發(fā)布時(shí)間：2018-07-12 10:08

  本文選題：阿爾茨海默氏病 + Aβ　； 參考：《山東大學(xué)》2012年博士論文

【摘要】：研究背景和研究目的 阿爾茨海默氏病(Alzheimer disease, AD)是神經(jīng)系統(tǒng)進(jìn)行性變性疾病,是癡呆最常見的病因。臨床上表現(xiàn)為記憶障礙、失語、失用、失認(rèn)、視空間能力損害、抽象思維和計(jì)算力損害、人格和行為的改變等。全球AD患者逐年增多,在發(fā)達(dá)國家老年人群中,已成為導(dǎo)致死亡的第四位原因。AD的發(fā)病機(jī)制迄今不明,亦無特效治療方法。因此明確AD發(fā)病機(jī)制,針對其不同環(huán)節(jié),尋求防治AD的新方法仍是目前亟需解決的重大問題。 AD可見顳、頂及前額葉萎縮。其主要病理特征：老年斑(senile plaques, SPs)、神經(jīng)原纖維纏結(jié)(neurofibrillary tangles, NFTs)、顳葉和海馬皮質(zhì)等部位神經(jīng)元丟失、顆�？张葑冃浴⒀艿矸蹣幼�。其中老年斑是含有β-淀粉樣蛋白(β-amyloid protein, Aβ)、早老素1、早老素2、a1抗糜蛋白酶、載脂蛋白E、α2巨球蛋白和泛素等的細(xì)胞外沉積物,神經(jīng)原纖維纏結(jié)是含有磷酸化tau蛋白(一種微管相關(guān)蛋白)和泛素的細(xì)胞內(nèi)沉積物,SPs、NFTs和神經(jīng)元丟失為AD特征性病理改變。Ap異常聚集是各種原因誘發(fā)AD的共同通路,由此研究Ap對神經(jīng)細(xì)胞的毒性作用和損傷機(jī)制以及阻止和消除Ap沉積已成為研究AD發(fā)病機(jī)制的重要靶點(diǎn)。 自噬(autophagy)是真核細(xì)胞中廣泛存在的降解/再循環(huán)系統(tǒng)。自噬可分為三種主要方式：大自噬、小自噬和分子伴侶介導(dǎo)的自噬,大自噬是最常見的自噬形式。自噬體-溶酶體系統(tǒng)是近年來研究Ap沉積的新方向。在發(fā)病早期的AD患者和8周齡的APP/PS1轉(zhuǎn)基因AD鼠腦內(nèi),均發(fā)現(xiàn)有大量的自噬體存在。細(xì)胞內(nèi)的Aβ可以由細(xì)胞內(nèi)自噬體-溶酶體中的β淀粉樣前體蛋白(β-amyloid precursor protein, APP)裂解產(chǎn)生,細(xì)胞外的Aβ亦可以激活自噬過程清除異常蛋白,但自噬的過度激活亦可導(dǎo)致細(xì)胞死亡。因此自噬在AD發(fā)病機(jī)制中的具體作用仍有待進(jìn)一步研究。 之前的研究發(fā)現(xiàn)一種新型丁內(nèi)酯衍生物3BD0(3-benzyl-5-((2-nitrophenoxy)methyl)-dihydrofuran-2(3H)-one)可以抑制血管內(nèi)皮自噬功能,防止細(xì)胞空泡化,抑制血管內(nèi)皮細(xì)胞凋亡,防止細(xì)胞老化。那么在神經(jīng)細(xì)胞中尤其是AD細(xì)胞模型中,是否也有同樣的作用?是否可以通過調(diào)節(jié)自噬對抗Aβ的毒性、減少Aβ的沉積來治療AD呢? 依據(jù)上述背景,本研究以化學(xué)小分子為工具,干預(yù)了AD細(xì)胞模型的自噬過程,進(jìn)而研究自噬在AD發(fā)病機(jī)制中的作用并尋找參與調(diào)控自噬的關(guān)鍵因子,這有助于明確AD的發(fā)病機(jī)制、尋找新的AD治療靶點(diǎn)和開發(fā)新型靶向藥物。 研究內(nèi)容 1.研究外源性的Aβ25-35對PC12細(xì)胞的毒性作用及是否能夠誘導(dǎo)細(xì)胞自噬。 2.研究3BDO是否可以保護(hù)PC12細(xì)胞拮抗Aβ的細(xì)胞毒性作用及作用機(jī)制。 