中文摘要：

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MCHII的影响及对CD4＋T细胞增殖分化及Thl／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MCHII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-Thl偏移的新的早产儿脑白质损伤模式。

英文摘要：

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 iniury 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（groupⅠ） ,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 CD 4＊ T cell proliferation and cytokines （ Th 1 and Th 2 ） 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 Thl 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 Thl 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＂