本文選題：雷帕霉素 + 腦缺血��； 參考：《上海交通大學(xué)》2014年博士論文

【摘要】：目的：1.研究腦缺血再灌注后不同時(shí)點(diǎn)線粒體自噬、線粒體氧化應(yīng)激水平、線粒體失功能的動(dòng)態(tài)變化。2.研究雷帕霉素（RAP）預(yù)處理對(duì)腦缺血再灌注后線粒體損傷的影響以及其具體作用機(jī)制。 方法：1.利用線栓法制備MCAO模型，結(jié)合電鏡、LC3免疫組化染色及western-blot定量分析手段，觀察再灌后不同時(shí)點(diǎn)線粒體自噬表達(dá)規(guī)律。分離純化大鼠缺血側(cè)皮層神經(jīng)細(xì)胞線粒體，并檢測(cè)缺血再灌注后不同時(shí)點(diǎn)線粒體MDA、ATP、JC-1、mtDNA水平，觀察腦缺血再灌注后線粒體相關(guān)功能及指標(biāo)的變化。2.通過(guò)缺血前30分鐘給予RAP0.8ng側(cè)腦室注射預(yù)處理，于再灌后24小時(shí)通過(guò)TTC染色、神經(jīng)行為評(píng)分、TUNEL染色來(lái)觀察RAP預(yù)處理的神經(jīng)保護(hù)作用，此外測(cè)定線粒體自噬、線粒體數(shù)目、線粒體MDA、ATP、JC-1等指標(biāo)。3.檢測(cè)RAP預(yù)處理后線粒體beclin1、p62表達(dá)的變化，檢測(cè)細(xì)胞p-Erk1/2表達(dá)的變化，檢測(cè)Cyt-c、Bax在線粒體蛋白和胞漿蛋白的表達(dá)差異。 結(jié)果：1.電鏡觀察到腦缺血再灌注誘導(dǎo)神經(jīng)元自噬及線粒體自噬顯著表達(dá)，western-blot定量分析結(jié)果顯示線粒體自噬在再灌后6小時(shí)即出現(xiàn)，再灌后24小時(shí)達(dá)到高峰，可持續(xù)到再灌后72小時(shí)。缺血再灌注導(dǎo)致線粒體MDA持續(xù)升高，線粒體膜電位持續(xù)下降，mtDNA拷貝數(shù)減少、ATP合成能力下降，但在再灌后24小時(shí)mtDNA拷貝數(shù)、ATP合成能力較再灌注后6小時(shí)有升高（P＞0.05）。2. RAP預(yù)處理組梗死體積、神經(jīng)行為評(píng)分均顯著優(yōu)于對(duì)照組（P＜0.05），RAP預(yù)處理組線粒體MDA水平明顯下降（P＜0.05），，線粒體功能改善（ATP合成能力提高，線粒體膜電位好轉(zhuǎn)，P＜0.01）。3.RAP預(yù)處理后線粒體自噬增加，神經(jīng)元線粒體數(shù)目及mtDNA拷貝數(shù)增加。4.RAP預(yù)處理組線粒體beclin1、p62表達(dá)增加，細(xì)胞p-Erk1/2下調(diào)， Bax線粒體轉(zhuǎn)位下降，細(xì)胞色素C釋放減輕，細(xì)胞凋亡指數(shù)顯著降低（P＜0.01）。 結(jié)論：1、線粒體自噬在腦缺血再灌注后表達(dá)顯著增高，是細(xì)胞對(duì)缺血再灌注氧化應(yīng)激損害的一種適應(yīng)性機(jī)制，2、RAP預(yù)處理對(duì)缺血再灌注有顯著的神經(jīng)保護(hù)作用，該作用與促進(jìn)線粒體自噬相關(guān)。強(qiáng)化的線粒體自噬能夠降低線粒體氧化應(yīng)激損傷，改善線粒體功能、增加線粒體數(shù)量。3.RAP預(yù)處理主要是通過(guò)刺激線粒體上調(diào)表達(dá)beclin1、p62來(lái)激活線粒體自噬，4.RAP預(yù)處理除激活線粒體自噬外還能抑制線粒體凋亡通路，可能與RAP抑制細(xì)胞內(nèi)Erk1/2磷酸化激活相關(guān)。
[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.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R743.3

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