小胶质细胞(MG) TLR 4介导T细胞获得性免疫炎症反应的机制

 目的 探讨以小胶质细胞(microglia,MG)为中心Toll样受体 4 (toll like receptor 4,TLR 4) 介导的T细胞获得性免疫炎症损伤机制在早产儿脑白质损伤中的作用。方法 分别分离纯化C3H/HeJ(tlr4基因缺失)小鼠和C57B/L (野生型) 小鼠脾脏CD4＋T细胞和大脑MG细胞并制作交互共培养模型。各组分别加以细菌脂多糖(lipopolysaccharide,LPS)为刺激因素,流式细胞术和细胞化学染色观察MG变化,检测CD4＋ T细胞增殖变化和ELISA方法检测其分泌功能;通过上调(LPS刺激)或下调TLR 4(tlr4基因缺失)表达对MG活化和共同免疫刺激因子MCH Ⅱ的影响及对CD4＋T细胞增殖分化及Th1/Th2极化方向的影响,阐明TLR 4在T细胞获得性免疫中的关键作用。结果 显微镜下观察,LPS刺激后tlr4基因缺失小鼠MG细胞胞体较小,突起细长,仍处于静息状态;相反,野生型组LPS刺激后出现较明显的细胞体积变大、胞体变大显圆满,突起增加呈分支状。LPS活化的MG其TLR 4和MCH Ⅱ蛋白表达显著上调与tlr4基因缺失小鼠比较有显著性差异(P<0.01)。此外,LPS刺激活化的CD4+ T淋巴细胞增殖明显,且伴有Th1型细胞因子显著升高和Th2型细胞因子的显著下降,与tlr4基因缺失小鼠比较均有显著性差异(P< 0.01)。TLR-4表达与CD4＋T细胞Th1细胞因子浓度间具有显著相关性(P<0.01)。结论 TLR-4介导MG活化在固有免疫和获得性免疫中发挥桥梁作用,并由此提出由Th1/Th2偏移向TLR4、Th1偏移的新的早产儿脑白质损伤模式。

Mechanisms of microglia(MG) toll like receptor-4(TLR-4) mediated T cell adaptive immune response in vitro

Objective To investigate the mechanisms of microglia(MG) toll like receptor-4(TLR-4) mediated CD4+T cell adaptive immune response in LPS induced premature white matter injury in vitro. Methods C3H/HeJ(tlr4 gene deletion) mouse,C57B/L(tlr4 wild type) mouse spleen CD4+T cells and MG were isolated and purified respectively as protocol and interactively co-cultured between these two types of cells [wild MG + wild T cell(group Ⅰ),wild MG + deleted T cell(group Ⅱ),deleted MG + deleted T(group Ⅲ) and deleted MG + wild T cell(group Ⅳ)].Every co-cultured group was stimulated by LPS in vitro.Flow cytometer,immuncytochemistry,Western blotting,and ELISA methods were used to detect MG activation and CD4+ T cell proliferation and cytokines(Th1 and Th2) production via up(LPS stimulation) and down regulation(tlr4 gene deletion) of TLR-4 expression on MG. Results In response to the stimulation of LPS,MG rapidly differentiated into the activated form with enlarged body and amoeboid-like in group I and group Ⅱ compared with ramified resting stage MG with bipolar and a small oval cell body in group Ⅲ and group Ⅳ.Compared with group Ⅲ and Ⅳ,LPS stimulation significantly enhanced the TLR-4 and co-stimulator MCH Ⅱ expression in a dose-dependent manner on MG of group Ⅰ and group Ⅱ(P<0.01).We also detected the significant increase of Th1 cytokines(P<0.01)and significant decrease of Th 2 cytokines(P<0.01) production following the dramatic proliferation of CD4+ T cell(P<0.01)in wild type group compared with tlr-4 deletion group and PBS control group,and there was a close relationship between the TLR-4 expression and CD4+ T cell proliferation and Th1 cytokines production(P<0.01). Conclusions Activated MG acts as a bridge between innate and adaptive immune response following LPS induced premature white matter injury in vitro and the turnover from "Th1/Th2 bias" to "TLR-4-Th1 bias" was proposed as a novel white matter injury paradigm.