【作者】 秦真侠；

【导师】 胡应和； 钱卓；

【作者基本信息】 华东师范大学 ， 生理学， 2009， 博士

【摘要】 β淀粉样肽（Aβ）是由42个氨基酸组成的肽(Aβ_1-42),它是其前体蛋白（APP）剪切后的产物。Aβ以一种不可溶解的沉积物形式积聚在阿尔茨海默病（Alzheimer’s disease,AD）患者大脑神经元的周围。增加的Aβ沉积物形式通过诱导神经元的死亡,进而改变AD的病理生理状态包括行为和认知方面的改变。已有报道神经元的细胞凋亡、钙稳态的失调和氧化胁迫与Aβ的神经毒性作用有关。但是有关Aβ神经毒性作用的机制尚未被研究透彻。人成神经瘤母细胞SH-SY5Y细胞是一个较好的神经元模型,它已被广泛用于建立AD模型,本实验利用功能基因组学、蛋白质组学和药理基因组学技术,如高密度基因芯片技术、实时荧光定量PCR技术、二维液相色谱技术、RNAi和免疫印迹技术分析研究Aβ诱导SH-SY5Y细胞凋亡的机制。1.β-淀粉样肽（1-42）诱导SH-SY5Y细胞发生凋亡的基因表达谱分析Aβ_1-42处理SH-SY5Y细胞。使用CCK-8对细胞活力进行测定。利用Hoechst33342和Annexin-Cy5/Calcein标记的方法,对细胞凋亡进行鉴定。利用安捷伦基因芯片进行表达谱分析。结果显示Aβ_1-42以一种浓度依赖性的方式抑制细胞生长,同时能够诱导细胞发生凋亡。芯片分析和实时荧光定量PCR结果显示Aβ_1-42处理细胞后,有46个基因的表达水平发生变化,其中包括Caspase-2、Caspase-3、Bax、TP53和TRAF1等基因。2.β-淀粉样肽（1-42）诱导SH-SY5Y细胞凋亡过程中的蛋白谱表达差异分析为了进一步从蛋白水平分析Aβ_1-42诱导神经元细胞凋亡的机制,本实验利用美国Beckman公司的ProteomeLab^TM PF 2D Protein Fractionation System对Aβ_1-42处理24小时前后的SH-SY5Y细胞进行蛋白表达差异的分析。结果显示有72个蛋白的表达水平发生变化,并利用MALDI-TOF-MS（matrix assisted laserdesorption/ionization time-of-flight mass spectrometry）鉴定出了三个蛋白包括组蛋白、真核翻译起始因子（eIF-5A）和蛋白质二硫键异构酶（PDIA6）。3.Caspase-2和TP53在β-淀粉样肽（1-42）诱导神经瘤母细胞凋亡过程中的作用我们发现在SH-SY5Y细胞中,Aβ_1-42能够诱导Caspase-2和TP53的mRNA表达水平的增加,但是有关这两个与细胞凋亡相关的基因在Aβ_1-42诱导SH-SY5Y细胞凋亡的作用机制尚需进一步研究。本实验利用转染外源Caspase-2或Caspase-2特异性的siRNA分别过表达Caspase-2或降低Caspase-2的表达;同时利用TP53特异性的抑制剂pifithrin-α和它的siRNA分别降低其蛋白活性和表达水平,从而研究Caspase-2和TP53在Aβ_1-42诱导SH-SY5Y细胞凋亡过程中的作用。结果表明,过表达Caspase-2诱导SH-SY5Y细胞凋亡,但并没有增强Aβ_1-42诱导的细胞凋亡;而抑制Caspase-2的表达并不能抑制Aβ_1-42诱导的神经元性细胞凋亡。同时TP53活性被抑制或表达水平被降低也不能抑制Aβ_1-42诱导的神经元性细胞凋亡。Aβ_1-42增加Bax/Bcl-xl的比例和Caspase-3的表达,然而,在降低Caspase-2表达水平的时候,同时将TP53的表达水平降低或者抑制其蛋白活性时,Aβ_1-42诱导的神经元性细胞凋亡能够被完全抑制。以上结果显示,Caspase-2和TP53是Aβ_1-42诱导细胞凋亡过程中的关键调节者。4.突变重组人胰高血糖素样肽-1抑制β淀粉样肽1-42诱导的细胞凋亡治疗阿尔茨海默病的选择的靶点之一是阻止β淀粉样肽（Aβ）引发的一系列的生物化学级联反应的发生。已有研究显示胰高血糖素样肽-1（GLP-1）是肠道分泌的一种内生性的促胰岛素释放肽,它能够与脑中的受体结合并具有神经保护效应。利用定点突变和基因重组的技术,我们得到了人突变重组的GLP-1（mGLP-1）。与天然GLP-1受体激动剂相比,这种突变重组的GLP-1呈现出较长的半衰期。本文旨在研究mGLP-1对β淀粉样肽1-42(Aβ_1-42)细胞毒性的影响并探讨其可能的机制。结果显示:当mGLP-1使用浓度在0.02ng/ml以上时能够促进SH-SY5Y细胞增殖,同时0.1ng/ml mGLP-1和0.5ng/ml的mGLP-1能够降低Aβ_1-42诱导的细胞凋亡。同时发现Aβ_1-42能够明显引发SH-SY5Y细胞内钙库中的钙离子外流,而mGLP-1有助于维持细胞内钙稳态。Aβ_1-42能够显著诱导SH-SY5Y细胞内TP53和Bax的表达,已有报道表明这两个基因参与细胞凋亡过程,而mGLP-1降低Aβ_1-42上调TP53和Bax表达的水平。在SH-SY5Y细胞中mGLP-1能够升高细胞质内cAMP浓度,我们鉴定mGLP-1可能通过与细胞中的GLP-1受体相结合从而产生cAMP,最后发挥作用。5.溶血磷脂酰胆碱对β淀粉样（1-42）肽诱导神经细胞凋亡的影响许多研究发现脂质平衡态的紊乱在AD发生过程中具有显著的影响作用。同时β淀粉样肽能够引起脂质过氧化反应。但是对于脂质过氧化反应产物和Aβ_1-42诱导神经元凋亡之间的关系还有待于研究。本实验的目的是要研究脂质过氧化的一个产物溶血磷脂酰胆碱（lysophosphatidylcholine,LPC）对Aβ_1-42诱导SH-SY5Y细胞死亡的影响。实验结果显示长时间用LPC处理SH-SY5Y细胞能够增强Aβ_1-42的神经毒性作用。Aβ_1-42上调Bax/Bcl-xl的比例,同时增加Caspase-3 mRNA和蛋白水平的表达,Caspase-3的蛋白活性也增加,而TRAF1的mRNA表达水平却被下调。当使用LPC和Aβ_1-42同时处理SH-SY5Y细胞时,LPC能够增强Aβ_1-42对这些基因表达的影响。已有报道LPC是一个孤儿G蛋白偶联受体G2A的一个特异性配体,所以我们研究LPC对SH-SY5Y细胞内钙离子浓度水平的影响。实验结果显示LPC能够增强Aβ_1-42诱导的细胞内钙离子浓度增加的程度。此外,我们发现Aβ_1-42能够显著增加SH-SY5Y细胞内G2A的表达,同时当G2A的表达被特异的siRNA抑制时,LPC对Aβ_1-42的神经毒性的影响也降低。这些实验结果表明LPC可能通过孤儿G蛋白偶联受体的信号途径影响Aβ_1-42诱导的细胞凋亡。更多还原

