【作者】 张丽；

【导师】 洪震；

【作者基本信息】 复旦大学 ， 神经病学， 2014， 博士

【摘要】 研究背景：小胶质细胞活化和炎性细胞因子释放参与缺血／缺氧后神经炎性损伤；microRNA调控大脑发育、介导了认知相关性疾病的发生与发展。目的：血管性认知障碍发生发展过程中,小胶质细胞活化和炎性细胞因子释放是否收受到miRNA的调控。方法：体外研究中,通过氧糖剥夺(OGD)诱导小胶质细胞活化,观察炎性因子和神经毒性介质表达变化；体内研究中,通过BCCAO和4-VO建立缺血模型,评估炎性细胞因子表达变化。然后通过miRNA芯片和表达谱芯片评估缺血/缺氧诱导小胶质细胞活化过程中差异表达miRNA,并构建miRNA与炎性细胞因子调控网络。体外评估miR-181c对炎性细胞因子表达调控作用,寻找并验证miR-181c直接调控靶基因；体内研究中,评估海马CA1区过表达miR-181c是否可延缓或阻断血管性认知障碍的发生发展。结果：体外OGD诱导小胶质细胞活化和炎性细胞因子释放,体内BCCAO 和 4-VO均可诱导海马CA1区小胶质细胞活化和炎性细胞因子过表达／分泌。我们构建了小胶质细胞活化过程中miRNA与炎性细胞因子调控网络,发现许多炎性细胞因子都受到miRNA调控。同时我们证实,缺血/缺氧诱导小胶质细胞miR-181c表达下调,miR-181c可直接调控下游靶基因TNF的表达,或通过调控TLR4及其下游NF-kB及炎性因子通路来调控神经元损伤。大鼠海马CA1区过表达miR-181c可抑制缺血后小胶质细胞活化、炎性细胞因子释放和神经元损伤,进而改善缺血后大鼠认知功能。结论：miRNA参与调控缺血／缺氧后小胶质细胞活化和炎性细胞因子释放。miR-181c通过调控炎性因子通路降低神经损伤,改善认知功能,未来有可能成为血管性认知障碍潜在干预靶点。更多还原

【Abstract】 Background:Post-ischemic microglial activation contributes to neuronal damage through the release of large amounts of proinflammatory cytokines. The involvement of microRNAs (miRNAs) in the pathogenesis of brain and central nervous system-related disorders has been previously studied.Aim:The aim of this study was to evaluate whether the production of proinflammatory cytokines is regulated by miRNAs.Methods:BV-2 and primary rat microglial cells were activated by exposure to oxygen-glucose deprivation (OGD). Cerebral ischemia was induced by the four-vessel occlusion (4-VO) and bilateral common carotid artery occlusion (BCCAO) in rats. Induction of proinflammatory and neurotoxic factors, such as tumor necrosis factor (TNF)-a, interleukin (IL)-1β and nitric oxide (NO), was assessed by ELISA, immunofluorence and Griess assay, respectively. Then the miRNA expression profiles of OGD-activated BV-2 cells were compared with the profiles of resting cells by miRNA microarray. BV-2 and primary rat microglia cells were transfected with miR-181c to evaluate its effects on TNF-a production following OGD. In addition, a luciferase reporter assay was conducted to confirm the direct targets of miR-181c.Results:OGD induced BV-2 microglial activation in vitro, as evidenced by overproduction of TNF-a, IL-1β and NO. Cerebral ischemia induced microglial activation and release of proinflammatory cytokines in the hippocampal CA1 region. We further found that OGD down-regulated miR-181c expression. We discovered that miR-181c could directly target the 3’-untranslated region (3’-UTR) of TNF-α mRNA, and suppress its mRNA and protein expressions. miR-181c could also inhibit NF-κB activation and the downstream production of proinflammatory mediators by suppressing TLR4 expression. Ectopic expression of miR-181c partially protected neurons from cell death caused by OGD-activated microglia. Lentiviral overexpression of miR-181c in CA1 subregion inhibited neuronal apoptosis and microglia activation following ischemia and cognitive impairment.Conclusions:Our data suggest an important role for microRNA in the regulation of microglial activation following ischemia/hypoxia and microglia-mediated neruonal injury, suggesting a novel miR-based intervention strategy for vascular cognitive impairment.更多还原