilage by pepsin digestion and purified, then identified by SDS-PAGE analysis. It was found that the bands of CII samples could be seen as same as the bands of the standard CII. Then its antiserum was prepared with the extracted human CII by immunizing mice, next
the level of antiserum was verified by ELISA method. The result was that anti-C II antibody could be detected in the third immunization .The results suggest that the extracted human C II has immunogenicity. The extracted protein was identified with human CII by western blot method.
In order to explore the significance of serum antibodies against human and bovine type II collagen in the patients with rheumatoid arthritis, HCII and BCII were used as coating antigen respectively, to detect the anti-C II antibodies by indirect ELISA in 30 RA patients, 30 patients with other rheumatic diseases and 34 normal individuals. The results were that: the positive rates of serum anti-C II antibodies reaction with HC II or BC II from the RA patients were 30% and 33% respectively, all higher than that of controls. The difference between RA patients and control groups was very notable. In anti-C II antibody positive RA patients positive rate of RF was more than that in anti-C II antibody negative RA patients.The serums reaction with HC II and BC II were all from the early RA patients. The results suggest that detection of anti-C II antibody may be used as a means for judging RA patients' conditions, the replacement of HCllby BCII can be used to study the immunological role of B cells in the pathogenesis of RA.
We isolated the peripheral blood mononuclear cells (PBMC) and synovial fluid mononuclear cells (SFMC) from RA patients by Lymphoprep density gradient centrifugation and cultured them with human CII and bovine C II respectively. Then a two-colour cytometric analysis was performed using a peridinin chorophyll protein(PerCP) conjugated anti-CD3 antibody in combination with phycoerythrin(PE) labeled anti-CD69 monoclonal antibody(mAb). Last,we investigated the expression of CD69 on CD3+ cells by flow cytometry (FCM) and believed the frequencies of CD3+ CD69+ T cells
6
as the frequencies of the CII -reactive T cells detected by FCM. The results of being stimulated with HCII were that: the frequency of the c II -reactive T cells in peripheral blood(PB) from 10 RA patients was (9.08 ?.42)%, significantly higher than controls', however that was (1.68?.47)%(P<0.05): the percent of the CII -reactive T cells in synovial fluid (SF) from 6 RA patients was (18.03 ?.62) %, higher than the percent of the CII -reactive T cells ((8.65 ?3.20 ) % ) (P <0. OS) in PB. The results of being stimulated with HC II ((9.08 ?4.42 ) % ) and stimulated with BCII ((10.83 ?. 37) %) had no difference. The results suggest that: CII -reactive T cells play an important role in the pathogenesis of RA,peripheral tolerance induction might be useful in the treatment of RA, BC II could take the place of HC II in the study of T cells correlation with the pathogenesis of RA.
The studies indicate that anti-C II antibody and CII -reactive T cells were all important in the pathogenesis of RA and bovine CII could take the place of human CII. The studies can provide the evidences for the treatment of RA by oral tolerance.
引文
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60 Yamamura Y, Gupta R, Morita Y, et al Effector function of resting T cells: activation of synovial fibroblasts. J Immunol,2001,166:2270-2275
61 Takemura S, Klimiuk PA, Braun A, et al. T cell activation in rheumatoid synovium is B cell dependent. J Immunol, 2001,167:4710-4718
62 吕爱平。关节软骨免疫与类风湿性关节炎——Ⅱ型胶原所致免疫性关节炎发病机理。中国中医基础医学杂志，2000，6(5)：1-5。
63 蔡春友 张维铭 孙保存，等．白细胞介素1对Ⅱ型胶原诱导类风湿性关节炎模型的影响。中华骨科杂志·基础研究．1990，10：614-616
64 Terao K,Hasty KA, Cremer MA,et al. Collagen-induced arthritis in mice:localization of an arthritogenic determinant to a fragment of the type Ⅱ collagen molecule. J Exp Med, 1985,162:637-646.