3.研究3BDO能否抑制AD轉(zhuǎn)基因細(xì)胞模型20E2細(xì)胞Aβ的生成以及其作用機(jī)制。 4.研究3BDO抑制自噬的分子機(jī)制。 研究方法 1.AD細(xì)胞模型的構(gòu)建、培養(yǎng)：PC12細(xì)胞(大鼠嗜鉻神經(jīng)瘤細(xì)胞)和SH-SY5Y細(xì)胞(人神經(jīng)母細(xì)胞瘤細(xì)胞)正常培養(yǎng),加入老化處理的Aβ25-35構(gòu)建AD細(xì)胞模型一；20E2細(xì)胞(HEK293細(xì)胞穩(wěn)定轉(zhuǎn)染瑞典突變APP基因)為AD細(xì)胞模型二。 2.細(xì)胞存活率的檢測：利用SRB方法檢測細(xì)胞存活率。 3.細(xì)胞凋亡的檢測：Hoechest33258染色結(jié)合熒光顯微鏡,觀察細(xì)胞核凝集。 4.細(xì)胞自噬的檢測： 4.1通過吖啶橙染色在熒光顯微鏡下觀察細(xì)胞中酸性膜泡的數(shù)量及分布。 4.2通過檢測LC3-Ⅱ蛋白水平檢測自噬是否被誘導(dǎo)。 5.ROS檢測：利用熒光探針(DCHF)結(jié)合激光掃描共聚焦顯微技術(shù)進(jìn)行。 6. Na+K+-ATP酶活性檢測：按照試劑盒說明檢測Na+K+-ATP酶活性。 7.線粒體膜電位檢測：利用熒光探針(JC-1)結(jié)合激光共聚焦顯微技術(shù)進(jìn)行。 8.細(xì)胞外Aβ1-40、Aβ1-42濃度檢測：利用ELISA方法參照說明書進(jìn)行檢測。 9. FLJ11812的表達(dá)分析：利用反轉(zhuǎn)錄PCR(RT-PCR)和瓊脂糖凝膠電泳相結(jié)合,檢測FLJ11812的水平變化。 10.細(xì)胞蛋白表達(dá)水平檢測：使用western blot法檢測LC3-II, p70s6k, p-p70s6k,p62, APP, IDE, NEP的蛋白水平。 研究結(jié)果 1丁內(nèi)酯衍生物3BDO保護(hù)PC12細(xì)胞拮抗Ap的細(xì)胞毒性作用及其作用機(jī)制 1.1Aβ25-35外源性干預(yù)PC12細(xì)胞可以引起細(xì)胞存活率降低,并呈劑量和時(shí)間依賴性。 1.23BDO預(yù)處理1小時(shí)后可以減少Aβ25-35引起的細(xì)胞存活率的降低,發(fā)揮保護(hù)作用。60μM和120μM的3BDO預(yù)處理1小時(shí)后可以抑制10μM Aβ引起的細(xì)胞存活率的下降。 1.33BDO可以減少Aβ25-35引起的ROS水平的增加。10μM Aβ處理2或4小時(shí)后,細(xì)胞內(nèi)ROS水平較對照組明顯增加,3BDO預(yù)處理1小時(shí)后可以減少Ap引起的細(xì)胞內(nèi)ROS的累積。 1.43BDO可以修復(fù)Aβ25-35引起的Na+K+-ATP酶活性的損傷。10μM Aβ25-35處理細(xì)胞4h后,Na+K+-ATP酶活性受到了抑制。120μM3BDO單獨(dú)處理細(xì)胞時(shí),該酶活性與對照組相比沒有明顯改變。120μM的3BDO預(yù)處理細(xì)胞1h再加入Aβ后,與Ap組相比Na+K+-ATP酶活性明顯上升。 1.53BDO對Aβ25-35引起的細(xì)胞線粒體膜電位的改變沒有明顯影響。10μM Aβ25-35處理細(xì)胞4h后,細(xì)胞線粒體膜電位與對照組相比明顯升高。而3BDO和Aβ共處理組線粒體膜電位與Ap處理組相比沒有明顯恢復(fù)。這表明3BDO對Aβ25-35引起的細(xì)胞線粒體膜電位的改變沒有明顯影響。 1.63BDO拮抗Ap細(xì)胞毒性的機(jī)制研究： 1.6.110μM Aβ25-35、120μM3BDO對細(xì)胞凋亡沒有明顯影響。但是3BDO可以減少Aβ25-35引起的細(xì)胞內(nèi)酸性膜泡的增加。