【Abstract】 Amyloid protein（Aβ）is a 42 amino acid peptide,derived fromβ-amyloid precursor protein that accumulates as an insoluble deposit around neurons in Alzheimer’s disease（AD）.Increased deposition of Aβis thought to contribute to the pathophysiology of AD by inducing neuronal cell death,consequences of which include alterations in AD patients’s behaviour and cognition.It has been reported that neuronal apoptosis,calcium dysregulation and oxidative stress are implicated in Aβneurotoxicity.However,the mechanisms,factors and genes involved in this neurotoxic effect have not been yet clearly identified.In the present study,functional genomics, proteomics and pharmacogenomics approach such as high-density microarray, fluorescence real-time quantitative PCR（QPCR）,two-dimensional liquid chromatography,RNAi and western blotting techniques were applied to analyze the mechanism ofβ-amyloid-induced apoptosis in cultured SH-SY5Y neuroblastoma cells,a well-characterized cell line that has been extensively used as a neuronal model in AD-related research.1.Gene expression profile in Aβ_1-42-treated SH-SY5Y neuroblastoma cells SH-SY5Y cells were treated with Aβ_1-42and cell viability was measured by CCK-8 kit.Apoptosis was detected by Hoechst33342 staining and Annexin V-Cy5/Calcein staining.The gene expression profile has been determined using the Agilent GeneChip Human 1A（V2）.Aβ_1-42inhibited cell growth in dose-dependent manner and induced neuronal apoptosis.The expression of fourty-six genes was altered including Caspase-2,Caspase-3,Bax,TP53 and TRAF1.2.Proteomic analysis of SH-SY5Y exposed to Aβ_1-42In order to provide a more complete picture of mechanism in Aβ_1-42-induced neuronal apoptosis,a proteomic analysis was carried out with Aβ_1-42treatment in SH-SY5Y cells.ProteomeLab^TMPF 2D Protein Fractionation System was used to compare the levels of proteins in cell lysates from SH-SY5Y cells exposed Aβ_1-42 （10μM）for 24h to those in control incubation.Seventy-two proteins were differentially expressed.Three proteins had been identified by matrix assisted laser desorption/ionization time-of-flight mass spectrometry（MALDI-TOF-MS）.Aβ_1-42 exposure of SH-SY5Y cells led to increased histone protein and decreased eIF5A and PDIA6.3.The role of Caspase-2 and TP53 in Aβ_1-42-induced neuronal apoptosis In order to investigate the role of Caspase-2 and TP53 in Aβ_1-42-induced SH-SY5Y cell apoptosis,exogenous Caspase-2 or siRNA for Caspase-2 was transfected into SH-SY5Y cells respectively to overexpress Caspase-2 or inhibit the expression of endogenous Caspase-2;Pifithrin-a was applied to block its transcription activity and siRNA for TP53 was transfected for reducing the TP53 expression. Overexpression of Caspase-2 caused apoptosis and exhibited no enhanced effect on apoptosis by Aβ_1-42.Apoptosis-induced by Aβ_1-42was not affected by reduction of Caspase-2.Apoptosis-induced by Aβ_1-42was also not repressed by pifithrin-αor siRNA for TP53.The ratio of Bax/Bcl-xl and the expression level of Caspase-3 was increased by Aβ_1-42.However,the effects of Aβ_1-42was abolished by pifithrin-αcombined with siRNA-mediated knockdown of Caspase-2,or by siRNA-mediated knockdown of Caspase-2 and TP53,demonstrating that both Caspase-2 and TP53 were required for Aβ_1-42-hiduced