【正文】 月 日 上海交通大學(xué)學(xué)位論文版權(quán)使用授權(quán)書(shū) 本學(xué)位論文作者完全了解學(xué)校有關(guān)保留、使用學(xué)位論文的規(guī)定，同意學(xué) 校保留并向國(guó)家有關(guān)部門(mén)或機(jī)構(gòu)送交論文的復(fù)印件和電子版，允 許論文 被查閱和借閱。10JC1410700） 上海市科委生物醫(yī)藥重點(diǎn)課題資助項(xiàng)目 （項(xiàng)目編號(hào)： 10411954200） 上海交通大學(xué)學(xué)位論文原創(chuàng)性聲明 本人鄭重聲明：所呈交的學(xué)位論文，是 本人在導(dǎo)師的指導(dǎo)下，獨(dú)立進(jìn)行 研究工作所取得的成果。除文中已經(jīng)注明引用的內(nèi)容外，本論文不包含 任何其他個(gè)人或集體已經(jīng)發(fā)表或撰寫(xiě)過(guò)的作品成果。本人授權(quán)上海交通大學(xué)可以將本學(xué)位論文的全部或部分 內(nèi)容編入有關(guān)數(shù)據(jù)庫(kù)進(jìn)行檢索，可以采用影印、縮印或掃描等復(fù)制手段 保存和匯編本學(xué)位論文。 方法 采用線栓法制作小鼠短暫性大腦中動(dòng)脈閉塞 (temporary middle cerebral artery occlusion， tMCAO)模型。在腦缺血 60分鐘后，分別再灌注 1小時(shí)， 3小時(shí)， 6小時(shí)， 12小時(shí)， 24小時(shí)， Western blot檢測(cè)缺血側(cè)皮質(zhì)和紋狀體自噬相 關(guān)蛋白 LC3 I、LC3 II和 Beclin 1的表達(dá)。與對(duì)照組相比， NAC干預(yù)后 LC3 II/ LC3 I 和 Beclin 1表達(dá)趨勢(shì)明顯升高。 關(guān)鍵詞 ： 缺血再灌注損傷，氧化應(yīng)激，自噬， LC3， Beclin 1， NAC II 上海交通大學(xué)碩士學(xué)位論文 THE REGULATION OF OXIDATIVE STREE ON AUTOPHAGY AFTER CEREBRAL ISCHEMIA REPERFUSION INJURY ABSTRACT Objective To discuss the activation of autophagy and changes of earlyphase autophagy in which the mechanisms of reactive oxygen species(ROS) involved. Discover the Antioxidant Nacetyl Lcysteine (NAC) or the autophagy inducer rapamycin in ischemic cerebrovascular disease of the application. Methods The temporary focal cerebral ischemia was induced by occlusion of the left middle cerebral artery by applying a modified intraluminal filament technique. Animals were divided into sham group and ischemia/reperfusion group (ischemia / reperfusion, I / R) (+ saline), the latter divided into temporary middle cerebral artery occlusion (tMCAO) group, tMCAO + NAC group and tMCAO + rapamycin group randomly. tMCAO + NAC group was treated by intraperitoneal injection of NAC after MCAO, then rapamycin was injected intracerebroventricular 20 min before MCAO. After 60 minutes in focal cerebral ischemia, the reperfusion was performed at 1, 3, 6, 12 and 24 h. The expression of LC3 I, LC3 II and Beclin 1 in the cortex and striatum after ischemiareperfusion were determined by immunoblotting analysis at each time point. The level of ROS was detected by staining technique with dihydroethidium (DHE). Effects of antioxidant NacetylLcysteine (NAC) or rapamycin on antophagy were evaluated using fluorescence microscope. The total infarct volume of slices was determined by 2,3,5triphenyltetrazolium chloride(TTC) staining after 24h reperfusion III 上海交通大學(xué)碩士學(xué)位論文 under the conditions of focal cerebral ischemia and durg adminstration Results Compared with the shamoperated group, the expression of Beclin1 and LC3 II/ LC3 I was increased at the point of 1h and 6h after focal ischemia reperfusion respectively, while upregulation treated by NAC. The results of DHE staining were that the level of ROS enhanced at 1h after focal ischemia reperfusion, while down regulated after received injections of NAC. According to TTC staining, the treatment with NAC or rapamycin prevented the expansion of the infarct. Conclusion The degree of autophagy and ROS were upregulated after focal ischemiareperfusion injury, which may be a kind of protection. Mutual influence happended between activation of oxidative stress and autophagy. Antioxidants or rapamycin possibly increased the expression of autophagy to protect neurons and attenuated ischemic neuron injury. KEY WORDS reperfusion injury, oxidative stress, autophagy, LC3,Beclin 1,NAC IV 上海交通大學(xué)碩士學(xué)位論文 目 錄 中文摘要 ....................................................................................................................... Ⅰ 英文摘要 ....................................................................................................................... Ⅲ 縮略語(yǔ)表 ....................................................................................................................... Ⅵ 論文正文 第一部分 : 腦缺血再灌注損傷后自噬相關(guān)蛋白的表達(dá)及調(diào)控機(jī)制 引言 ........................................................................................................................... 1 材 料與方法 ............................................................................................................... 7 實(shí)驗(yàn)結(jié)果 ................................................................................................................. 16 討論 ......................................................................................................................... 23 參考文獻(xiàn) ................................................................................................................. 28 第二部分：局部腦缺血再灌注損傷后 ROS激活及對(duì)自噬調(diào)控 引言 ......................................................................................................................... 31 材料與方法 ............................................................................................................. 36 實(shí)驗(yàn)結(jié)果 ................................................................................................................. 40 討論 ......................................................................................................................... 53 參考文獻(xiàn) ................................................................................................................. 57 附錄 ............................................................................................................................. 60 致謝 .............................................................................................................................71 攻讀學(xué)位期間發(fā)表的學(xué)術(shù)論文目錄 ......................................................................... 72 V 上海交通大學(xué)碩士學(xué)位論文 縮略語(yǔ)表 縮略詞 英文全稱(chēng) 中文全稱(chēng) AMPK 539。嚴(yán)重的后遺癥及很高的死亡率一 直困擾著醫(yī)療界，多年來(lái)缺乏新型的有效治療藥物。凋亡的主要形態(tài)學(xué)特征表現(xiàn)為細(xì)胞皺縮，染色質(zhì) 邊集，核 DNA 降解，殘余的細(xì)胞被吞噬細(xì)胞消化，被稱(chēng)為 Ⅰ 型程序性細(xì)胞死亡。針對(duì)目前自噬檢測(cè)缺乏統(tǒng) 一標(biāo)準(zhǔn)的問(wèn)題，《 Autophagy》雜志在