我們利用Hoechst染色法檢測細(xì)胞有無凋亡現(xiàn)象。10μM Aβ25-35、120μM3BDO及共處理組細(xì)胞凋亡與對照組相比沒有明顯差別,說明低濃度的Aβ25-35引起的細(xì)胞存活率的下降并不是由于增加細(xì)胞凋亡引起的。隨后我們利用吖啶橙染色觀察細(xì)胞內(nèi)酸性膜泡的變化來初步鑒定細(xì)胞自噬的變化。10μM Aβ25-35處理細(xì)胞4h后,細(xì)胞內(nèi)酸性膜泡的累積與對照組相比明顯增加,而3BDO預(yù)處理1h后,Aβ引起的細(xì)胞內(nèi)酸性膜泡的累積被抑制了 1.6.23BDO可以減少Aβ25-35引起的LC3-Ⅱ的增加。LC3-Ⅱ已經(jīng)作為一種自噬小體特異性標(biāo)記物被廣泛應(yīng)用于自噬的檢測。為了研究Aβ25-35和3BDO對于自噬小體數(shù)量的影響,我們用Western blot法檢測了細(xì)胞中LC3加工的情況。結(jié)果顯示3BDO能夠顯著地抑制Aβ引起的LC3-Ⅱ的積累。 23BDO抑制AD轉(zhuǎn)基因細(xì)胞模型20E2細(xì)胞外Aβ的生成及其作用機(jī)制的研究 2.13BDO可以減少SH-SY5Y細(xì)胞及20E2細(xì)胞外Aβ1-40的濃度。我們提取含有同一數(shù)量級細(xì)胞的細(xì)胞培養(yǎng)液上清進(jìn)行ELISA檢測。實(shí)驗(yàn)結(jié)果發(fā)現(xiàn),3BDO可以抑制SH-SY5Y細(xì)胞、20E2細(xì)胞Aβ1-40的產(chǎn)生,而對HEK293細(xì)胞沒有明顯影響。 2.23BDO可以降低20E2細(xì)胞外Aβ1-42的水平,而對SH-SY5Y細(xì)胞及HEK293細(xì)胞外Aβ1-42無明顯影響。 2.33BDO對細(xì)胞內(nèi)APP水平無明顯影響。我們利用Western blot方法檢測了細(xì)胞內(nèi)APP水平有無變化。3BDO處理前后20E2和SH-SY5Y細(xì)胞中APP水平?jīng)]有變化,說明3BDO并不是通過降低APP的水平來減少細(xì)胞外Ap的水平。 2.43BDO可以抑制HEK293細(xì)胞、SH-SY5Y細(xì)胞、20E2細(xì)胞LC3-Ⅱ蛋白水平。120μM3BD0處理24小時(shí)后,三種細(xì)胞的LC3-Ⅱ蛋白水平均受到了抑制,20E2細(xì)胞、SH-SY5Y細(xì)胞p62水平升高,進(jìn)一步說明了3BDO抑制了這兩個(gè)細(xì)胞內(nèi)的自噬水平。我們初步推斷3BDO可能通過抑制自噬抑制了其內(nèi)Aβ的生成。 2.53BDO可以升高細(xì)胞內(nèi)胰島素降解酶(IDE)及腎胰島素殘基溶解酶(NEP)水平。120μM3BD0處理24小時(shí)后,20E2細(xì)胞中NEP和IDE含量與對照組相比水平增加,我們推斷3BDO還可能通過增加Aβ降解酶增加Aβ的清除的方式減少Ap的胞外累積。 33BDO抑制自噬的分子機(jī)制研究 3.13BDO是以mTOR依賴的方式調(diào)控Aβ誘導(dǎo)的自噬過程。mTOR介導(dǎo)的信號(hào)通路是自噬過程的經(jīng)典通路,mTOR是其中的關(guān)鍵分子。我們利用Western blot法檢測mTOR下游底物p70S6K的磷酸化水平的變化檢測其活性。同時(shí)利用雷帕霉素作為陽性對照處理細(xì)胞。結(jié)果顯示雷帕霉素及Aβ均可抑制mTOR活性激活自噬,60μM及120μM3BDO可通過激活mTOR活性抑制自噬。 3.23BDO可以升高Aβ引起的SH-SY5Y細(xì)胞內(nèi)FLJ11812基因水平的下降。10μMAβ25-35處理SH-SY5Y細(xì)胞4小時(shí)后,FLJ11812cDNA水平下調(diào),3BDO預(yù)處理1小時(shí)后可以上調(diào)其表達(dá)。