| 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, nextthe 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 cells6as 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. |
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