apoptosis.4.Mutated recombinant human glucagon-like peptide-1 protects SH-SY5Y cells from apoptosis induced by Aβ_1-42A therapeutic target for AD is to block the cascade reaction induced by Aβ.It has been demonstrated that Glucagon-like peptide-1（GLP-1）,which is an endogenous insulinotropic peptide secreted from the gut,binds to its receptor in the brain and possesses neuroprotective effects.Using site-directed mutagenesis and gene recombination techniques,we generated a mutated recombinant human Glucagon-Like Peptide-1（mGLP-1）which has longer half-life as compared with native GLP-1.This present work aims to examine whether mGLP-1 attenuates Aβ_1-42-mediated cytotoxicity in SH-SY5Y cells and to explore the possible mechanisms.Our data indicate that≥0.02ng/mL of mGLP-1 facilitated cell proliferation and 0.1ng/ml and 0.5ng/ml of mGLP-1 rescued SH-SY5Y cells from Aβ_1-42-induced apoptosis.Moreover,Aβ_1-42treatment dramatically stimulated the release of Ca²⁺from internal calcium stores in SH-SY5Y cells,while mGLP-1 helped to maintain the intracellular Ca²⁺homeostasis.Aβ_1-42also significantly increased the expression level of TP53 and Bax genes which are involved in apoptotic pathways, and mGLP-1 decreasedAβ_1-42-induced up-regulation of TP53 and Bax.Since mGLP-1 treatment elevated cytosolic cAMP concentration in SH-SY5Y cells,we postulate that mGLP-1 may exert its influence via binding to GLP-1 receptors in SH-SY5Y cells and stimulating the production of cAMP.These results suggest that mGLP-1 exhibited neuronal protection properties,and could potentially be a novel therapeutic agent for intervention in Alzheimer’s disease.5.Effects of lysophosphatidylcholine onβ-amyloid-induced neuronal apoptosisDysfunctional lipid homeostasis has a significant causative impact on the initiation and progression of AD.It has been known thatβ-amyloid can induce lipid peroxidation.However,the relationship between the lipid peroxidation and Aβ_1-42 induced neuronal apoptosis remains to be elucidated.We have investigated the effects of lysophosphatidylcholine（LPC）,a product of lipid peroxidation,on Aβ_1-42-induced SH-SY5Y cell apoptosis.Our results showed that long-term exposure of SH-SY5Y cells to LPC augmented the neurotoxicity of Aβ_1-42.Furthermore,the Bax/Bcl-xl ratio and the expression levels as well as the activity of Caspase-3 were elevated,while the expression level of TRAF1 was reduced after LPC treatment.Since LPC was reported as a specific ligand for an orphan G-protein coupled receptor G2A,we investigated LPC-mediated changes of calcium level in SH-SY5Y cells.Our results demonstrated that LPC could enhance Aβ_1-42-induced elevation of intracellular concentration of calcium.Interestingly,Aβ_1-42could significantly increase the expression of G2A in SH-SY5Y cells,while the knockdown of G2A using siRNA could reduce the effects of LPC on Aβ_1-42-induced neurotoxicity.These results suggested that the effects of LPC on Aβ_1-42-induced apoptosis could be through the signal pathways of the orphan G-protein coupled receptor.更多还原