类风湿性关节炎患者抗胶原蛋白Ⅱ抗体及其自身反应性T淋巴细胞的检测

Anti-CⅡ antibody and autoreactive T cells in patients with rheumatoid arthritis

目的:分析类风湿性关节炎(RA)患者T淋巴细胞免疫应答及抗胶原蛋白Ⅱ(CⅡ)抗体的异常表现。方法:应用流式细胞仪分析22例RA及6例骨关节炎(OA)患者外周血和关节滑膜液(SF)中T淋巴细胞免疫表型和趋化因子受体的表达。选用HSP-65和胶原蛋白Ⅱ(CⅡ)抗原诱导和分析患者自身反应性T淋巴细胞克隆频率,采用经典的酶联免疫吸附试验(ELISA)检测患者外周血和SF中抗CⅡ抗体以及IFN-γ、IL-10、IL-4和IL-12的含量。结果:RA患者关节SF中CD4+/CD8+T淋巴细胞的比值为1.09,显著低于外周血中CD4+/CD8+T淋巴细胞比值(2.02);SF中CD3+/CD25+双阳性的活化T淋巴细胞占(15.9±9.6)%。SF中T淋巴细胞表面趋化因子受体CCR5表达较低,与外周血相比无统计学意义,但趋化因子受体CD25+/CXCR3+表达量较高,为(16.0±4.0)%,远远高于外周血T淋巴细胞[仅为(0.5±0.3)%](P<0.01)。ELISA检测结果表明RA患者关节SF中抗CⅡ抗体阳性率明显高于其血清以及OA患者SF和血清;RA患者SF中IFN-γ、IL-12的含量高于外周血。来自RA患者病灶关节SF单个核细胞(SFMC)较外周血单个核细胞(PBMC)对HSP-65抗原和CⅡ的诱导具有更高的自身反应性T淋巴细胞应答频率(分别为8.5×10-6和11.5×10-6)。结论:RA患者关节SF中存在高活化状态的T淋巴细胞,其对CⅡ的高克隆反应频率以及SF中高表达抗CⅡ抗体,均提示自身反应性T淋巴细胞的浸润是造成患者病灶区组织损伤的关键因素。

Objective To analyze the abnormal T cell immune-response and level of anti-collagen Ⅱ（CⅡ） antibody in patients with rheumatoid arthritis.Methods Twenty-two rheumatoid arthritis（RA） patients and 6 osteoarthritis（OA） patients were enrolled.Peripheral blood（PB） and synovial fluid（SF） were collected for determining the immunophenotype of T lymphocytes and expressions of chemokine receptors by cytometry.HSP-65 and collagen protein Ⅱ antigen（CⅡ Ag） were used for inducing and analyzing the autoreactive T cell clone frequency,and ELISA was used to measure the CⅡantibody and INF-γ,IL-10,IL-4 and IL-12 levels.Results The ratio of CD4＋/CD8＋ in SF was 1.09,significantly lower than that in PB（2.02）,and the percentage of CD3＋/CD25＋ double positive T cell was（15.9 ± 9.6） %.The expression of CCR5 on SF T cell was low and similar to that on PB T cells.CXCR3 expression on SF T cells was（16.0±4.0）%,much higher than that on PB T cells [（0.5±0.3）%,P0.01）.ELISA showed a higher positive rate of anti-CⅡ Ab in SF than in PB and in SF and PB of OA patients;the levels of IFN-γ,IL-12 in SF were significantly higher than those in PB.The autoreactive T cell response frequencies of SFMC（SF monocytes） to HSP-65 and CⅡ Ag（8.5×10-6 and 11.5×10-6）were higher than those of PBMC（PB monocytes）.Conclusions High activated T cells existed in SF of RA patients,which has a high clone response frequency to CⅡ Ag,and the high level of anti-CⅡ antibody in SF all suggests that the infiltration of autoreactive T cells is a key factor in the tissue injury of RA joints.