65 Rowley MG, Williamson DJ,Mackay IR.Evidence for local synthesis of antibodies to denatured collagen in the synovium in rheumatoid arthritis. Arthritis Rheum, 1987,30:1420-1425
66 Londei M, Savill CM, Averhoef A, et al. Persistence of collagen type Ⅱ specific T-cell clones in the synovial membrane of a patient with rheumatoid arthritis.Proc Natl Acad Sci USA,1989,86:636-640
67 Park SH, Min DJ, Cho ML, et al. Shift toward T helper 1 cytokines by type Ⅱ collagen-reactive T cells in patients with rheumatoid arthritis. Arthritis Rheum, 2001, 44:561-569
68 He X, Kang AH, Stuart JM.Accumulation of T cells reactive to type Ⅱ collagen in synovial fluid of patients with rheumatoid arthritis. J Rheumatol,2000,27:589-593
69 Sekine T, Kato T, Masuko-Hongo K, et al. Type Ⅱ collagen is a target antigen of clonally expanded T cells in the synovium of patients with rheumatoid arthritis. Ann Rheum Dis, 1999,58:446-450
70 陈声林，熊思东，俞顺章，口服耐受机制的研究进展。2000，20(3)187-189
71 Mowat AM, Viney JL. The anatomical basis of intestinal immunity. Immunol Rev, 1997, 156:145-166
72 Myers LK, Stuart JM, Seyer JM, et al. Identification of an immunosuppressive epitope of type Ⅱ collagen that confers protection aginst collagen-induced arthritis. J Exp Med, 1989,170,1999-2010.
73 Myers LK, Terato K, Seyer JM, et al. Characterization of a toleragenic T cell epitope of type Ⅱ collagen and its relevance to collagen-induced arthritis,J Immunol, 1992,149:1439-1443.
74 Malmstrom V, Ho KK, Lun J, et al. Arthritis susceptibility in mice expressing human type Ⅱ collagen in cartilage. Scand J Immunol, 1997,45(6):670-675.
75 朱平，冷南，王彦宏，等．口服Ⅱ型胶原蛋白诱导免疫耐受治疗佐剂关节炎的研究．。细胞与分子免疫学杂志，1999，15(4)：305-307．
76 帅宗文，丁长海，徐建华．口服Ⅱ型胶原治疗类风湿关节炎的临床研究．中国医师杂志，2001，7：510-512
77 Ausar SF, Beltramo DM, Castagna LF，et al. Treatment of rheumatoid arthritis by oral administration of bovine tracheal type Ⅱ collagen.. Rheumatol Int,2001,20:138-144
78 Hirabayashi Y, Kurata H, Funato H, et al. Comparison of intranasal inoculation of influenza HA vaccine combined with cholera toxin B subunit with oral or parenteral vaccination. Vaccine, 1990,8(3):243-248
79 McSorley SJ, Rask C, Pichot R, et al. Selective tolerization of Th1-like cells after nasal administration of a cholera toxoid-LACK conjugate. Eur J Immunol 1998,28(2):424-432
80 Derry CJ, Harper N, Davies DH, et al. Importance of dose of type Ⅱ collagen in suppression of collagen-induced arthritis by nasal tolerance. Arthritis Rheum ,2001, 44:1917-1927
81 Fujii K, Tsuji M, Murota K, et al. An improved enzyme-linked immunosorbent assay of anti-collagen antibodies in human serum. J Immunol Methods,1989, 124(11): 63-70.
82 Clague RB, Morgan K, Reynolds Ⅰ, et al. The prevalence of serum IgG antibodies to type Ⅱ collagen in American patients with rheumatoid arthritis. Br J Rheumatol, 1994,33: 336-338.