說明FLJ11812可能參與了3BDO對抗Aβ毒性的作用機(jī)制中。 3.3過表達(dá)FLJ11812后,細(xì)胞整體自噬水平受到了抑制。在正常實(shí)驗(yàn)組及過表達(dá)FLJ11812的實(shí)驗(yàn)組中,3BDO都可以抑制Aβ引起的LC3-Ⅱ的增加。在過表達(dá)FLJ11812之后細(xì)胞內(nèi)的LC3-Ⅱ水平與對照組相比均減少。在正常實(shí)驗(yàn)組及過表達(dá)FLJ11812的實(shí)驗(yàn)組中,3BDO都可以上調(diào)Aβ引起的p62的降低,進(jìn)一步說明了3BDO可以抑制自噬。實(shí)驗(yàn)結(jié)果說明過表達(dá)FLJ11812可以抑制細(xì)胞內(nèi)LC3-Ⅱ的積累,上調(diào)p62水平,抑制自噬過程。 3.4利用siRNA干擾掉FLJ11812的表達(dá)后,自噬標(biāo)記蛋白LC3-Ⅱ含量無明顯變化。 結(jié)論 1. Aβ可以產(chǎn)生細(xì)胞毒性作用,減少細(xì)胞存活率,呈時(shí)間和劑量依賴性。 2.3BDO可以抑制Ap引起的細(xì)胞存活率的降低,減少Ap引起的細(xì)胞內(nèi)ROS水平的增加,修復(fù)Aβ引起的細(xì)胞內(nèi)Na+K+-ATP酶活性的損傷,但對Ap引起的細(xì)胞線粒體膜電位的改變沒有明顯影響。 3.3BDO可以增加細(xì)胞存活率是通過抑制細(xì)胞自噬而對細(xì)胞凋亡沒有明顯影響。 4.3BDO可以可能通過抑制AD轉(zhuǎn)基因細(xì)胞20E2細(xì)胞自噬水平減少Ap的生成,并且可以抑制20E2細(xì)胞內(nèi)Ap降解酶NEP的表達(dá)增加Aβ的降解,減少細(xì)胞外Aβ1-40、Aβ1-42的產(chǎn)生。 5.3BDO是通過上調(diào)mTOR信號(hào)通路抑制自噬,同時(shí)FLJ11812在3BDO調(diào)控自噬的過程中發(fā)揮了重要作用,3BDO通過上調(diào)FLJ11812的表達(dá)抑制自噬。
[Abstract]:Research background and purpose
Alzheimer disease (AD) is a progressive neurodegenerative disease of the nervous system. It is the most common cause of dementia. It is characterized by memory disorders, aphasia, loss of use, apathy, visual impairment, abstract thinking and computational power impairment, and changes in personality and behavior. The number of AD patients worldwide increased year by year in the elderly population of developed countries. The pathogenesis of.AD, the fourth cause of death, has so far been unknown and there is no special therapeutic method. Therefore, it is still a major problem to be solved to find a new mechanism for the pathogenesis of AD and to seek a new method to prevent and control AD.
