脑源性神经营养因子（BDNF）与Notch信号通路在肺炎链球菌脑膜炎神经生发调控中的交互作用

The occurrence of neurological sequelae is as high as 30% to 52% in streptococcus pneumoniae meningitis survivors, which remains a challenge in the clinical field. Our previous studies have shown that BDNF and its receptor TrkB were strongly expressed in dentate gyrus (DG) in streptococcus pneumoniae meningitis. Their expression, however, was significantly down-regualted after antibiotic treatment, and the number of neurons in cortex and hippocampus was significantly decreased. After adjuvant therapy of exogenous BDNF, the number of neurons and the TrkB expression at the mRNA level were significantly increased. Our recent studies have shown that BDNF could improve the proliferation and differentiation of neural stem cells in hippocampus following streptococcus pneumoniae meningitis and enhance its self-repair ability. However, its mechanism is still unclear. We hypothesize that the repair ability of BDNF might be associated with the Notch signaling pathway that is fundamental for neural stem cells development. Thus, in this study, we will construct a neural stem cell culturing model in vitro and a streptococcus pneumonia meningitis animal model in vivo to explore the interaction between BDNF and Notch signaling pathway on regulating neurogenesis in streptococcus pneumoniae meningitis. Our results may provide a new theoretical basis for prevention and treatment of neurological sequelae after streptococcus pneumoniae meningitis.

细菌性脑膜炎存活者中神经系统后遗症发生率仍高达30%-50%，一直是临床难以克服的难题。我们前期研究发现，肺炎链球菌脑膜炎皮层和海马BDNF及其受体TrkB表达升高，而抗生素治疗后，他们表达明显降低，同时存活神经元显著减少。外源性BDNF辅佐治疗后，皮层和海马存活神经元明显增多，TrkBmRNA表达增高。我们最近研究还发现，BDNF能促进肺炎链球菌脑膜炎内源性神经干细胞增殖和分化，提高自身修复能力，但其机制尚不清楚。我们假设BDNF提高神经再生修复能力与神经干细胞的重要调控通路Notch有关。因此，本项目分别通过体外神经干细胞培养和肺炎链球菌脑膜炎模型，研究外源性BDNF与Notch信号通路对神经生发调控的交互作用，阐明BDNF对肺炎链球菌脑膜炎神经生发调控机制，为降低肺炎链球菌脑膜炎后遗症带来更多的契机，以致有效地防治肺炎链球菌脑膜炎神经系统后遗症。

目的：.尽管强有力的抗生素不断应用，肺炎链球菌脑膜炎神经系统后遗症发生仍居高不下。我们研究发现，肺炎链球菌脑膜炎神经系统受损后，内源性神经修复系统存在限制，其原因需进一步探讨。Notch1信号参与调控神经炎症反应，并调控神经损伤后的神经干细胞增殖与分化。肺炎链球菌脑膜炎中Notch通路可能参与了炎症反应与神经再生修复。因此，本研究探讨1.在肺炎链球菌脑膜炎亚急性及慢性期DG区神经干细胞及前体细胞的增殖及分化。2.Notch信号通路在神经干细胞及前体细胞的表达以及在肺炎链球菌脑膜炎中,Notch信号通路的表达变化。3.在肺炎链球菌脑膜炎，Notch信号通路对DG区神经干细胞及前体细胞的增殖及分化的影响。.方法：.采用3周龄SD大鼠，构建肺炎链球菌脑膜炎模型，分别通过免疫荧光双标技术，分别观察脑膜炎7天、14天、28天时海马中EdU 或Ki67及, Nestin, DCX, NeuN, GFAP,和NG2的阳性细胞数目，评估海马中的神经再生及胶质再生。在构建模型后，通过western blot以及免疫荧光技术，分别观察脑膜炎中不同时间点Notch通路的活性及其表达部位。在模型中，分别给予Notch通路激活剂/抑制剂或安慰剂治疗，通过免疫荧光技术，分别观察脑膜炎不同时间点海马中的神经再生及胶质再生。.结果：.第一部分：脑膜炎大鼠海马Edu+/Nestin+、Edu+/NG2+、Edu+/DCX+、Edu+/GFAP+均较对照组增加（p<0.05）。Edu+/MAP2+数在对照组与脑膜炎第14、28天无明显差异（p>0.05）。第二部分：脑膜炎24h后Notch 1信号通路被短暂激活伴反应性胶质增生，Notch通路活性在慢性期下降。第三部分：Jagged 1（Notch 1激活剂）处理组大鼠海马内Nestin、NG2、DCX、GFAP细胞及与Edu或Ki67双标的增殖细胞数较PBS组增加（p<0.05）。IMR-1（Notch 1抑制剂）处理组大鼠海马内Nestin、NG2、DCX细胞数及其与Edu或Ki67双标的增殖细胞数目较DMSO组降低（p<0.05），PBS处理的脑膜炎大鼠分化的Edu&MAP2细胞数明显高于Jagged 1处理的脑膜炎大鼠（p<0.05）。但Edu&MAP2/Edu比例未见明显增加。