【作者】 赵建华；

【导师】 孙圣刚；

【作者基本信息】 华中科技大学 ， 神经病学， 2007， 博士

【摘要】 第一部分大鼠局灶脑缺血预处理诱导脑缺血耐受和HSP70的表达目的研究局部脑缺血预处理(ischemic preconditioning[PC])、HSP70的表达和对随后发生的局灶脑缺血的保护作用。方法应用改进的Longa’s法,建立大鼠大脑中动脉阻塞(MCAO)的局部脑缺血预处理诱导脑缺血耐受(ischemic tolerance[IT])模型。MCAO20min作为PC,分别在再灌注12h、1d、3d、5d、7d、14d后造成永久性MCAO(PMCAO)并与未行PC的假手术组对照。检测以下指标: TTC染色测量梗死体积;神经功能缺失评分;HE染色观察组织结构变化;同时应用原位杂交、免疫组织化学染色和RT-PCR、Western blotting观察PC后HSP70表达的变化。每小组动物5只。结果PC组1d、3d、5d、7d与未行PC组比较,PMCAO后24h脑梗死体积明显减小(P<0.01),神经功能缺损明显减轻(P<0.05);PC后不同时间点与假手术组比较缺血区HSP70mRNA(12h、1d、3d、5d)(P<0.01)和蛋白(1d、3d、5d、7d)的表达明显增加(P<0.05)。结论PC可以充分诱导其后1~7d的脑缺血耐受,同时PC诱导HSP70表达,可能与缺血耐受的产生有关。第二部分HSP70在脑缺血预处理诱导脑缺血耐受中的作用和对细胞凋亡的影响目的探讨HSP70蛋白合成在脑缺血预处理(PC)诱导脑缺血耐受(IT)中的作用和对细胞凋亡的影响。方法应用改进的Longa’s法建立大鼠大脑中动脉阻塞(MCAO)的局部脑缺血预处理诱导脑缺血耐受(IT)模型。MCAO20min作为PC, PC后24h给予永久性MCAO(PMCAO),并与未进行PC假手术组比较。免疫印记(Western blotting)法测量PC24h后HSP70的变化,TUNEL法、流式细胞仪(flow cytometry,FCM)检测大鼠前脑皮质细胞的凋亡;同时观察在PC前或PC后PMCAO前给予蛋白合成抑制剂放线菌酮对PC后24h HSP70、PMCAO后24h脑梗死体积、神经功能评分和细胞凋亡的影响。每组动物5只。结果PC组PMCAO后24h脑梗死体积明显减小(P<0.01),神经功能评分降低(P<0.05)、细胞凋亡数和凋亡率降低(P<0.05);PC前给予放线菌酮消除了以上影响,但在PC后较长时间而在PMCAO之前给予则没有以上影响;HSP70在PC后1d明显表达,而在PC前30min给予放线菌酮抑制了HSP70的表达(P<0.05)。结论HSP70的合成在PC诱导脑缺血耐受中发挥重要作用,可能是通过抑制神经细胞凋亡实现的。第三部分HSP70在缺血预处理诱导脑缺血耐受中抑制细胞凋亡的机制目的探讨HSP70在缺血预处理诱导脑缺血耐受中抑制细胞凋亡的机制。方法应用改进的Longa’s法建立大鼠大脑中动脉阻塞(MCAO)局部脑缺血预处理诱导脑缺血耐受(IT)模型。MCAO20min作为PC, PC后24h给予永久性MCAO(PMCAO),并与未进行PC假手术组比较。免疫组织化学和免疫印记(Western blotting)法测量PMCAO24h后c-fos、bcl-2和caspase-3的变化,同时观察在PC前给予蛋白合成抑制剂放线菌酮对c-fos、bcl-2和caspase-3的影响。结果PC-PMCAO组PMCAO后24h c-fos、bcl-2表达增加,caspase-3表达减少,与假手术(SS-PMCAO)组比较均有显著行差异(P<0.05)。PC前给予放线菌酮消除了以上影响。结论HSP70在PC诱导脑缺血耐受中抗神经细胞凋亡的作用可能是通过促进再缺血后c-fos bcl-2的表达、抑制caspase-3的表达实现的。第四部分氧-糖剥夺预处理和HSP70表达在诱导体外培养神经细胞缺血耐受中的作用目的研究氧-糖剥夺预处理和HSP70表达在诱导体外培养神经细胞缺血耐受中的作用方法原代培养的大鼠皮层神经细胞进行非致死性氧-糖剥夺(Oxygen-glucose deprivation[OGD])20min作为预处理(ischemic preconditioning[PC]),分别在PC后12h、24h、48h、72h进行第二次致死性OGD50min,建立体外缺血耐受模型。致死行OGD后24h测定乳酸脱氢酶(lactate dehydrogenase[LDH])检测神经元损伤的程度、台盼兰染色检测神细胞死亡率;RT-PCR、免疫组织化学、Western blotting测定PC后HSP70的表达;同时测定在PC前30min或PC后较长时间而在致死性OGD前应用放线菌酮对神经细胞损伤和在PC前30min应用放线菌酮对HSP70蛋白的影响。结果与致死性OGD组比较PC-OGD组OGD后24h、48h LDH水平明显减低(P<0.05)、神经细胞死亡数减少(P<0.05),同时PC后12h、24h、48h HSP70mRNA的表达增加(P<0.05),24h、48h HSP70蛋白的表达增加(P<0.01),而PC后12h、72h组则无变化。在PC前应用放线菌酮消除了PC-OGD组OGD后24h上述指标的变化,而在PC后较长时间应用放线菌酮对此则无影响。同时PC前应用放线菌酮抑制了HSP70蛋白的表达(P<0.05)。结论20min OGD预处理成功的诱导了体外皮层神经细胞的缺血耐受,这种作用可能与HSP70的高表达有关。第五部分氧-糖剥夺预处理对体外培养大鼠神经细胞凋亡的保护作用及机制目的探讨氧-糖剥夺预处理对体外培养神经细胞凋亡的保护作用及机制方法原代培养的大鼠皮层神经细胞进行非致死行的氧-糖剥夺(Oxygen-glucose deprivation[OGD])20min作为预处理(ischemic preconditioning[PC]),在PC后24h进行第二次OGD50min,建立体外缺血耐受模型。流式细胞仪、TUNEL法,吖啶橙荧光染色测定致死行OGD后24h细胞凋亡率和凋亡细胞数。免疫组织化学和免疫印迹测定致死行OGD后24h bcl-2、caspase-3的表达。结果与对照组相比PC-OGD组致死性OGD后24h神经细胞凋亡百分率和凋亡阳性细胞均减少(P<0.05),同时发现其致死性OGD24h后bcl-2表达增加(P<0.05)而caspase -3表达减少(P<0.05)。结论PC对体外培养大鼠神经细胞凋亡有保护作用,这种作用可能是通过促进随后发生的致死性OGD后bcl-2表达、抑制caspase -3表达实现的。更多还原

