雷帕霉素对脑缺血再灌注后线粒体损伤的影响

发布时间：2018-04-18 16:19

本文选题：雷帕霉素 + 脑缺血　；参考：《上海交通大学》2014年博士论文

【摘要】：目的：1.研究脑缺血再灌注后不同时点线粒体自噬、线粒体氧化应激水平、线粒体失功能的动态变化。2.研究雷帕霉素（RAP）预处理对脑缺血再灌注后线粒体损伤的影响以及其具体作用机制。方法：1.利用线栓法制备MCAO模型，结合电镜、LC3免疫组化染色及western-blot定量分析手段，观察再灌后不同时点线粒体自噬表达规律。分离纯化大鼠缺血侧皮层神经细胞线粒体，并检测缺血再灌注后不同时点线粒体MDA、ATP、JC-1、mtDNA水平，观察脑缺血再灌注后线粒体相关功能及指标的变化。2.通过缺血前30分钟给予RAP0.8ng侧脑室注射预处理，于再灌后24小时通过TTC染色、神经行为评分、TUNEL染色来观察RAP预处理的神经保护作用，此外测定线粒体自噬、线粒体数目、线粒体MDA、ATP、JC-1等指标。3.检测RAP预处理后线粒体beclin1、p62表达的变化，检测细胞p-Erk1/2表达的变化，检测Cyt-c、Bax在线粒体蛋白和胞浆蛋白的表达差异。结果：1.电镜观察到脑缺血再灌注诱导神经元自噬及线粒体自噬显著表达，western-blot定量分析结果显示线粒体自噬在再灌后6小时即出现，再灌后24小时达到高峰，可持续到再灌后72小时。缺血再灌注导致线粒体MDA持续升高，线粒体膜电位持续下降，mtDNA拷贝数减少、ATP合成能力下降，但在再灌后24小时mtDNA拷贝数、ATP合成能力较再灌注后6小时有升高（P＞0.05）。2. RAP预处理组梗死体积、神经行为评分均显著优于对照组（P＜0.05），RAP预处理组线粒体MDA水平明显下降（P＜0.05），，线粒体功能改善（ATP合成能力提高，线粒体膜电位好转，P＜0.01）。3.RAP预处理后线粒体自噬增加，神经元线粒体数目及mtDNA拷贝数增加。4.RAP预处理组线粒体beclin1、p62表达增加，细胞p-Erk1/2下调， Bax线粒体转位下降，细胞色素C释放减轻，细胞凋亡指数显著降低（P＜0.01）。结论：1、线粒体自噬在脑缺血再灌注后表达显著增高，是细胞对缺血再灌注氧化应激损害的一种适应性机制，2、RAP预处理对缺血再灌注有显著的神经保护作用，该作用与促进线粒体自噬相关。强化的线粒体自噬能够降低线粒体氧化应激损伤，改善线粒体功能、增加线粒体数量。3.RAP预处理主要是通过刺激线粒体上调表达beclin1、p62来激活线粒体自噬，4.RAP预处理除激活线粒体自噬外还能抑制线粒体凋亡通路，可能与RAP抑制细胞内Erk1/2磷酸化激活相关。
[Abstract]:Purpose 1.To study the dynamic changes of mitochondrial autophagy, mitochondrial oxidative stress and mitochondrial dysfunction at different time points after cerebral ischemia-reperfusion.To study the effect of rapamycin rapamycin (rapamycin) preconditioning on mitochondrial injury after cerebral ischemia reperfusion and its mechanism.Method 1: 1.The MCAO model was established by the method of thread embolization, and the expression of mitochondrial autophagy at different time points after reperfusion was observed by immunohistochemical staining and western-blot quantitative analysis.The mitochondria of ischemic cortical neurons in rats were isolated and purified, and the level of mitochondrial MDA-ATPase JC-1mtDNA was detected at different time points after ischemia-reperfusion, and the changes of mitochondrial function and indexes after cerebral ischemia-reperfusion were observed.The neuroprotective effects of RAP preconditioning were observed by TTC staining and Tunel staining at 24 hours after reperfusion, 30 minutes before ischemia, 30 minutes before ischemia, and 24 hours after reperfusion, and the number of mitochondria autophagy and mitochondria were measured.Mitochondrial MDAA ATP JC-1.After pretreatment with RAP, the changes of mitochondrial beclin1 p62 expression, p-Erk1/2 expression and the difference of Cyt-cu Bax expression in mitochondria and cytosolic proteins were detected.The result is 1: 1.The results of Western blot quantitative analysis of neuronal autophagy and mitochondrial autophagy induced by cerebral ischemia-reperfusion showed that mitochondrial autophagy appeared at 6 hours after reperfusion and reached its peak at 24 hours after reperfusion and lasted until 72 hours after reperfusion.Ischemia-reperfusion resulted in a continuous increase of mitochondrial MDA and a continuous decrease in mitochondrial membrane potential (mtDNA) copy number. However, 24 hours after reperfusion, the mtDNA copy number and ATP synthesis ability of mitochondria were significantly increased compared with that at 6 hours after reperfusion (P > 0.05).The infarct volume and neurobehavioral score in RAP pretreatment group were significantly higher than those in control group (P < 0.05). The level of mitochondrial MDA decreased significantly (P < 0.05), and the mitochondrial function was improved (P < 0.05), and the mitochondrial autophagy was increased after pretreatment with 0.01).3.RAP (P < 0.05).The number of mitochondria and the number of copies of mtDNA in neurons were increased. In rap pretreatment group, the expression of beclin1 p62 was increased, the expression of p-Erk1/2 was down-regulated, the mitochondrial transposition of Bax was decreased, the release of cytochrome C was alleviated, and the apoptosis index was significantly decreased (P < 0.01).Conclusion the expression of mitochondrial autophagy is significantly increased after cerebral ischemia-reperfusion. It is an adaptive mechanism of cell against oxidative stress injury induced by ischemia-reperfusion injury. Rap preconditioning has a significant neuroprotective effect on ischemia-reperfusion injury.This effect is related to the promotion of mitochondrial autophagy.Enhanced mitochondrial autophagy can reduce mitochondrial oxidative stress damage and improve mitochondrial function.Increasing the number of mitochondria .3.RAP pretreatment can activate mitochondrial autophagy by stimulating the up-regulation of mitochondrial expression beclin1p62. Besides activating mitochondrial autophagy, rap pretreatment can inhibit mitochondrial apoptosis pathway, which may be related to the inhibition of Erk1/2 phosphorylation by RAP.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R743.3

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