【作者】 曹旭；

【导师】 孙圣刚； 褚晓凡；

【作者基本信息】 华中科技大学 ， 神经病学， 2010， 博士

【摘要】 目的观察星形胶质细胞短暂性缺血再灌注损伤过程中胞质和胞核的NF-E2相关因子2(Nrf2)表达变化,分析其核转位情况与细胞氧化损伤水平的相关性。方法用缺血缺氧4h再灌注24h诱导原代培养的大鼠大脑皮质星形胶质细胞损伤,酶标仪检测各时间点细胞活性氧簇(ROS)和谷胱甘肽(GSH)水平,MTT比色法测定细胞存活率,蛋白免疫印迹分析(Western blot)分别检测星形胶质细胞胞质和胞核内Nrf2表达水平。结果随着缺血再灌注时间的延长,细胞ROS逐渐增加,GSH含量和存活率逐渐下降(P<0.05),但再灌注12小时与24小时比较,细胞ROS, GSH和存活率无显著性差异(P>0.05)；缺血再灌注早期(再灌注0.5h),胞浆Nrf2表达减少(P<0.05),胞核Nrf2略增高但无统计学差异,而继续再灌注损伤,胞浆和胞核Nrf2均减少,再灌注12小时与24小时比,胞浆和胞核Nrf2表达无显著性差异(P>0.05)。Pearson相关分析显示短暂性缺血再灌注损伤后,胞浆Nrf2表达量与细胞内ROS呈负相关,与GSH含量和细胞存活率呈正相关。结论Nrf2核转位与短暂性缺血再灌注损伤造成星形胶质细胞氧化应激损伤具有相关性。目的构建RNA干扰真核表达载体,并对原代培养星形胶质细胞进行转染,验证该方法能否有效抑制细胞Nrf2的表达。方法选取特异性针对Nrf2基因的寡核苷酸序列,构建重组载体,用绿色荧光蛋白标记并转染原代培养大鼠星形胶质细胞。通过流式细胞术检测绿色荧光来评估转染效率。利用RT-PCR和western blot技术分别检测转染后星形胶质细胞Nrf2 mRNA和蛋白表达水平改变情况以评估RNA干扰效果。结果构建RNAi真核表达载体并转染星形胶质细胞后,流式细胞术检测提示转染后24h-96h细胞转染率分别为52.19%、59.72%、61.43%、70.61%和62.16%,转染72h瞬时转染效率最高。收获该转染时间点细胞经RT-PCR及western blot显示,siRNA转染可有效抑制星形胶质细胞Nrf2 mRNA和蛋白水平的表达(P<0.05)。结论成功构建Nrf2 RNA干扰表达载体并转染星形胶质细胞能有效抑制细胞Nrf2基因表达,为进一步研究Nrf2在星形胶质细胞脑缺血损伤模型中的作用提供了重要的实验材料。目的探讨1,5-二咖啡酰奎宁酸(1,5-dicaffeoylquinic acid,1,5-diCQA)对星形胶质细胞脑缺血模型的保护作用及其可能的机制。方法用缺糖缺氧再灌注损伤(oxygen and glucose deprivation/reperfusion, OGD/R)处理原代培养星形胶质细胞作为脑缺血的体外模型,实验分为对照组,缺血再灌注组和1,5-diCQA预处理+缺血再灌注组,根据1,5-diCQA预处理的浓度,将后者又分为5,10、20、50、100μmol/L四组。用MTT法测定各组细胞存活率,酶标仪检测细胞ROS含量和GSH水平,western blot检测各组细胞Nrf2蛋白表达水平。为验证1,5-diCQA的保护效应是否依赖于Nrf2核转位,我们又观察了1,5-diCQA预处理对缺血再灌注诱导Nrf2 siRNA组星形胶质细胞损伤的GCL活性、细胞存活率和GSH含量的干预作用。结果星形胶质细胞经缺血再灌注损伤后,与正常对照组相比,细胞存活率下降,ROS生成增多,GSH耗竭(P<0.05)；不同浓度的1,5-diCQA预处理可以减轻缺血再灌注导致的细胞损伤,且在一定范围内具有量-效关系；50μmol/L的1,5-diCQA预处理可以显著增加缺血再灌注星形胶质细胞的Nrf2核转位和GCL活性；上述保护效应不见于Nrf2 siRNA组星形胶质细胞。结论1,5-diCQA能改善缺血再灌注诱导的星形胶质细胞氧化应激损伤和细胞死亡,该效应依赖于1,5-diCQA对Nrf2核转位的促进作用。目的持续性缺糖缺氧(oxygen-glucose deprivation, OGD)处理原代培养星形胶质细胞以模拟脑缺血损伤,观察细胞能量变化及死亡途径并探讨其相关机制。方法将原代星形胶质细胞分为对照组、OGD组、100μmol/L PD150606预处理+OGD组。分别观察各组星形胶质细胞形态学变化,能量代谢变化,胀亡和凋亡百分比,western blot方法观测各组细胞paxillin、vinculin、vimentin及GFAP四种细胞骨架蛋白表达水平。结果电镜显示OGD导致细胞出现凋亡和胀亡；OGD使细胞ATP水平呈时间依赖性下降,而凋亡和胀亡细胞比例增高,OGD处理4小时,ATP水平下降低于正常值的35%,此时胀亡成为星形胶质细胞主要死亡途径；持续OGD使星形胶质细胞paxillin、vinculin、vimentin表达逐渐减少,而GFAP先增多后减少,calpain抑制剂PD150606预处理能减少OGD导致的上述蛋白降解,并延迟细胞胀亡。结论持续性OGD损伤可导致星形胶质细胞凋亡和胀亡；当细胞ATP耗竭到一定阈值(正常值的35%)以下,细胞主要经胀亡途径死亡；calpain调节的细胞骨架蛋白水解参与星形胶质细胞胀亡。更多还原