【Abstract】 PartⅠProtective effects of focal ischemical preconditioning and HSP70 expression on middle cerebral artery occlusion in ischemic tolerance ratsObjective To investigate protective effect of a short duration focal ischemic preconditioning(PC) and the HSP70 expression after PC on the subsequent permanet middle cerebral artery occlusion( MCAO) in rat brain.Methods Temporary MCAO (20 minutes) was used for PC , various periods of reperfusion (ie, 12 hours and 1,3 5, 7, and 14days) were allowed after PC and before permanent MCAO (PMCAO) (n=5 per group) establish ischemic tolerance (IT) compared with non-PC (sham-surgery) rats (n=5 per group) by Longa’s . Infarct size, neurological deficits were measured after 24hours PMCAO by the triphenyltetrazoliumchloride(TTC) and neurological examination respectively. The hitological damage was evaluated by HE staining. The expression of HSP70 was analyzed by in situ hybridization, RT-PCR and immunohistochemistry, Western blotting at 12hours, 1, 3, 5, 7, and 14 adys after PC or non-PC(sham-surgery) .Results Hemispheric infarct was significantly (P<0.01) reduced only if PC was performed 1 , 3, 5 and 7 days before PMCAO compared with non-PC (sham-surgery) rats. PC significantly (P<0.05) reduced neurological deficits (similar to reductions in infarct size). PC produced no brain injury but did significantly (P<0.01) increased HSP70 gene at 12hours,1 , 3 , and 5days and protein (P<0.05) at 1 , 3 , 5 , and 7days respectively compared with non-PC (sham-surgery) rats.Conclusion The results suggest that PC is a powerful inducer of ischemic brain tolerance as reflected preservation of brain tissue and motor function. The protective effects occur at the PC brain site 1 to 7days after PC. Meanwhile ,we found that PC increase HSP70 gene expression (12hours to 5 day) and HSP70 protein expression (1 to 7 day) after PC, PartⅡExpression of HSP70 protein following focal ischemic preconditioning and neural apoptosis on middle cerebral artery occlusion in ischemic tolerance ratsObjective To systematically evaluate the importance of protein synthesis in ischemic preconditioning( PC )-induced ischemic tolerance ( IT ) and neural apoptosis in ratsMethods Temporary middle cerebral artery occlusion(MCAO) by Longa (20 minutes) was used for PC (ischemic precondioning). 1day of reperfusion was allowed after PC and before permanent MCAO to establish ischemic tolerance (IT) compared with non-PC rats (n=5 per group). Infarct size, neurological deficits were measured 24hours after PMCAO by the triphenyltetrazoliumchloride(TTC) and neurological examination respectively. Samples of brain were taken 24hours after PC for the determination of HSP70 expression by Western blot analysis and the apoptotic neural cells were detected 24hours after PMCAO by TdT-mediated dUTP nick end labeling (TUNEL) techenique and flow cytometry . The effects of the protein synthesis inhibitor (cycloheximide administered just before PC or administered long after PC but just before PMCAO on IT ) were also determined (n=5 per group).Results Hemispheric infarct 24hours after PMCAO was significantly(P<0.01) reduced only if PC was performed 1day, and PC significantly (P<0.05) reduced neurological deficits (similar to reductions in infarct size). The number of TUNEL positive cells and the apoptotic cell percentages of rat neocortex by flow cytometry were also significantly (P<0.05) reduced only if PC was performed. Cycloheximide