本文關(guān)鍵詞： 丙戊酸鈉(VPA) 腦缺血 星形膠質(zhì)細(xì)胞 膠質(zhì)瘢痕 自噬　出處：《蘇州大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:腦缺血發(fā)生后,炎癥反應(yīng)會促使星形膠質(zhì)細(xì)胞反應(yīng)性增生并形成膠質(zhì)瘢痕。膠質(zhì)瘢痕的形成阻礙了損傷區(qū)域新的神經(jīng)環(huán)路的建立,影響中風(fēng)后期神經(jīng)功能的恢復(fù)。有文獻報道丙戊酸鈉(valproate,VPA)對缺血性腦損傷有保護作用,但其對星形膠質(zhì)細(xì)胞反應(yīng)性增生及膠質(zhì)瘢痕的作用尚缺少研究。本課題旨在探索和研究VPA對缺血性腦中風(fēng)誘導(dǎo)的膠質(zhì)瘢痕的作用及其機制。方法:體內(nèi)建立大鼠短暫性大腦中動脈阻塞(transient middle cerebral artery occlusion,tMCAO)模型,每天腹腔注射VPA(250mg/kg),連續(xù)28天,每周進行一次行為學(xué)檢測,28天后斷頭取腦檢測腦萎縮體積;體外建立原代培養(yǎng)新生大鼠皮層星形膠質(zhì)細(xì)胞糖氧剝奪(oxygen-glucose deprivation,OGD)再復(fù)氧模型,在體外模擬膠質(zhì)瘢痕的形成過程;乳酸脫氫酶(lactate dehydrogenase,LDH)和壞死凋亡檢測VPA對OGD誘導(dǎo)的星形膠質(zhì)細(xì)胞損傷的保護作用;Western Blotting和免疫熒光法檢測VPA對膠質(zhì)瘢痕標(biāo)志性蛋白GFAP,neurocan,phosphacan表達的影響以及膠質(zhì)瘢痕與自噬的相互關(guān)系;雷帕霉素和sh RNA ATG5慢病毒轉(zhuǎn)染分別用于激活和抑制自噬。結(jié)果:在大鼠腦缺血再灌注模型中,給予VPA之后,能顯著降低大鼠的腦萎縮體積,改善損傷后期的行為學(xué)癥狀。在星形膠質(zhì)細(xì)胞OGD和OGD再灌注模型上,VPA均可降低星形膠質(zhì)細(xì)胞的LDH漏出率(P?0.05,P?0.01)及減少細(xì)胞壞死。免疫熒光和Western Blotting結(jié)果也顯示原代星形膠質(zhì)細(xì)胞經(jīng)過OGD6h再灌24h處理后膠質(zhì)瘢痕標(biāo)志性蛋白neurocan,phosphacan,GFAP表達增加,給予VPA(1mM)后三者表達均下調(diào)。進一步研究發(fā)現(xiàn),在膠質(zhì)瘢痕形成過程中自噬通路相關(guān)蛋白LC3Ⅱ,p-ULK1,Cathepsin D表達下調(diào)(P0.05,P?0.01),p62上調(diào),給予VPA(1mM)之后,以上蛋白均上調(diào)(p0.05,P?0.01),p62下調(diào)(P?0.01),自噬激活。同時我們用雷帕霉素和shRNA ATG5慢病毒轉(zhuǎn)染分別激活和抑制自噬,結(jié)果證實激活自噬可顯著抑制膠質(zhì)瘢痕形成,阻斷自噬后VPA不能抑制膠質(zhì)瘢痕形成。進一步研究發(fā)現(xiàn),VPA還能上調(diào)膠質(zhì)瘢痕形成過程中acetyl-histone H3,H4和乙酰化ULK1(ac-ULK1)的蛋白水平,提示VPA激活自噬的機制可能與其增加ac-ULK1表達有關(guān)。結(jié)論:長期給予VPA能夠顯著降低tMCAO再灌注模型大鼠腦萎縮體積,對缺血性腦損傷大鼠后期的神經(jīng)功能恢復(fù)有明顯的改善作用;VPA對OGD和OGD再灌注誘導(dǎo)的原代星形膠質(zhì)細(xì)胞損傷具有保護作用;VPA抑制缺血性腦中風(fēng)誘導(dǎo)的膠質(zhì)瘢痕形成,這可能與其上調(diào)acetyl-histone H3,H4和ac-ULK1的表達水平,從而激活自噬有關(guān)。
[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，

本文編號：1541971