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目的:探讨RAGE及其配体S100B在实验性自身免疫性重症肌无力(EAMG)中对T细胞的作用及其相关机制。方法:选取体重160-180g的Lewis大鼠,并将其随机分为EAMG模型组和CFA对照组。EAMG模型组大鼠通过尾根部注入200μL含有R-ACh R97-116肽段的免疫乳剂,并于初始免疫后的第30天尾根部追加免疫一次;CFA对照组大鼠为免疫乳剂中不含有R-ACh R97-116肽段。采用流式细胞术检测和比较两组大鼠CD4+T细胞上RAGE的表达情况;ELISA方法检测淋巴细胞培养上清中IFN-γ、IL-4、IL-17、TGF-β、IL-6和S100B的表达水平;采用S100B体外干预淋巴细胞,进一步检测S100B对EAMG大鼠T细胞增殖、亚型分布、细胞因子分泌的影响。结果:在疾病发生的晚期时相(初次免疫后第45天),EAMG组淋巴细胞CD4+T细胞上RAGE的表达明显高于CFA组(P<0.001),血清中RAGE的配体S100B的表达也高于CFA组(P<0.001);体外加入S100B干预能促进T淋巴细胞的增殖,与未干预组相比较差异显著(P<0.05);S100B刺激后,Th1细胞和Th17细胞的百分比进一步增高,Th2细胞和Treg细胞百分比下降(PTh1<0.05,PTh17<0.05,PTh2>0.05,PTreg<0.05),IFN-γ和IL-17的表达上调(PIFN-γ<0.05,PIL-17<0.05),而IL-4和TGF-β表达下降(PTGF-β<0.01,PIL-4>0.05),Th17细胞的调控因子IL-6表达升高(PIL-6<0.05)。结论:RAGE及其配体S100B参与EAMG的发病过程,在晚期时相中表现出明显的致病性;RAGE与S100B相互作用可以上调T细胞的致病性作用,加重四种辅助性T细胞之间的网络失衡。
Objective: To investigate the effect of RAGE and its ligand S100B on T cells in experimental autoimmune myasthenia gravis (EAMG) and its related mechanism. Methods: Lewis rats weighing 160-180 g were selected and randomly divided into EAMG model group and CFA control group. EAMG model rats were injected 200μL of R-ACh R97-116 peptide immuno-emulsion through the root of the tail and immunized once more at the tail of root on the 30th day after initial immunization. The rats in the CFA control group did not contain R -ACh R97-116 peptide. The expression of RAGE on CD4 + T cells in both groups was detected by flow cytometry and the levels of IFN-γ, IL-4, IL-17, TGF-β, IL-6 S100B and S100B in vitro. S100B was used to infect lymphocytes in vitro to further investigate the effect of S100B on T cell proliferation, subtypes distribution and cytokine secretion in EAMG rats. Results: The expression of RAGE on CD4 + T lymphocytes in EAMG group was significantly higher than that in CFA group (P <0.001) at the late phase of disease (45 days after the first immunization), and the expression of RAGE ligand S100B in serum (P <0.001). The addition of S100B could promote the proliferation of T lymphocytes in vitro, which was significantly different from that of non-intervention group (P <0.05). After S100B stimulation, the percentage of Th1 cells and Th17 cells were increased , The percentage of Th2 cells and Treg cells were decreased (PTh1 <0.05, PTh17 <0.05, PTh2> 0.05, PTreg <0.05) IL-4 and TGF-βdecreased (PIL-6 <0.05, PGF-| P <0.01, PIL-6 <0.05). CONCLUSION: RAGE and its ligand S100B are involved in the pathogenesis of EAMG and show obvious pathogenicity in late phase. The interaction between RAGE and S100B can up-regulate T-cell pathogenicity and aggravate four kinds of helper T cells The network between the imbalance.