【Abstract】 Objective To investigate the expression and the effect of NF-E2-related factor2 on oxidative stress in astrocytes induced by ischemia/reperfusion.Methods Oxygen-glucose-deprivation and (OGD)/reperfusion to induce the injury of primary cultured rat astrocytes, and ROS level, cellular GSH content, changes of survival rate and the expression of Nrf2 in cytoplasm and nucleus were observed respectively.Results With the prolongation of OGD/reperfusion, the cellular ROS level was gradually increased, GSH content was delption and the cell viability was decreased. However, at the early stage of injury (reperfusion 0.5h), the expression of Nrf2 was decreased in cytoplasm but increased in nuclear. With the injury continuing, Nrf2 in both cytoplasm and nuclear were decreased. After 12h reperfusion, the expression of Nrf2 showed a tendency toward stabilization.Conclusion The Nrf2 nuclear translocation possessed correlation with the oxidative stress in astrocytes induced by OGD/reperfusion.Objective To identify the effect of Nrf2 siRNA transfaction in astrocytes for the silenceof target gene.Methods The small interfering RNA specific targeted to Nrf2 gene was constructed by chemical synthesis and was transfected into primary culture astrocytes using lipofectamine2000. Transfection efficiency was detected using fluorescence microscopy ans flow cytometry. The expression of Nrf2 mRNA and protein in the tansfected astrocytes was identified by RT-PCR and western blot analysis respectively.Results The eukaryotic expression vector of Nrf2 was constructed and tansfected primary culture astrocytes. The transfection efficiency was 52.19%、59.72%、61.43%、 70.61% and 62.16% respectively in 24,48,72 and 96h. RT-PCR and western blot analysis showed that Nrf2 siRNA can efficiently suppress Nrf2 mRNA and protein expression (P＜0.05).Conclusions Transfect Nrf2 siRNA into astrocytes can effectively inhibite Nrf2 mRNA and protein expression, prepare a condition for subsequent research of medical intervention in ischemia model of astrocytes.Objective To investigate the protective effects of 1,5-diCQA preconditioning on the injury of astrocytes induced by OGD/reperfusion and the related mechanisms.Methods Oxygen-glucose-deprivation and (OGD)/reperfusion to induce the injury of primary cultured rat astrocytes. The cells were divided into:the control group, OGD/reperfusion group,1,5-diCQA+OGD/reperfusion group. The release of LDH, ROS level, cellular GSH content, changes of survival rate and the expression of Nrf2 in cytoplasm and nucleus, glutamate-cysteine ligase (GCL) activity were observed respectively. The indicators mentioned above were also detected in Nrf2 siRNA tansfected astrocytes to verificated whether the protective effects of 1,5-diCQA depend on the activation of Nrf2/ARE pathway.Results 1,5-diCQA pretreatment significantly suppressed cell death, reduced the production of reactive oxygen species, prevented glutathione (GSH) depletion, increased the activity of glutamate-cysteine ligase (GCL) and triggered Nrf2 nuclear translocation in astrocytes induced by 4 h OGD and 24 h reperfusion. Interestingly, these protective effects were greatly attenuated in Nrf2 siRNA-transfected cells.Conclusions 1,5-diCQA has antioxidant signaling properties that upregulate GSH synthesis by stimulating the Nrf2 pathway in astrocytes and protects them from cell death in an in vitro model of ischemia/reperfusion.Objective To study the effect of persistent oxygen-glucose-deprivation (OGD) on the death pathway of primary culture rat astrocytes and the relevant mechanisms.Methods The primary culture rat astrocytes were divided into three groups:①the control group:cells maintained in high glucose DMEM supplemented with 20% fetal calf serum;②the cells treated with OGD;③pretreatment with PD 150606 (100μmol/L) before OGD. the changes of the cellular morphology, the energy metabolism of astrocytes, and the percentages of apoptosis or oncosis of the astrocytes induced by OGD were observed respectively, and Western blot was adoped to assay the protein expression of several cytoskeletal proteins, including paxillin, vinculin, vimentin and GFAP.Results Electron microscopy revealed the coexistence of ultrastructural features of both apoptosis and oncosis in individual cells. The cellular ATP content was gradually decreased (P＜0.05) and the percentages of apoptotic and oncotic cells were increased over the OGD time(P＜0.05). After 4 h OGD, the ATP depletion below 35% of control and oncosis became the main pathway of astrocytes death. OGD led to a significant decrease in paxillin, vinculin, vimentin protein levels in a time-dependent manner (P<0.05) while GFAP appeared markedly more than the control at 0.5 h OGD treatment (P＜0.05), then decreased later. Pre-treatment with 100μmol/L Calpain inhibitor PD150606 led to decrease in the loss of cytoskeleton-associated proteins, and delayed the LDH release of astrocytes associated OGD damage.Conclusion Astrocytes induced by persistent OGD would be go though apoptosis and oncosis, and there was a narrow range of ATP threshold (<35%of the control) that determines astrocytes oncotic death induced by persistent OGD; calpain-mediated hydrolysis of the cytoskeleton-associated proteins may contribute to astrocytes oncosis.更多还原