类风湿性关节炎滑液刺激T细胞表达GADD45β机制的研究

Study on role of GADD45β in pathogenesis of rheumatoid arthritis:GADD45β expression profile and involvement in IFN-γ product

目的研究GADD45β在类风湿性关节炎病灶局部组织细胞中的表达、与炎症因子间的关系以及对Th1细胞活化的作用,以探讨GADD45β在RA等自身免疫病发病机制中的作用。方法用Real-Time PCR检测RA患者滑膜组织单个核细胞(SFMC)、滑膜液单个核细胞(STMC)和外周血单个核细胞(PBMC)以及SFMC、PBMC中CD4/CD8+T细胞(磁珠法分离)中GADD45β的表达格局;用RA患者滑膜液(RA SF)刺激体外培养的健康人PBMC,用Real-Time PCR检测GADD45β和IFN-γ mRNA表达情况;SiRNA感染GADD45β基因表达。流式细胞仪检测T细胞亚群。ELISA检测细胞因子。结果RA SFMC中GADD45β的表达水平明显高于其STMC和PBMC及健康人PBMC,且以SF中浸润的CD4+T细胞为主;RA SF可以上调健康人PBMC(主要是CD4+T细胞)中GADD45β和IFN-γ表达;用SiRNA干扰GADD45β基因转录后IFN-γ分泌减少。结论GADD45β在RA患者滑膜组织和滑液单个核细胞中表达明显上调,并且参与SF刺激的T细胞产生IFN-γ。

Objective To study the role of growth arrest and DNA damage-inducible beta(GADD45β) in pathogenesis of rheumatoid arthritis(RA) and the relationship between GADD45β and inflammatory cytokines.Methods The expression of GADD45β mRNA in synovial fluid mononuclear cells(SFMC),synovium mononuclear cells(STMC) and peripheral blood mononuclear cells(PBMC) of RA patients were analyzed by real-time PCR,as well as CD4/CD8+T cells were purified from SFMC and PBMC using Dynalbeads.PBMC derived from healthy volunteers were used as control.The cytokine concentration was analyzed by ELISA.In stimulation test,PBMC from healthy individuals were cultured with SF from RA patients.Special GADD45β RNA was used to silence GADD45β expression in T cells.The expression of GADD45β and IFN-γ mRNA were analyzed by real-time PCR.Results The expression of GADD45β gene in synovium tissues from RA patients was higher than that from healthy controls.SFMC derived from RA patients expressed a higher level of GADD45β mRNA than paired STMC and PBMC.RA-SF could induce the expression of GADD45β in PBMC from healthy individuals.Moreover,GADD45β mainly expressed in CD4+T cells either healthy individual or RA patients under SF stimulation.In the test,GADD45β expression in CD4+T cells is higher than healthy controls.Further,the concentration and mRNA of IFN-γ decreased in GADD45β knock down CD4+T cells.Conclusion The expression of GADD45β mRNA in SFMC,particularly in CD4+ T cells from SF,is up-regulated in RA patients.And RA-SF could stimulate the expression of GADD45β in PBMC of healthy individuals.GADD45β mignt mediate the expression of IFN-γ production in RA patients.