83 Noyori K, Koshino T, Takagi T, et al. Binding characteristics of antitype Ⅱ collagen antibody to the surface of diseased human cartilage as a probe for tissue damage. J Rheumatol, 1994,21:293-296
84 Jasin HE, Noyori K, Takagi T, et al. Characteristics of anti-type Ⅱ collagen antibody binding to articular cartilage. Arthritis Rheum, 1993,36:651-659
85 刘升云，曾庆馀，陈素标，等．抗Ⅱ型胶原抗体的检测及其在类风湿关节炎中的意义．中华风湿病学杂志，2000，3：150-152．
86 林冰，吴东海，张荣富，等。类风湿关节炎患者抗天然Ⅱ型胶原抗体的测定及临床意义。中国康复医学杂志，1997，12(4)170-173
87 Kim WU, Yoo WH, Park W, et al. IgG antibodies to type Ⅱ collagen reflect inflammatory activity in patients with rheumatoid arthritis. J Rheumatol,2000, 27:575-581
88 Cook AD, Stockman A, Brand CA, et al. Antibodies to type Ⅱ collagen and HLA disease susceptibility markers in rheumatoid arthritis. Arthritis Rheum,1999, 42:2569-2576
89 Wernhoff P, Unger C,Bajtner E,et al. Identification of conformationdependent epitopes and Ⅴ gene selection in the B cell response to type Ⅱcollagen in the DA rat. Int Immunol,2001,13:909-919
90 Terato K, DeArmey DA, Ye XJ, et al. The mechanism of autoantibody formation to cartilage in rheumatoid arthritis: possible cross-reaction of antibodies to dietary collagen with autologous type n collagen. Clin Immunol Immunopathol, 1996,79: 142-150．
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58 Bhardqaj N, Friedman S M, Cole BC, et al. Dendritic cells are potent antigen-presenting cells for microbial superantigens. J Exp Med. 1992,175:267-273
59 Harris ED Jr. Rheumatoid arthritis: pathophysiology and implications for therapy. N Engl J Med. 1990,322:1277-1289
60 Yamamura Y, Gupta R, Morita Y, et al Effector function of resting T cells: activation of synovial fibroblasts. J Immunol,2001,166:2270-2275
61 Takemura S, Klimiuk PA, Braun A, et al. T cell activation in rheumatoid synovium is B cell dependent. J Immunol, 2001,167:4710-4718
62 吕爱平。关节软骨免疫与类风湿性关节炎——Ⅱ型胶原所致免疫性关节炎发病机理。中国中医基础医学杂志，2000，6(5)：1-5。
63 蔡春友 张维铭 孙保存，等．白细胞介素1对Ⅱ型胶原诱导类风湿性关节炎模型的影响。中华骨科杂志·基础研究．1990，10：614-616
64 Terao K,Hasty KA, Cremer MA,et al. Collagen-induced arthritis in mice:localization of an arthritogenic determinant to a fragment of the type Ⅱ collagen molecule. J Exp Med, 1985,162:637-646.
65 Rowley MG, Williamson DJ,Mackay IR.Evidence for local synthesis of antibodies to denatured collagen in the synovium in rheumatoid arthritis. Arthritis Rheum, 1987,30:1420-1425
66 Londei M, Savill CM, Averhoef A, et al. Persistence of collagen type Ⅱ specific T-cell clones in the synovial membrane of a patient with rheumatoid arthritis.Proc Natl Acad Sci USA,1989,86:636-640
67 Park SH, Min DJ, Cho ML, et al. Shift toward T helper 1 cytokines by type Ⅱ collagen-reactive T cells in patients with rheumatoid arthritis. Arthritis Rheum, 2001, 44:561-569
68 He X, Kang AH, Stuart JM.Accumulation of T cells reactive to type Ⅱ collagen in synovial fluid of patients with rheumatoid arthritis. J Rheumatol,2000,27:589-593
69 Sekine T, Kato T, Masuko-Hongo K, et al. Type Ⅱ collagen is a target antigen of clonally expanded T cells in the synovium of patients with rheumatoid arthritis. Ann Rheum Dis, 1999,58:446-450
70 陈声林，熊思东，俞顺章，口服耐受机制的研究进展。2000，20(3)187-189
71 Mowat AM, Viney JL. The anatomical basis of intestinal immunity. Immunol Rev, 1997, 156:145-166
72 Myers LK, Stuart JM, Seyer JM, et al. Identification of an immunosuppressive epitope of type Ⅱ collagen that confers protection aginst collagen-induced arthritis. J Exp Med, 1989,170,1999-2010.
