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

【摘要】：目的:阿爾茨海默病(Alzheimer's disease,AD)是一種進(jìn)行性中樞神經(jīng)系統(tǒng)變性病,其病因及發(fā)病機(jī)制尚未闡明。前期工作提示,AD可能與線粒體自噬相關(guān),但是生物學(xué)效應(yīng)和機(jī)制均不清楚。本文旨在研究Aβ誘導(dǎo)的線粒體自噬及其生物學(xué)效應(yīng)。方法:(1)質(zhì)粒的構(gòu)建。首先從神經(jīng)瘤母細(xì)胞(N2a)中提取總RNA,反轉(zhuǎn)錄聚合酶鏈?zhǔn)椒磻?yīng)(reverse transcription polymerase chain reaction,RT-PCR)得到微管相關(guān)蛋白 1 輕鏈 3(microtubule-associatedprotein 1 lightchain3,MAP1LC3或LC3)基因,構(gòu)建pEGFP-C1-LC3和pEGFP-N1-LC3質(zhì)粒;(2)Aβ誘導(dǎo)線粒體自噬生物學(xué)效應(yīng)的檢測(cè)。利用熒光顯微鏡研究Aβ誘導(dǎo)自噬EGFP-LC3熒光斑點(diǎn)的效應(yīng),并研究了劑量、時(shí)間和Aβ聚集程度的依賴性;Western blot檢測(cè)LC3BⅡ/LC3BI 和 p62 水平;通過(guò)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(jié)構(gòu);(3)線粒體動(dòng)力相關(guān)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(xì)胞,qPCR確定Drp1的RNA水平變化;Western blot檢測(cè)Drp1蛋白表達(dá)量的變化;通過(guò)siRNA下調(diào)Drp1表達(dá)水平,用Western blot檢測(cè)Drp1、LC3BⅡ/LC3BⅠ和p62表達(dá)量的變化;用TEM觀察線粒體形態(tài)及結(jié)構(gòu)的變化;用MTT檢測(cè)伴隨的細(xì)胞活力改變的情況。結(jié)果:(1)獲得了質(zhì)粒pEGFP-C1-LC3和pEGFP-N1-LC3克隆,并成功轉(zhuǎn)染神經(jīng)膠質(zhì)瘤U87MG細(xì)胞;(2)明確了 Aβ誘導(dǎo)線粒體自噬的生物學(xué)效應(yīng)。Aβ處理pEGFP-C1-LC3轉(zhuǎn)染的U87 MG細(xì)胞,熒光顯微鏡檢測(cè)發(fā)現(xiàn)Aβ可以誘導(dǎo)細(xì)胞出現(xiàn)明顯的LC3熒光斑點(diǎn),并且具有劑量、時(shí)間以及聚集程度的依賴性;在超微結(jié)構(gòu)水平,TEM提示Aβ誘導(dǎo)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(duì)照組相比,LC3BⅡ/LC3BⅠ的比值增加,同時(shí)p62的表達(dá)水平也增加了,提示3MA在抑制自噬的同時(shí),也抑制了 LC3B的降解;(3)Drp1調(diào)控Aβ42線粒體自噬的機(jī)制。首先,qPCR及Western blot結(jié)果顯示,Aβ處理細(xì)胞后,Drp1在RNA和蛋白水平都是下降的;TEM結(jié)果顯示,與空白對(duì)照組相比,Aβ42誘導(dǎo)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(dāng)特異性siRNA降低Drp1的表達(dá)水平后,發(fā)現(xiàn)LC3BⅡ/LC3BⅠ的比值增加,p62的表達(dá)量下降,說(shuō)明Drp1的下調(diào)促進(jìn)了自噬通量;最后,MTT結(jié)果顯示,與空白對(duì)照組相比,Aβ42誘導(dǎo)線粒體自噬對(duì)細(xì)胞具有毒性作用。結(jié)論:本研究確定了 Aβ42誘導(dǎo)線粒體自噬模型條件,并在細(xì)胞水平證實(shí)了Aβ42誘導(dǎo)線粒體自噬的生物學(xué)效應(yīng),進(jìn)而研究了 Drp1與Aβ42誘導(dǎo)的線粒體自噬之間調(diào)控的關(guān)系,為進(jìn)一步探討線粒體自噬在AD病理中的作用機(jī)制奠定了基礎(chǔ)。
[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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