AD shows temporal, top and prefrontal atrophy. The main pathological features are senile plaque (senile plaques, SPs), neurofibrillary tangles (neurofibrillary tangles, NFTs), loss of neurons in the temporal and hippocampal cortex, granular vacuolation, and amyloidosis of blood vessels. The senile plaques contain beta amyloid (beta -amyloid protein, A beta), early Elochin 1, proin 2, A1 anti chymotrypsin, apolipoprotein E, alpha 2 giant globulin and ubiquitin and other extracellular sediments, neurofibrillary tangles containing phosphorylated tau protein (a microtubule related protein) and ubiquitin intracellular sediments, SPs, NFTs, and neuronal loss of AD characteristic pathological changes of.Ap abnormal aggregation are various causes inducing AD Therefore, studying the toxicity and damage mechanism of Ap to neurons and preventing and eliminating Ap deposition have become an important target for studying the pathogenesis of AD.
Autophagy (autophagy) is a widespread degradation / recirculation system in eukaryotic cells. Autophagy can be divided into three main ways: autophagy, small autophagy and autophagy mediated by chaperone. Autophagy is the most common form of autophagy. Autophagosome system is a new direction for the study of Ap deposition in recent years. In early onset AD patients and 8 weeks of age A large number of autophagosomes are found in the brain of APP/PS1 transgenic AD mice. The intracellular A beta can be produced by the lysis of beta amyloid precursor protein (beta -amyloid precursor protein, APP) in the autophagosome, and the extracellular A beta can activate autophagy to clear the ISO protein, but the excessive activation of autophagy can also lead to cells. Therefore, the specific role of autophagy in the pathogenesis of AD remains to be further studied.
Previous studies have found that a new butylene derivative, 3BD0 (((2-nitrophenoxy) methyl) -dihydrofuran-2 (3H) -one, can inhibit autophagy, prevent vacuolization of cells, inhibit apoptosis of vascular endothelial cells and prevent cell aging. Then, it is also the same in the neural cells, especially in the AD cell model. Can we treat AD by regulating autophagy against the toxicity of A beta and reducing the deposition of A beta?
Based on the above background, this study used small molecules as a tool to intervene the autophagy process of the AD cell model, and then study the role of autophagy in the pathogenesis of AD and find the key factors involved in the regulation of autophagy. This is helpful to clarify the pathogenesis of AD, to find new targets for the treatment of AD and to develop new target drugs.
research contents
1. to study the toxic effects of exogenous A beta 25-35 on PC12 cells and whether they can induce autophagy.
2. to study whether 3BDO can protect PC12 cells against the cytotoxic effect of A beta and its mechanism.
3. to study whether 3BDO can inhibit the production of A beta in 20E2 cells of AD transgenic cells and its mechanism.
4. study the molecular mechanism of 3BDO inhibition of autophagy.
research method
The construction of 1.AD cell model, culture: normal culture of PC12 cells (rat chromaffin neuroma cells) and SH-SY5Y cells (human neuroblastoma cells), adding the aging treated A beta 25-35 to construct a AD cell model, and 20E2 cells (HEK293 cells stably transfected with Swedish mutant APP base) is a AD cell model of two.
2. detection of cell viability: SRB assay was used to detect cell viability.
3. apoptosis detection: Hoechest33258 staining combined with fluorescence microscopy to observe nuclear agglutination.
4. cell autophagy: detection of autophagy:
4.1 the number and distribution of acid membrane vesicles in the cells were observed under acridine orange staining under fluorescence microscope.
4.2 to detect whether autophagy is induced by detecting the level of LC3- II protein.
5.ROS detection: using fluorescence probe (DCHF) combined with laser scanning confocal microscopy.
