脑缺血再灌注诱导的海马齿状回区神经发生相关信号通路的研究

发布时间：2018-06-16 04:39

本文选题：脑缺血 + 神经发生　；参考：《南京医科大学》2009年硕士论文

【摘要】：【研究背景和目的】: 成年哺乳动物脑内存在着神经干细胞,一定条件下可以发生分裂、增殖,称为神经发生(neurogenesis)现象。脑损伤、缺血等病理过程能激发海马齿状回区神经干细胞发生分裂、增殖。研究表明短暂全脑缺血后,产生的新生神经细胞发生沿着一定通路迁移,并与周围的正常细胞建立突触联系发挥部分功能,为中枢神经损伤后神经元的修复提供了可能。因此,探索脑缺血后调控神经发生的细胞内信号通路具有十分重要的意义。 Src是一种钙离子激活的细胞内非受体酪氨酸激酶,在多种组织细胞的增殖、分化、迁移中发挥重要作用。本研究探讨其在脑缺血再灌注诱导的大鼠海马齿状回区神经发生中的调节作用及其具体机制。尼莫地平是一种双氢吡啶类钙拮抗剂,因其能选择性扩张脑血管而被广泛应用于缺血性脑损伤等疾病的治疗,但是否影响神经发生过程,目前报道较少。为此,本实验观察尼莫地平给药后对脑缺血海马齿状回神经发生的影响,并探讨其相关机制。【研究方法】: 1.非受体酪氨酸激酶Src在脑缺血再灌注诱导的大鼠海马齿状回区神经发生中的作用机制研究: 采用四动脉阻断法诱导大鼠全脑缺血,缺血20 min前脑室内注射Src的抑制剂SU6656或ERK的抑制剂U0126;免疫组化Brdu标记法检测脑内海马神经发生;Western Blot法检测海马组织磷酸化Src(p-Src)、Src蛋白的表达,磷酸化Raf(p-Raf)、Raf蛋白的表达,磷酸化ERK(p-ERK)、ERK蛋白的表达,磷酸化CREB(p-CREB)、CREB蛋白的表达.尼氏体染色检测海马神经的细胞损伤。 2.尼莫地平对脑缺血海马齿状回神经发生的影响及其相关机制的研究: 采用四动脉阻断法诱导大鼠全脑缺血,缺血20min前静脉注射尼莫地平或脑室内注射细胞外信号调节激酶ERK的抑制剂U0126;免疫组化Brdu标记法检测脑内海马神经发生;Western Blot法检测海马组织磷酸化ERK(p-ERK)、ERK蛋白的表达。【研究结果】: 1. Src在脑缺血再灌注24小时后大鼠齿状回区持续激活;预先给予Src特异性抑制剂SU6656,能有效的减少再灌注7天后齿状回Brdu阳性细胞的数量,抑制再灌注24小时、72小时齿状回区Raf蛋白在酪氨酸340/341位点的激活以及ERK、CREB蛋白的磷酸化水平;U0126,ERK通路的特异性抑制剂,同样抑制了再灌注7天后齿状回新生细胞的数量,下调了ERK、CREB蛋白磷酸化表达,而不影响Src、Raf的表达。 2.尼莫地平抑制脑缺血再灌注后海马部位的神经发生的同时也抑制了脑缺血再灌注后海马齿状回p-ERK的表达;海马部位神经发生在U0126组与U0126+尼莫地平联合给药组差异无统计学意义。【研究结论】: 1. Src通过调节Raf/ERK/CREB信号通路在脑缺血再灌注后海马区神经发生中发挥正向调节作用。 2.尼莫地平显著抑制脑缺血再灌注后海马齿状回的神经发生,其机制与下调p-ERK表达密切相关。
[Abstract]:Background and objective: there are neural stem cells in the brain of adult mammals, which can divide and proliferate under certain conditions. Brain injury, ischemia and other pathological processes can stimulate the division and proliferation of neural stem cells in dentate gyrus. The results show that after transient global cerebral ischemia, the neonate neurons migrate along a certain pathway, and establish synaptic connections with the surrounding normal cells to play a part of the function, which provides the possibility for the repair of neurons after central nervous system injury. Therefore, it is of great significance to explore the intracellular signal pathway regulating neurogenesis after cerebral ischemia. Src is a calcium activated intracellular tyrosine kinase, which proliferates and differentiates in a variety of histocytes. Migration plays an important role. The aim of this study was to investigate the regulatory effect and its mechanism of neurogenesis in dentate gyrus of rats induced by cerebral ischemia and reperfusion. Nimodipine is a dihydropyridine calcium antagonist, which has been widely used in the treatment of ischemic brain injury due to its selective dilatation of cerebral vessels. Therefore, the effect of nimodipine on the development of dentate gyrus after cerebral ischemia was observed, and the related mechanism was discussed. [study method]: 1. The role of non-receptor tyrosine kinase Src in the neurogenesis of dentate gyrus induced by cerebral ischemia-reperfusion in rats: a four-artery occlusion method was used to induce global cerebral ischemia in rats. SRC inhibitor SU6656 or ERK inhibitor U0126was injected into forebrain for 20 min after ischemia, and the expression of phosphorylated Srcnp-Srcnc-Src protein was detected by immunohistochemical Brdu labeling method, and the expression of RAF protein in hippocampal tissue was detected by immunohistochemical Brdu labeling method. The expression of phosphorylated ERKP-ERK protein and phosphorylated CREBB-CREBN-CREB protein. The neuronal damage of hippocampal nerve was detected by Nissl body staining. 2. Effects of nimodipine on dentate gyrus neurogenesis following cerebral ischemia and its related mechanisms: global cerebral ischemia was induced by four-artery occlusion in rats. Nimodipine was injected intravenously before ischemic 20min or U0126, an inhibitor of extracellular signal regulated kinase (ERK), and the expression of phosphorylated ERK- ERK- ERK- ERK protein in hippocampus was detected by immunohistochemical Brdu labeling method. [research results]: 1. SRC was continuously activated in dentate gyrus of rats 24 hours after cerebral ischemia-reperfusion, and the number of Brdu positive cells in dentate gyrus was reduced after 7 days of reperfusion by pretreatment with SRC-specific inhibitor SU6656. Inhibition of activation of Raf protein in dentate gyrus at site 340 / 341 and phosphorylation level of ERKN CREB protein at site 340 / 341 and specific inhibitor of U0126 ERK pathway at 24 h and 72 h after reperfusion also inhibited the number of nascent cells in dentate gyrus 7 days after reperfusion. The phosphorylation of ERKG CREB protein was down-regulated, but the expression of Srctr Raf was not affected. 2. Nimodipine inhibited the neurogenesis of hippocampus after cerebral ischemia and reperfusion, and also inhibited the expression of p-ERK in dentate gyrus after cerebral ischemia and reperfusion. There was no significant difference between U0126 group and U0126 nimodipine group. [conclusion]: 1. Src plays a positive regulatory role in hippocampal neurogenesis after cerebral ischemia-reperfusion by regulating the Rafr / ERK / CREB signaling pathway. 2. Nimodipine significantly inhibited the neurogenesis of dentate gyrus after cerebral ischemia and reperfusion, and its mechanism was closely related to the down-regulation of p-ERK expression.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R363

【共引文献】