73 Myers LK, Terato K, Seyer JM, et al. Characterization of a toleragenic T cell epitope of type Ⅱ collagen and its relevance to collagen-induced arthritis,J Immunol, 1992,149:1439-1443.
74 Malmstrom V, Ho KK, Lun J, et al. Arthritis susceptibility in mice expressing human type Ⅱ collagen in cartilage. Scand J Immunol, 1997,45(6):670-675.
75 朱平，冷南，王彦宏，等．口服Ⅱ型胶原蛋白诱导免疫耐受治疗佐剂关节炎的研究．。细胞与分子免疫学杂志，1999，15(4)：305-307．
76 帅宗文，丁长海，徐建华．口服Ⅱ型胶原治疗类风湿关节炎的临床研究．中国医师杂志，2001，7：510-512
77 Ausar SF, Beltramo DM, Castagna LF，et al. Treatment of rheumatoid arthritis by oral administration of bovine tracheal type Ⅱ collagen.. Rheumatol Int,2001,20:138-144
78 Hirabayashi Y, Kurata H, Funato H, et al. Comparison of intranasal inoculation of influenza HA vaccine combined with cholera toxin B subunit with oral or parenteral vaccination. Vaccine, 1990,8(3):243-248
79 McSorley SJ, Rask C, Pichot R, et al. Selective tolerization of Th1-like cells after nasal administration of a cholera toxoid-LACK conjugate. Eur J Immunol 1998,28(2):424-432
80 Derry CJ, Harper N, Davies DH, et al. Importance of dose of type Ⅱ collagen in suppression of collagen-induced arthritis by nasal tolerance. Arthritis Rheum ,2001, 44:1917-1927
81 Fujii K, Tsuji M, Murota K, et al. An improved enzyme-linked immunosorbent assay of anti-collagen antibodies in human serum. J Immunol Methods,1989, 124(11): 63-70.
82 Clague RB, Morgan K, Reynolds Ⅰ, et al. The prevalence of serum IgG antibodies to type Ⅱ collagen in American patients with rheumatoid arthritis. Br J Rheumatol, 1994,33: 336-338.
83 Noyori K, Koshino T, Takagi T, et al. Binding characteristics of antitype Ⅱ collagen antibody to the surface of diseased human cartilage as a probe for tissue damage. J Rheumatol, 1994,21:293-296
84 Jasin HE, Noyori K, Takagi T, et al. Characteristics of anti-type Ⅱ collagen antibody binding to articular cartilage. Arthritis Rheum, 1993,36:651-659
85 刘升云，曾庆馀，陈素标，等．抗Ⅱ型胶原抗体的检测及其在类风湿关节炎中的意义．中华风湿病学杂志，2000，3：150-152．
86 林冰，吴东海，张荣富，等。类风湿关节炎患者抗天然Ⅱ型胶原抗体的测定及临床意义。中国康复医学杂志，1997，12(4)170-173
87 Kim WU, Yoo WH, Park W, et al. IgG antibodies to type Ⅱ collagen reflect inflammatory activity in patients with rheumatoid arthritis. J Rheumatol,2000, 27:575-581
88 Cook AD, Stockman A, Brand CA, et al. Antibodies to type Ⅱ collagen and HLA disease susceptibility markers in rheumatoid arthritis. Arthritis Rheum,1999, 42:2569-2576
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