丙戊酸钠对缺血性脑中风诱导的胶质瘢痕的抑制作用及其机制

发布时间：2018-02-27 08:48

  本文关键词： 丙戊酸钠(VPA) 脑缺血 星形胶质细胞 胶质瘢痕 自噬　出处：《苏州大学》2016年硕士论文　论文类型：学位论文

【摘要】：目的:脑缺血发生后,炎症反应会促使星形胶质细胞反应性增生并形成胶质瘢痕。胶质瘢痕的形成阻碍了损伤区域新的神经环路的建立,影响中风后期神经功能的恢复。有文献报道丙戊酸钠(valproate,VPA)对缺血性脑损伤有保护作用,但其对星形胶质细胞反应性增生及胶质瘢痕的作用尚缺少研究。本课题旨在探索和研究VPA对缺血性脑中风诱导的胶质瘢痕的作用及其机制。方法:体内建立大鼠短暂性大脑中动脉阻塞(transient middle cerebral artery occlusion,tMCAO)模型,每天腹腔注射VPA(250mg/kg),连续28天,每周进行一次行为学检测,28天后断头取脑检测脑萎缩体积;体外建立原代培养新生大鼠皮层星形胶质细胞糖氧剥夺(oxygen-glucose deprivation,OGD)再复氧模型,在体外模拟胶质瘢痕的形成过程;乳酸脱氢酶(lactate dehydrogenase,LDH)和坏死凋亡检测VPA对OGD诱导的星形胶质细胞损伤的保护作用;Western Blotting和免疫荧光法检测VPA对胶质瘢痕标志性蛋白GFAP,neurocan,phosphacan表达的影响以及胶质瘢痕与自噬的相互关系;雷帕霉素和sh RNA ATG5慢病毒转染分别用于激活和抑制自噬。结果:在大鼠脑缺血再灌注模型中,给予VPA之后,能显著降低大鼠的脑萎缩体积,改善损伤后期的行为学症状。在星形胶质细胞OGD和OGD再灌注模型上,VPA均可降低星形胶质细胞的LDH漏出率(P?0.05,P?0.01)及减少细胞坏死。免疫荧光和Western Blotting结果也显示原代星形胶质细胞经过OGD6h再灌24h处理后胶质瘢痕标志性蛋白neurocan,phosphacan,GFAP表达增加,给予VPA(1mM)后三者表达均下调。进一步研究发现,在胶质瘢痕形成过程中自噬通路相关蛋白LC3Ⅱ,p-ULK1,Cathepsin D表达下调(P0.05,P?0.01),p62上调,给予VPA(1mM)之后,以上蛋白均上调(p0.05,P?0.01),p62下调(P?0.01),自噬激活。同时我们用雷帕霉素和shRNA ATG5慢病毒转染分别激活和抑制自噬,结果证实激活自噬可显著抑制胶质瘢痕形成,阻断自噬后VPA不能抑制胶质瘢痕形成。进一步研究发现,VPA还能上调胶质瘢痕形成过程中acetyl-histone H3,H4和乙酰化ULK1(ac-ULK1)的蛋白水平,提示VPA激活自噬的机制可能与其增加ac-ULK1表达有关。结论:长期给予VPA能够显著降低tMCAO再灌注模型大鼠脑萎缩体积,对缺血性脑损伤大鼠后期的神经功能恢复有明显的改善作用;VPA对OGD和OGD再灌注诱导的原代星形胶质细胞损伤具有保护作用;VPA抑制缺血性脑中风诱导的胶质瘢痕形成,这可能与其上调acetyl-histone H3,H4和ac-ULK1的表达水平,从而激活自噬有关。
[Abstract]:Objective: after cerebral ischemia, inflammatory reaction can promote reactive proliferation of astrocytes and form glial scar. The formation of glial scar hinders the establishment of a new neural loop in the injured area. It has been reported that valproate VPA has protective effect on ischemic brain injury. However, the effects of VPA on astrocyte reactive hyperplasia and glial scar were not studied. The purpose of this study was to explore and study the effect of VPA on glial scar induced by ischemic stroke and its mechanism. Methods: the rat model was established in vivo. Transient middle cerebral artery occlusion (MCAO) model, Every day, 250 mg 路kg ~ (-1) of VPA was injected intraperitoneally for 28 days. Brain atrophy volume was measured after 28 days of decapitation. In vitro, oxygen-glucose deprivation oxygen-glucose deprivation (OGD) reoxygenation model of primary cultured rat cortical astrocytes was established. The formation process of glial scar was simulated in vitro. The protective effect of VPA on astrocyte injury induced by OGD was detected by lactate dehydrogenate dehydrogenate (LDHs) and apoptosis. The effects of VPA on the expression of glial scar iconic protein GFAP neurocanine phosphacan and the relationship between glial scar and autophagy were detected by Western Blotting and immunofluorescence. Rapamycin and sh RNA ATG5 lentivirus transfection were used to activate and inhibit autophagy respectively. Results: in rat model of cerebral ischemia-reperfusion, the volume of brain atrophy was significantly decreased after VPA administration. In astrocyte OGD and OGD reperfusion model, VPA can reduce the LDH leakage rate of astrocytes. 0.05,P? The results of immunofluorescence and Western Blotting also showed that the expression of glial scar iconic protein neurocanine phosphacanine (GFAP) was increased after 24 hours of OGD6h reperfusion, and the expression of GFAP was down-regulated after administration of VPA-1mM). During the process of glial scar formation, the expression of autophagy pathway related protein LC3 鈪，

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