抗细胞膜DNA抗体检测方法的建立及对系统性红斑狼疮诊断价值的临床应用研究

A new method for detecting of autoantibodies to cell membrane associated DNA and its value for the diagnosis of systemic lupus erythematosus

目的应用问接免疫荧光法（IIF）检测抗细胞膜DNA（cmDNA）抗体，探讨抗cmDNA抗体对系统性红斑狼疮（SLE）的诊断价值。比较以人B淋巴细胞株Raji、人早幼粒白血病细胞株HL60为底物，抗cmDNA抗体在SLE中的检测效果。方法选取306例SLE患者，192例非SLE疾病对照组，包括脊柱关节病（sDA）52例，类风湿关节炎（RA）70例，原发干燥综合征（pSS）30例，其他结缔组织病（CTD）40例。50名健康志愿者作为对照组。采用IIF法观察培养的人B淋巴细胞株Raji、人早幼粒白血病细胞株HL60的细胞膜的荧光图形；用IIF法检测抗核抗体、抗双链DNA（dsDNA）抗体；联合应用免疫双扩散法和蛋白印迹法检测抗Sm抗体，酶联免疫吸附试验（ELISA）法测定抗核小体抗体（AnuA）。配对资料比较用McNemarr检验，相关性分析采用Spearman相关及Logistic回归分析。结果以Raji及HL602种细胞株为底物检测SLE患者抗cmDNA抗体，敏感性分别为72．5％、76．1％，特异性分别为91．7％、86．8％，差异均无统计学意义（P〉0．05）。抗cmDNA抗体的敏感性明显高于抗Sm抗体及抗dsDNA抗体，差异有统计学意义（P〈0．01）；特异性与抗dsDNA抗体相似（P〉0．05），但低于抗Sm抗体及AnuA（P〈0．01）。抗cmDNA抗体分别与抗dsDNA抗体、抗Sm抗体及AnuA联合检测在SLE诊断中的敏感性均明显高于上述自身抗体单独检测，差异有统计学意义（P〈O．05）。抗CmDNA抗体与SLE患者的黏膜溃疡之间有相关性（OR=2．343，P=O．029）。抗cmDNA抗体与红细胞沉降率（ESR）有相关性（OR=1．031，P=0．012）。抗cmDNA抗体与SLE疾病活动指数（SLEDAI）评分无相关性（r=0．070，P=0．600）。本文研究还证实Raji细胞株较HL60细胞株更易复苏、荧光图形亮度更强，表达效果更好。结论抗cmDNA抗体对SLE诊断的敏感性高，特异性强，可能成为SLE诊断的相对特异性抗体之一。抗cmDNA抗体与抗dsDNA抗体、抗Sm抗体及AnuA联合检测可提高对SLE诊断的敏感性。选择Raji细胞株为底物检测抗cmDNA抗体较HL60细胞株更有优势。

Objective To compare the significance of anti-cmDNA antibody in systemic lupus erythematosus （SLE） patients detected with IIF on human＇s B lymphoma cell line Raji and promyelocytic line HL60. The diagnostic value of anti-emDNA antibody in SLE was also explored. Methods Three hundred and six patients with SLE were included in this study. As control groups, we included 192 patients with other rheumatic diseases and 50 healthy controls. The testing method for anti-cmDNA antibody was set up. The assessment of the significance of anti-cmDNA antibody in SLE detected with IIF on cell line Raji and HL60 was carried out and the diagnostic value of anti-cmDNA antibody in SLE was investigated. ANA and antidsDNA antibody were measured by IIF at the same time. Anti-Sm was measured by immuno-diffusion and Western blotting. AnuA was tested by enzyme linked immunosorbent assay. The statistical methods used in this study including McNemar X2 test,Spearman related test and Logistic regression analysis. Results The fluorescence brightness of Raji cell line was stronger than HL60 cell line.There was no statistically significant difference in the sensitivity and specificity of anti-cmDNA antibody in SLE detected with IIF with Raji or HL60 cell lines （P〉0.05）. The sensitivity of anti-cmDNA antibody detected with IIF on Raji cell line was higher than anti-dsDNA antibody and anti-Sm antibody （P〈0.01）, while the specificity of anti-cmDNA antibody was similar to anti-dsDNA antibody （P〉0.05） and was lower than anti-Sm antibody （P〈0.01）. The sensitivity of anti-cmDNA antibody was similar to AnuA （P〉0.05） and the specificity was lower than AnuA （P〈0.01）. The sensitivity of ANA was higher than anti-cmDNA antibody （P〈0.01） and the specificity was much lower than anti-cmDNA antibody （P〈0.01）. The sensitivities of anti-dsDNA antibody, anti-Sm antibody and AnuA were much higher when combined with anti-dsDNA antibody than any one antibody only （P〈0.05）. Anti-cmDNA antibody was correlated with mucosa ulcer in SLE patients （OR=2.343, P=0.029）. The ESR of SLE patients was also correlated with anti-cmDNA antibody（OR = 1.031, P=0.012）. Anti-cmDNA antibody was not correlated with SLEDAI （r=0.070, P=0.600）. Conclusion Raji cell line is better than HL60 cell line in detecting anti-cmDNA antibody with IIF. Anti-cmDNA antibody has higher sensitivity and specificity in SLE. Combined detection of anti-cmDNA antibody and other autoantibodies can further improve the diagnostic accuracy of SLE.