摘要
目的 优化并建立CCP/AST分泌干扰素γ（IFN-γ）与白细胞介素4（IL-4）的ELISpot方法,以探讨CCP/AST细胞在RA发生、发展中的作用与临床意义.方法 以CCP抗原肽为特异性刺激物,FLAG肽为对照,用ELISpot检测64例RA患者和64例非RA患者及30名健康人PBMC分泌IFN-γ、IL-4的阳性SFC频数与比值,并评估其在RA疾病中的诊断价值,同时对上述3项指标与RA患者的关节症状及其他指标间的相关性进行分析.结果 RA患者中特异性分泌IFN-γ和IL-4-的SFC分别为39（12～77）个/3×105PBMC和1（1～3）个/3×105PBMC,阳性率分别为81.3%和18.8%;IFN-γ与IL-4的SFC频数比值为15（5～39）,阳性率78.1%.其中IFN-γ的SFC和两者频数比值均显著高于非RA疾病（Z分别为-7.458、-7.019,P均＜0.01）和健康对照组（Z分别为-6.643、-5.760,P均＜0.01）,且这2项指标在抗CCP抗体阳性与阴性的RA组中均显著高于系统性红斑狼疮（Z分别为-6.573、-6.098、-4.552、-4.726,P均＜0.01）、强直性脊柱炎（Z分别为-3.520、-3.326、-2.950、-2.126,P均＜0.01或0.05）、其他自身免疫病（Z分别为-4.838、-4.418、-3.681、-3.839,P均＜0.01）和健康对照组（Z分别为-6.553、-5.578、-4.635、-4.163,P均＜0.01）.用IFN-γ-SFC、IL-4-SFC和IFN-γ-SFC/IL-4-SFC频数比值3项指标联合检测RA的ROC曲线下面积和Youden指数分别为0.910和0.747,其诊断RA的敏感度和特异度分别为87.5%和87.2%,阳性和阴性预测值分别为82.4%和91.1%.相关分析结果显示,IFN-γ-SFC/IL-4-SFC频数比值不仅与抗CCP水平显著相关（r=0.393,P＜0.01）,而且与患者的关节症状,如肿痛关节数（r=0.429,P＜0.01）、破坏关节数（r=0.463,P＜0.01）、RF（r=0.166,P＜0.01）及ESR（r=0.199,P＜0.05）亦显著相关.结论 RA患者体内广泛存在CCP/AST的活化和异常,并呈CCP抗原特异性Th1细胞优势状态,这为客观了解RA瓜氨酸化蛋白介导的特异性细胞免疫应答状态及细胞因子网络调节功能提供了新的试验依据,并具有潜在诊断和临床应用价值.
Objective To optimize and establish ELISpot assay for CCP/AST which could secrete IFN-γ and IL-4, and explore the role and clinical significance of CCP/AST cells in occurrence and development of RA disease. Methods CCP was used as specific-stimulator with FLAG peptide as a control,the frequencies of positive SFC which could specifically secrete IFN-γ and IL-4 in 64 cases of RA, 64 cases of non-RA autoinunune diseases and 30 cases of healthy individuals were tested by ELISpot technique. The diagnostic value of CCP/AST cells was evaluated in patients with RA disease. Meanwhile, the relationships among the indexes above and patient joint symptoms as well as other laboratory parameters were further analyzed and discussed. Results The results showed that the mediam numbers of IFN-γ-SFC and IL-4-SFC were 39（12-77）/3 x 105 PBMC and 1 （1-3）/3 × 105 PBMC in RA patients, the positive rates were 81.3% and 18. 8% respectively. The median value of IFN-γ-SFC/IL-4-SFC ratio was of 15（5-39）, the positive rate was of 78. 1%. Both IFN-γ-SFC and ratio of IFN-γ-SFC/IL-4-SFC were significantly higher than those of non-RA diseases （Z = - 7. 458, - 7. 019, P 〈 0. 01 ） and healthy control （ Z = - 6. 643, - 5. 760, P 〈0. 01 ）, also both these parameters in RA patients with positive anti-CCP antibody and negative anti-CCP antibody were significantly higher than those patients with systemic lupus erythematosns （ Z = - 6. 573, - 6. 098, - 4. 552, - 4. 726, P 〈 0. 01 ）, ankylosing spondylitis （ Z = - 3. 520, - 3. 326, - 2. 950,-2. 126, P〈0. 01 or 0. 05）, other autoimmune diseases （Z = -4. 838, -4. 418, - 3. 681, -3. 839,P 〈 0. 01 ） and healthy controls （ Z = - 6. 553, - 5. 578, - 4. 635, - 4. 163, P 〈 0. 01 ）. Combining IFN-γ-SFC, IL-4-SFC with IFN-γ-SFC/IL-4-SFC for RA diagnosis, the area under curve of receiver operating characteristic（ ROCAUC） and Youden index were 0. 910 and 0. 747. The diagnostic sensitivity and specificity were 87. 5% and 87.2%. Positive and negative predictive values were 82. 4% and 91.1%, respectively. Correlation analysis showed that ratio of IFN-γ-SFC/IL-4-SFC was not only significantly correlated with antiCCP antibody（r =0.393, P 〈0.01）, but also correlated with joint symptoms in patients such as with number of joints swelling-pain （ r = 0. 429 , P 〈 0. 01 ）, number of joints damage （ r = 0. 463, P 〈 0. 01 ）,rheumatoid factor （r = 0. 166, P 〈 0.01） and erythrocyte sedimentation rate （r=0. 199,P〈0.05）.Conclusions There is widely existence of CCP/AST cells activation and abnormity in RA patients,indicating higher frequency of CCP-specific Th1 cells. These results provides a new experimental evidence for an objective understanding the function of specific cellular immune responses and cytokine network regulation mediated by citrullinated proteins in RA. So, the assay for CCP/AST cells has potential values in RA diagnosis and clinical application.
出处
《中华检验医学杂志》
CAS
CSCD
北大核心
2010年第8期728-734,共7页
Chinese Journal of Laboratory Medicine
基金
中国博士后科学基金特别资助项目（200801375）
中国博士后科学基金资助项目（20060400940）
江苏省博士后科学基金资助项目（0602008C）
