中期因子对实验性脑脊髓膜炎小鼠自身反应性Th17细胞活化的影响

Role of Midkine in activation of autoreactive Th17 cells on EAE mouse

目的观察中期因子对实验性脑脊髓膜炎小鼠自身反应性Th17细胞活化的影响。方法利用RT-PCR方法检测小胶质细胞系6-3细胞前炎症细胞因子IL-1β、IL-6和TNF-α及炎性介质i NOS m RNA表达,ELISA方法检测培养上清中上述细胞因子的表达水平,Greiss方法检测6-3细胞培养上清中NO水平;使用C57BL/6小鼠建立实验性脑脊髓膜炎小鼠动物模型,免疫磁珠纯化CD4+T细胞,与6-3细胞共培养,ELISA方法检测培养体系中培养上清IL-17的水平。结果与生理盐水对照相比,中期因子以剂量依赖方式促进6-3细胞表达IL-1β、IL-6和TNF-α及分泌NO的水平(P<0.05),中期因子适体抑制上述细胞因子及NO的表达(P<0.05)。体内实验结果表明中期因子适体抑制实验性脑脊髓膜炎小鼠MOG35-55特异性Th17细胞产生IL-17的水平(P<0.05)。结论中期因子通过活化小胶质细胞促进自身反应性Th17细胞活化。中期因子适体抑制小胶质细胞活化及实验性脑脊髓膜炎小鼠MOG35-55特异性Th17细胞活化。

Objective To investigate the role of Midkine in the activation of microglia and the influence on the activation of autoreactive Th17 cells on mouse model with experimental autoimmune encephalomyelitis(EAE). Methods 6-3 microglia cell line was cultured in the presence of various concentrations of Midkine, expression of proinflammatory cytokines and inflammtory mediator IL-1β, IL-6 and TNF-α and i NOS were assessed by RT-PCR and ELISA methods, NO production was detected by Greiss reaction method. EAE mice was induced by MOG35-55-inoculation, followed by PTX injection. CD4+T cells were purified at the peak of EAE by MACS CD4+ T cells were co-cultured with MOG35-55 pulsed 6-3 cells in the presence of various concentrations of Midkine aptamer. The supernatants of culture was qualified by ELISA. Results Compared with negative control, Midkine significantly increased the production of IL-1β, IL-6, TNF-α and NO production by 6-3 cells as dose-dependent manner(P<0.05). Midkine aptamer inhibited the production of above cytokines and NO production by 6-3 cells(P<0.05). Furthermore, MOG35-55 specific Th17 cells that co-cultured with MOG35-55 pulsed 6-3 cells produced elevated levels of IL-17. In the presence of Midkine aptamer, IL-17 production was inhibited(P<0.05). Conclusions Midkine promotes the activation of autoreactive Th17 cells through activation of microglia, Midkine aptamer effectively inhibit the activation of microglia and MOG35-55 specific Th17 cells on EAE model. These results suggest that Midkine plays critical role in the pathogenesis of multiples scelerosis, targeting Midkine by Midkine aptamer in combination with effective chemotherapy provides novel strategy for MS therapy.