eliminated (P<0.05) ischemic PC-induced IT effects on brain injury , neurological deficits and the number of TUNEL positive cells apoptotic and cell percentages if administered before PC but not if administered long after PC but before PMCAO. PC produced no brain injury but did increased HSP70 protein at 1 day after PC . Cycloheximide eliminated (P<0.05) the effects if administered just before PC but not if administered long after PC but just before PMCAO on IT.Conclusions PC induces IT that is dependent on de novo protein HSP70 synthesis which suppress the neural apoptosis following severe ischemia.PartⅢMechanism of HSP70 protein following focal ischemical preconditioning suppresing the neural apoptosis following severe ischemia in ischemic tolerance ratsObjective To explore the mechanism of HSP70 protein following focal ischemical preconditioning suppresing the neural apoptosis following severe ischemia.Methods Temporary middle cerebral artery occlusion(MCAO) by Longa (20 minutes) was used for PC (ischemic precondioning). 1d of reperfusion was allowed after PC and before permanent MCAO to establish ischemic tolerance (IT) compared with non-PC (sham-surgery) rats (n=5 per group). Samples of brain were taken 24hours after PMCAO for the determination of c-fos, bcl-2 and caspase-3 expression by immunohistochemistry and Western blot analysis. The effects of the protein synthesis inhibitor (cycloheximide administered just before PC on IT ) were also determined (n=5 per group).Results The expression of c-fos and bcl-2 protein significantly (P<0.05) increased 24hours after PMCAO if PC was performed 1day compared with non-PC. PC rats also exhibited a significant (P<0.05) reduction in caspase-3 protein 24hours after PMCAO. Cycloheximide eliminated (P<0.05) the effects if administered just before PC on ITConclusion HSP70 suppresing the neural apoptosis following severe ischemia on IT is associated with upregulation of the expression of c-fos and bcl-2 protein and reduction of caspase-3 protein in cells in ischemic region. PartⅣNon-lethal oxygen-glucose deprivation-induced to lerance in cultured rat cortical neurones and critical role of HSP70Objective To explore the protective effect of non-lethal oxygen-glucose deprivation(OGD)-induced tolerance in cultured rat cortical neurones and changes in expression of HSP70 after non-lethal OGDMethods Primary cultures of rat cortical neurones were exposed to non-lethal oxygen–glucose deprivation (OGD), i.e. ischaemic preconditioning(PC), for 20 min. At a designated period after the PC stimulus (12, 24, 48, or 72 hours), the cultures were again deprived of lethal OGD for 50 min to establish ischemic tolerance (IT) compared with non-PC. Neuronal injury was assessed by measurement of lactate dehydrogenase (LDH) efflux into the medium 24 hours after lethal OGD. The expression of HSP70 was analyzed by RT-PCR and immunohistochemistry, Western blotting at 12, 24, 48, and 72hours after PC or non-PC(control). In addition, cell death was confirmed in some experiments by the trypan blue exclusion assay. The effects of the protein synthesis inhibitor (cycloheximide