【作者】 张薇；

【导师】 周文斌；

【作者基本信息】 中南大学 ， 神经病学， 2012， 硕士

【摘要】 目的：吉兰-巴雷综合征(GBS)是一种急性炎症性脱髓鞘性周围神经多发性神经病,至今其确切发病机制仍不清楚。研究发现,CD4+T细胞及其相关细胞因子在GBS发病过程中起着重要的作用。调节性T细胞(Tregs)是维持免疫耐受的作用细胞,在GBS患者急性期外周血中表达下调。IL-35是IL-12家族的新成员,能够促进Tregs的功能。本实验旨在检测IL-35在GBS患者血浆中的水平,并探讨其体外干预GBS患者CD4+T细胞对CD4+T细胞亚群功能的影响。方法：收集GBS患者组及正常对照组外周血标本提取血浆、总蛋白和总RNA：采用酶联免疫吸附测定(ELISA)法检测血浆中IL-35的水平；利用Western blot方法观察GBS患者组较正常对照组STATs蛋白的表达变化；使用实时荧光定量PCR法对比GBS患者组与正常对照组CD4+T细胞相关转录因子和细胞因子mRNA的表达水平。分选出GBS患者及正常对照的CD4+T细胞,给予人融合性蛋白IL-35体外干预培养3天,观察IL-35对STATs蛋白及转录因子、细胞因子mRNA的表达影响。结果：GBS患者组血浆中IL-35的表达较正常组降低,具有显著性差异(p<0.01)。与正常对照组相比,GBS患者组STAT1、STAT3、 STAT4蛋白及T-bet、RORγt、IFN-γ、IL-17A mRNA表达升高,有明显差异(p<0.05),STAT5、STAT6蛋白及GATA3、Foxp3、IL-4、TGF-β1mRNA表达降低,有统计学意义(p<0.01)。IL-35体外干预CD4+T细胞结果：与GBS空白组相比,GBS患者p35-Fc嵌合体干预组STATs蛋白及CD4+T细胞相关转录因子及细胞因子表达无明显变化(p>0.05)；与GBS空白组相比,GBS患者IL-35干预组STAT1、STAT5、STAT6蛋白及T-bet、GATA3、Foxp3、IFN-γ、IL-4、 TGF-pmRNA表达升高,具有显著性差异(p<0.05),STAT3、STAT4蛋白及RORγt、IL-17AmRNA表达降低,具有显著性差异(p<0.01)。结论：IL-35在GBS患者外周血血浆中的水平降低：IL-35对GBS患者Th1/Th2细胞轴可能存在双重作用,对Th17细胞有抑制作用,对Tregs细胞有促进作用,为GBS的治疗提供了新的靶点。更多还原

【Abstract】 Objective Guillain-Barre syndrome (GBS) is an acute inflammatory demyelinating polyneuropathy of peripheral nerves. Until now, the exact mechanism of GBS pathogenesis is elusive. Recens evidence suggests that CD4+T cells and related cytokines play important roles in the pathogenic mechanism of GBS. The number of regulatory T cells (Tregs) which are the effector cells keeping immune tolerance in check decreased in acute phase of GBS. IL-35, a novel member of the IL-12cytokine family, can promote the function of Tregs. This experiment was designed to detect the level of IL-35expressed in plasma of GBS patients, and to investigate corresponding function changes of CD4+T cells subsets after CD4+T cells from GBS patients were cultured with IL-35in vitro.Methods Collect peripheral blood of patients with GBS and normal controls for extracting plasma, total protein and total RNA:Detect the level of IL-35in plasma by the enzyme-linked immunosorbent assay (ELISA) method; Use the method of Western blot to observe the different expression of STATs between GBS patients and normal controls; Compare their different expression levels of CD4+T cells related transcription factors and cytokines mRNA by realtime fluorescence quantitative PCR. Separate CD4+T cells of GBS Patients and normal controls, then culture the cells dealing with the fusion protein IL-35in vitro three days, at last analyse the influence of IL-35on the expression of STATs proteins as well as transcription factors and cytokines mRNA, which as indicated above.Results Compared with normal controls, IL-35expressed in plasma decreased in GBS patients with significant differences (p<0.01). The expression levels of STAT1, STAT3, STAT4protein and T-bet, ROR γt, IFN-γ, IL-17A mRNA increased in the group of GBS, having sinificant differences with normal control group (p<0.05). Whereas, the expression of STAT5, STAT6protein and GATA3, Foxp3, IL-4,TGF-β1mRNA reduced in the group of GBS in contrast with normal control group, with statistically significance (p<0.01). The results of IL-35intervention CD4+T cells in vitro are as follows:With GBS blank group, there were no chang in the expression of STATs proteins, CD4+T cells associated transcription factors and cytokines mRNA in GBS p35-Fc chimera interventional group (p>0.05); Compared with GBS blank group, the levels of STAT5, STAT6protein and T-bet, GATA3, Foxp3, IFN-γ, IL-4, IL-17A, TGF-β1mRNA expression increased in GBS IL-35intervention group with statistically significance (p<0.01), while STAT3, STAT4protein and RORγt, IL-17A mRNA expression were reduced, having significant differences (p<0.01). Conclusions The level of IL-35decreased in plasma of GBS patients peripheral blood; IL-35might not only have dual effects on Th1/Th2paradigm of GBS patients in vitro, but also have an inhibited role in Th17but promoted the function of Tregs, which may indicate a new target for the treatment of GBS.更多还原