6. Na+K+-ATP enzyme activity assay: Na+K+-ATP enzyme activity was detected according to the kit.
7. mitochondrial membrane potential detection: using fluorescence probe (JC-1) combined with laser confocal microscopy.
8. detection of extracellular A beta 1-40 and A beta 1-42 concentration: ELISA method and reference manual for detection.
9. expression analysis of FLJ11812: reverse transcription PCR (RT-PCR) and agarose gel electrophoresis were used to detect the level of FLJ11812.
10. detection of cellular protein expression: the protein levels of LC3-II, P70S6K, p-p70s6k, p62, APP, IDE and NEP were detected by Western blot.
Research results
1 butylene derivative 3BDO protects PC12 cells against Ap cytotoxicity and its mechanism
Exogenous 1.1A beta 25-35 interfered with PC12 cells, which could induce cell viability to decrease in a dose and time dependent manner.
1.23BDO pretreatment could reduce the decrease of cell survival rate caused by A beta 25-35 after 1 hours of pretreatment, and the survival rate of cells induced by 10 u M A beta could be inhibited after 1 hours of 3BDO preconditioning with protective effect of.60 Mu and 120 mu M.
1.33BDO can reduce the level of ROS induced by A beta 25-35. After 2 or 4 hours of.10 - M A beta treatment, the intracellular ROS level is significantly higher than that of the control group. 3BDO preconditioning can reduce the accumulation of ROS in the cells caused by Ap after 1 hours.
When 1.43BDO could repair the Na+K+-ATP enzyme activity induced by A beta 25-35, the activity of.10 mu M A beta 25-35 treated cell 4h, the activity of Na+K+-ATP enzyme was inhibited by.120 micron M3BDO alone, and the activity of the enzyme did not significantly change the activity of.120 micron M pretreated cells, and the activity of the enzyme was obviously higher than that of the control group. Rise.
The mitochondrial membrane potential of cell mitochondrial membrane potential induced by A beta 25-35 was not significantly affected by.10 mu M A beta 25-35 treated cell 4h, and the mitochondrial membrane potential of cell mitochondria was significantly higher than that of the control group. The mitochondrial membrane potential of 3BDO and A beta co processing group was not significantly restored to the Ap treatment group. This indicates that 3BDO has a mitochondrial membrane caused by A beta 25-35. The change of potential was not significantly affected.
The mechanism of 1.63BDO to antagonize the toxicity of Ap cells:
1.6.110 micron M A beta 25-35120 M3BDO has no obvious effect on cell apoptosis. But 3BDO can reduce the increase of intracellular acidic vesicles induced by A beta 25-35. We use Hoechst staining method to detect cell apoptosis in.10 u M A beta 25-35120 micron M3BDO and there is no significant difference in cell apoptosis compared with the control group, indicating low concentration. The decrease of cell survival rate caused by A beta 25-35 was not caused by increasing cell apoptosis. Then we used acridine orange staining to observe the changes in intracellular acid vesicles to preliminarily identify the changes in cell autophagy by.10 mu M A beta 25-35 treatment of cell 4h, the accumulation of intracellular acid vesicles was significantly increased compared with the control group, while 3BDO was predisposed. After 1h, the accumulation of intracellular acidic membrane vesicles induced by A beta was inhibited.
1.6.23BDO can reduce the increase of LC3- II induced by A beta 25-35.LC3- II has been widely used as an autophagic specific marker for autophagy detection. In order to study the effect of A beta 25-35 and 3BDO on the number of autophagic corpuscles, we detected LC3 addition in the cells by Western blot method. The results showed that 3BDO could be significant. Inhibition of the accumulation of LC3- II induced by A beta.
Inhibitory effect of 23BDO on the formation of extracellular 20E2 A in AD transgenic cell lines and its mechanism
2.13BDO can reduce the concentration of A beta 1-40 outside the SH-SY5Y cells and 20E2 cells. We extract the cell culture supernatant containing the same number of cells for ELISA detection. The results showed that 3BDO could inhibit the production of A beta 1-40 in SH-SY5Y cells and 20E2 cells, but had no obvious effect on HEK293 cells.
