本文選題：阿爾茨海默病 + β淀粉樣蛋白　； 參考：《北京交通大學》2017年碩士論文

【摘要】：目的:阿爾茨海默病(Alzheimer's disease,AD)是一種進行性中樞神經(jīng)系統(tǒng)變性病,其病因及發(fā)病機制尚未闡明。前期工作提示,AD可能與線粒體自噬相關,但是生物學效應和機制均不清楚。本文旨在研究Aβ誘導的線粒體自噬及其生物學效應。方法:(1)質粒的構建。首先從神經(jīng)瘤母細胞(N2a)中提取總RNA,反轉錄聚合酶鏈式反應(reverse transcription polymerase chain reaction,RT-PCR)得到微管相關蛋白 1 輕鏈 3(microtubule-associatedprotein 1 lightchain3,MAP1LC3或LC3)基因,構建pEGFP-C1-LC3和pEGFP-N1-LC3質粒;(2)Aβ誘導線粒體自噬生物學效應的檢測。利用熒光顯微鏡研究Aβ誘導自噬EGFP-LC3熒光斑點的效應,并研究了劑量、時間和Aβ聚集程度的依賴性;Western blot檢測LC3BⅡ/LC3BI 和 p62 水平;通過透射電鏡(transmission electron microscopy,TEM)及免疫電鏡(immunoelectron microscopy,IEM)確定自噬小體的超微結構;(3)線粒體動力相關蛋白1(dynamin-related protein 1,Drp1)調(diào)控Aβ線粒體自噬的研究。以10 μmol/L的Aβ4242處理U87 MG細胞,qPCR確定Drp1的RNA水平變化;Western blot檢測Drp1蛋白表達量的變化;通過siRNA下調(diào)Drp1表達水平,用Western blot檢測Drp1、LC3BⅡ/LC3BⅠ和p62表達量的變化;用TEM觀察線粒體形態(tài)及結構的變化;用MTT檢測伴隨的細胞活力改變的情況。結果:(1)獲得了質粒pEGFP-C1-LC3和pEGFP-N1-LC3克隆,并成功轉染神經(jīng)膠質瘤U87MG細胞;(2)明確了 Aβ誘導線粒體自噬的生物學效應。Aβ處理pEGFP-C1-LC3轉染的U87 MG細胞,熒光顯微鏡檢測發(fā)現(xiàn)Aβ可以誘導細胞出現(xiàn)明顯的LC3熒光斑點,并且具有劑量、時間以及聚集程度的依賴性;在超微結構水平,TEM提示Aβ誘導線粒體自噬的出現(xiàn);經(jīng)3MA阻斷早期線粒體自噬后,與空白對照組相比,LC3BⅡ/LC3BⅠ的比值增加,同時p62的表達水平也增加了,提示3MA在抑制自噬的同時,也抑制了 LC3B的降解;(3)Drp1調(diào)控Aβ42線粒體自噬的機制。首先,qPCR及Western blot結果顯示,Aβ處理細胞后,Drp1在RNA和蛋白水平都是下降的;TEM結果顯示,與空白對照組相比,Aβ42誘導線粒體自噬,線粒體的形態(tài)較大且伴有線粒體損傷(膜損傷、線粒體嵴腫脹等)。其次,當特異性siRNA降低Drp1的表達水平后,發(fā)現(xiàn)LC3BⅡ/LC3BⅠ的比值增加,p62的表達量下降,說明Drp1的下調(diào)促進了自噬通量;最后,MTT結果顯示,與空白對照組相比,Aβ42誘導線粒體自噬對細胞具有毒性作用。結論:本研究確定了 Aβ42誘導線粒體自噬模型條件,并在細胞水平證實了Aβ42誘導線粒體自噬的生物學效應,進而研究了 Drp1與Aβ42誘導的線粒體自噬之間調(diào)控的關系,為進一步探討線粒體自噬在AD病理中的作用機制奠定了基礎。
[Abstract]:Objective: Alzheimer's disease (AD) is a progressive central nervous system disease and its etiology and pathogenesis have not been elucidated.Previous work suggests that AD may be associated with mitochondrial autophagy, but the biological effects and mechanisms are unclear.The aim of this study was to study A 尾 -induced mitochondrial autophagy and its biological effects.Methods the plasmid was constructed.Firstly, total RNAs were extracted from neuroblastoma cells (N2a) and reverse transcription polymerase chain reactions-reverse polymerase chain reaction (RT-PCR) were used to obtain the microtubule-associated protein 1 light chain 3(microtubule-associatedprotein 1 lightchain3MAP1LC3 or LC3) gene. PEGFP-C1-LC3 and pEGFP-N1-LC3 plasmids were constructed to detect the biological effects of mitochondrial autophagy induced by pEGFP-C1-LC3 and pEGFP-N1-LC3 plasmids.The effects of A 尾 -induced fluorescent spots on autophagy EGFP-LC3 were studied by fluorescence microscope. The concentration of LC3B 鈪，

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