administered just before PC on IT ) were also determined.Results All PC stimuli resulted in a significant(P<0.05) decrease in LDH activity and cell death 24h after OGD if PC was performed 24hours or 48hours. Neuroprotection was lost if the time between the preconditioning and severe insult were decreased to 12 hours or increased to 72 hours . PC produced no cell injury but did increased HSP70 protein.Western blot analysis and immunohistochemistry demonstrated that there was significant(P<0.01) difference between HSP70 protein levels at PC 24hours or 48hours but was no significant difference at PC 12 hours or 72 hours and very significant (P<0.05) between HSP70 gene levels at PC 12hours, 24hours or 48hours but was no significant difference at PC 72 hours compared with non-PC respectively.These effects were abolished (P<0.05) by the protein synthesis inhibitor cycloheximide if administered just before PC on IT.Conclusion A non-lethal oxygen–glucose deprivation (OGD) induces ischaemic tolerance in the cultured neurons. The protective effects occur at 24 to 48hours after PC. Meanwhile, we found that the protective effects is dependent de novo protein synthesis HSP70 in the present model system.PartV The protective effect and mechanism of PC suppresing the neural apoptosis on non-lethal oxygen-glucose deprivation-induced tolerance in cultured rat cortical neuronesObjective To explore the protective effect and mechanism of PC suppresing the neural apoptosis following lethal oxygen-glucose deprivation -induced tolerance in cultured rat cortical neurones.Methods Primary cultures of rat cortical neurones were exposed to non-lethal oxygen–glucose deprivation (OGD), i.e. ischaemic preconditioning(PC), for 20 min. At 24 hours after the PC stimulus , the cultures were again deprived of lethal OGD for 50 min to establish ischemic tolerance (IT) compared with non-PC. The apoptotic neural cells were detected 24 hours after lethal OGD by TdT-mediated dUTP nick end labeling techenique (TUNEL),acridine orange staining and flow cytometry .The bcl-2 protein expression,and caspase -3 protein expression were confirmed respectively by immunohistochemistry and Western blot analysis 24 hours after lethal OGD stimuli. The effects of the protein synthesis inhibitor (cycloheximide administered just before PC or administered long after PC but just before lethal OGD on IT ) were also determined.Results The number of TUNEL positive cells and the acridine Orange staining positive cells were significantly (P<0.05) reduced and the apoptotic cells percentages of rat neocortex by flow cytometry were also significantly (P<0.05) reduced only 24 hours after lethal OGD if PC was performed . The bcl-2 protein were significantly (P<0.05) increased 24 hours after lethal OGD compared with non-PC(control) , PC also exhibited a significant (P<0.05) reduction in caspase-3 protein 24 hours after lethal OGD .Conclusion The non-lethal OGD for PC may supprese the neural apoptosis following lethal OGD on IT, which is associated with upregulation of the expression of bcl-2 protein and reduction of caspase-3 protein in the present model system.更多还原