2.23BDO can reduce the level of extracellular A beta 1-42 in 20E2 cells, but has no significant effect on A cells and SH-SY5Y cells in SH-SY5Y cells and HEK293 cells.
2.33BDO had no obvious effect on the intracellular APP level. We used Western blot to detect the level of APP in cells without change of.3BDO before and after.3BDO treatment. The APP level in 20E2 and SH-SY5Y cells did not change, indicating that 3BDO did not reduce the level of extracellular Ap by reducing APP.
2.43BDO can inhibit HEK293 cells, SH-SY5Y cells, and 20E2 cell LC3- II protein level.120 mu M3BD0 after 24 hours treatment, the level of LC3- II protein of three cells is inhibited, 20E2 cells, SH-SY5Y cells p62 level, further demonstrate that 3BDO inhibits the level of autophagy within the two cells. We preliminarily infer that it may pass inhibition. Autophagy inhibits the formation of A beta in it.
2.53BDO could increase the level of intracellular insulin degrading enzyme (IDE) and renal insulin residue dissolving enzyme (NEP) level for 24 hours. After 24 hours, the content of NEP and IDE in 20E2 cells increased compared with that of the control group. We infer that 3BDO may also reduce the extracellular accumulation of Ap by increasing A beta degrading enzyme and increasing A beta clearance.
Study on the molecular mechanism of inhibition of autophagy by 33BDO
3.13BDO is a mTOR dependent manner to regulate the process of autophagy induced by A beta,.MTOR mediated signaling pathway is the classic pathway of autophagy. MTOR is the key molecule. We use Western blot to detect the activity of the phosphorylation level of p70S6K in the lower reaches of mTOR, and use rapamycin as a positive control treatment. Cells showed that rapamycin and A beta could inhibit mTOR activity and activate autophagy. 60 M and 120 M3BDO could inhibit autophagy by activating mTOR activity.
3.23BDO can increase the level of FLJ11812 gene in SH-SY5Y cells induced by A beta, and.10 u MA beta 25-35 can reduce the level of FLJ11812cDNA after 4 hours of SH-SY5Y cells. 3BDO preconditioning can increase its expression after 1 hours. It indicates that FLJ11812 may be involved in 3BDO antagonism to A beta toxicity.
3.3 after overexpression of FLJ11812, the level of autophagy was inhibited. In the normal experimental group and in the experimental group that overexpressed FLJ11812, 3BDO could inhibit the increase of LC3- II induced by A beta. The level of LC3- II in the cells after overexpression of FLJ11812 was reduced compared with the control group. In the normal experimental group and in the experimental group that overexpressed FLJ11812, 3BDO can all increase the decrease of p62 induced by A beta, and further demonstrate that 3BDO can inhibit autophagy. The results show that overexpression of FLJ11812 can inhibit the accumulation of LC3- II in cells, up-regulate the p62 level and inhibit the autophagy process.
3.4 there was no significant change in the content of autophagy labelled protein LC3- II after interfering with the expression of FLJ11812 by siRNA.
conclusion
1. A beta can produce cytotoxicity and reduce cell viability in a time and dose-dependent manner.
2.3BDO can inhibit the decrease of cell survival rate caused by Ap, reduce the increase of intracellular ROS level caused by Ap and repair the damage of intracellular Na+K+-ATP activity induced by A beta, but it has no significant effect on the changes of mitochondrial membrane potential induced by Ap.
3.3BDO can increase cell survival rate by inhibiting autophagy and has no obvious effect on cell apoptosis.
4.3BDO can reduce the formation of Ap by inhibiting the autophagy level of 20E2 cells of AD transgenic cells, and can inhibit the expression of Ap degrading enzyme NEP in 20E2 cells to increase the degradation of A beta, and reduce the production of A beta 1-40, A beta 1-42.
5.3BDO inhibits autophagy by up-regulating the mTOR signaling pathway, while FLJ11812 plays an important role in 3BDO regulation of autophagy, and 3BDO inhibits autophagy by up regulation of FLJ11812 expression.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R749.16

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相關(guān)期刊論文 前1條

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本文編號(hào)：2116803