维生素D结合蛋白在类风湿关节炎滑膜中表达的研究

Expression of vitamin D-binding protein in synovial tissues of rheumatoid arthritis

目的本研究通过蛋白质组学的方法比较类风湿关节炎（RA）、骨关节炎及强直性脊柱炎（AS）滑膜组织中表达的蛋白，筛选RA滑膜中表达特异性下调的蛋白，并通过单核苷酸多态性（SNPs）分析方法分析靶蛋白编码基因对RA的遗传易感性。方法提取RA（10例）、骨关节炎（10例）及AS（10例）滑膜组织总蛋白，每类疾病的样本等比例混合，然后用2．D凝胶电泳进行分析，对RA样本中表达量明显低于骨关节炎和As的蛋白进行飞行时间质谱鉴定，检测结果用蛋白印迹法验证。用Taqman方法对中国人群中267例RA患者和160名健康对照的维生素D结合蛋白（VDBP）编码基因的标签SNPs进行基因分型，并扩大样本量（389例RA和371名健康对照）验证分型结果。采用单因素方差分析和Fisher确切概率法进行检验。结果蛋白质组学研究发现，在RA患者滑膜中VDBP表达量明显下降，蛋白印迹法也验证了这一结果。基因分型研究发现，SNP位点rs2282679与RA密切相关（P=-0．026794）。进一步扩大样本实验也得出了一致结果，rs2282679与RA有相关性[比值比（OR）=0．678639，95％可信区间（凹）0．541113～0．851118，P=O．000776]。结论与骨关节炎和AS患者相比，VDBP在RA患者滑膜中表达量明显下降。VDBP基因上的SNP位点rs2282679与RA有相关性。VDBP在RA中的表达下调及其基因的遗传效应显示VDBP可能参与RA的病理过程。

Objective To screen the proteins with decreased expression in the synovial tissues of rheumatoid arthritis （RA） patients by comparing their expression profiles with that of osteoarthitis （OA） and ankylosing spondylitis （AS） patients by a proteomic approach, and to explore the association of reduced expression with disease susceptibility by a single nucleotide polymorphism （SNP） analysis. Methods Proteins extracted from the synovial membranes （n=10 for each disease） were separated by 2-D electrophoresis. The proteins with significantly decreased expression in the RA samples were subjected to MALDI-TOF-MS. The results were verified using Western blotting. Tag SNPs located in the targeted gene were assessed using the Taqman assay in a cohort of 267 Chinese patients with RA and ]60 healthy controls. The genotypJng results were confirmed in a large cohort of 389 patients with RA and 371 healthy controls. SPSS I1.5 software package was used for one way ANOVA and Fisher＇s exact test. Results The expression of vitamin D-binding protein （VDBP） in the synovial membranes from patients with RA was significantly decreased when examined by proteomic approach. This result was confirmed by Western blotting analysis. The rs2282679 was significantly associated with RA （P=0.026 794）. This result was confirmed in a large cohort of RA（OR=0.678 639, 95%CI 0.541 113-0.85] i]8, P=-0.000 776）. Conclusion Compared with samples from patients with OA and AS, RA patients＇ synovial tissues have low VDBP expression when examined by the proteomic method. The tag SNP rs2282679 located in VDBP is significantly associated with RA. The decreased expression and the geneticeffect of VDBP in RA suggest that a novel involved in the arthritis process of RA.