APP N端片段的神经营养作用

APP是β－淀粉样肽的前体蛋白,由695－770个氨基酸经,春N端水解产物可分沁至细胞外环境。AP具有促进神经细胞生长作用,其中319－335肽段即APP17肽能提高动物的学习记忆能力。其他APP片段是否具有神经营养功能未见报道。本研究通过观察APP N端片段对人神经母细胞瘤株SY5Y生长的影响以及对实验性糖尿病动物行为的影响,希望APP促进神经细胞生长的其他肽段。用化学合肥APP N端多肽片段,以SY5Y细胞MTT代谢率、细胞计数、LDH漏出率和实验性糖尿病小鼠水迷宫试验结果为观察指标。结果APP64肽、29肽、11肽均有促进SY5Y细胞生长的作用,APP11肽可提高糖尿病动物水迷宫测试成绩,说明可溶性APP的N端可能具有神经营养作用,我们认为保持此作用的最短片段为APP11肽,此肽段的发现为进一步研究APP的构效关系奠定了基础。     

表达APPswe_(695)基因细胞系的构建及Aβ分泌水平的分析

为探究β-淀粉样前体蛋白(amyloid-βprecursor protein, APP)在阿尔茨海默病(Alzheimer’s disease, AD)发病过程中的作用及构建应用于AD发病机理研究的实验细胞模型,该研究构建了过表达APP695瑞典型突变体(APPswe_(695))的SH-SY5Y细胞系(APPswe_(695)细胞)并分析了该细胞系中Aβ的分泌水平。采用慢病毒介导转染方法,将APPswe_(695)表达质粒转染至SH-SY5Y细胞,抗性药物筛选阳性转染细胞。分别采用RT-PCR、Western blot技术验证APPswe_(695) mRNA、APP蛋白的表达, ELISA分析Aβ1-40和Aβ1-42的分泌水平。结果显示,转染慢病毒包装的APPswe_(695)质粒后,细胞的APPswe_(695)mRNA表达呈现阳性;与野生型细胞和转染质粒空白细胞相比,转染APPswe_(695)基因细胞表达APP695的瑞典型突变蛋白(APPswe_(695))。APPswe_(695)具有与内源性APP770相同的细胞分布。转染APPswe_(695)基因后,细胞内分泌Aβ水平增加(P0.05)而细胞外液中Aβ含量并没有显著变化。由此说明, APPswe_(695)细胞能够表达被转染的APPswe_(695)基因, APPswe_(695)细胞倾向于产生更多的胞内而不是胞外Aβ,APPswe_(695)细胞可应用于阿尔茨海默病发病机理及药物治疗的研究。     

APP5肽对糖尿病小鼠学习记忆功能与海马神经元蛋白表达的影响

目的研究APP5肽对糖尿病模型小鼠学习记忆能力及海马神经元蛋白表达的影响。方法用链脲佐菌素诱发小鼠糖尿病模型,应用APP5肽（0.0014 mg/kg）皮下注射治疗,5周后进行Morris水迷宫试验;小鼠脑组织海马做Akt、PI3K、P-CREB、Bcl-2、Bax、CytoC免疫组织化学染色;另一部分鼠脑海马,做Bcl-2、Bax抗体蛋白免疫印记。结果（1）水迷宫试验：糖尿病模型小鼠到达站台游动时间比正常对照组延长（P〈0.01）;而APP5肽皮下注射治疗组较DM组动物分别缩短（P〈0.01）。（2）神经免疫组织化学实验和Western blot：给予APP5肽糖尿病小鼠与对照组小鼠海马组织内神经元表达细胞存活相关蛋白及抗凋亡相关蛋白PI3K、Akt、P-CREB、Bcl-2阳性细胞数相似,明显高于糖尿病小鼠（P〈0.01）;APP5肽给予糖尿病小鼠与对照组小鼠表达凋亡蛋白Bax、cytoC阳性细胞数相似,明显少于糖尿病小鼠（P〈0.01）。Western blot结果相同。结论糖尿病小鼠海马神经元表达细胞存活相关蛋白下降,神经元表达细胞凋亡相关蛋白增加,导致其学习记忆能力下降。APP5肽应用可以使上述蛋白恢复到接近正常,从而改善糖尿病小鼠学习记忆能力。     

β-淀粉样肽前体蛋白的结构及生物活性

β－淀粉样肽前体蛋白是ＡＤ患者脑内神经炎斑的主要成分－－β－淀粉样肽的代谢前体。其基因定位于人第２１号染色体,经可变剪接可产生１０种转录物。β－淀粉样肽前体蛋折广泛表达于几乎所有的神经元和非神经元组织,具有一个较长的细胞外肽链、单一跨膜区及一个短的胞内区域。研究表明,它具有神经营养、调节细胞粘附及抑制丝氨酸蛋白酶等多种生物活性。现有资料还提示β－淀粉样肽前体蛋白可能是细胞因子（或其类似物）的受体。     

小肽在原核和真核细胞中抑制α-synuclein异常折叠的研究

目的:研究在大肠杆菌中能够有效抑制α-synuclein异常折叠的小肽,在人神经母细胞瘤SH-SY5Y中的抑制作用.方法:基于α-Syn疏水区域设计的5种透膜小肽在大肠杆菌中能够有效抑制α-synuclein异常折叠;构建真核表达质粒CMV-SynGFP-IRES-Pn并转入SH-SY5Y细胞,通过测定细胞荧光强度分析小肽抑制α-synuclein异常折叠情况,由此评价大肠杆菌筛选系统确定的小肽在真核SH-SY5Y细胞中的有效性.结果:在大肠杆菌中能有效抑制α-Syn(129A)异常折叠的三种小肽R7P1、R7P3、R7P4,在SH-SY5Y中的作用效果也十分明显,分别比对照肽ASIlD提高了114%、98%和98%.对于α-Syn(53T),R7P1和R7P2在SH-SY5Y中分别比对照提高了90%和44%;然而在两种细胞中,5种透膜小肽对野生型α-Syn(WT)的作用与对照相比差距不明显.     

β-淀粉样肽诱导小胶质细胞培养上清致海马神经元损伤模型的建立

目的：建立β淀粉样肽（Aβ1-40）诱导激活小胶质细胞的上清致海马神经元损伤的细胞模型,并初步研究神经元损伤的机制。方法：用不同浓度的可溶性Aβ1-40诱导激活小胶质细胞,光镜下观察不同时间点的细胞形态,ELISA检测其分泌的肿瘤坏死因子仪;用激活后的小胶质细胞条件培养基刺激海马神经元,光镜下观察细胞形态,Western blot检测刺激后海马神经元内诱导型一氧化氮合酶（iNOS）和硝基酪氨酸（NT）的表达水平,ELISA检测海马神经元内胱冬蛋白酶-3（caspase-3）活性来评价神经元的损伤程度。结果：终浓度为10μmol／L的Aβ1-40与小胶质细胞孵育24h后,取上清液加到培养的海马神经元,孵育24-72h,海马神经元较对照组形态有明显变化;经Western blot检测,神经元内iNOS、NT表达明显增加;ELISA检测神经元内caspase-3活性明显增高。结论：小胶质细胞被Aβ1-40激活后,其释放物有明显的致神经元损伤效应,表明建立了神经元损伤模型。     

具有八肽重复区突变的朊蛋白其抗氧化性降低

抗氧化性被认为是细胞朊蛋白的主要生理功能之一,研究显示它的抗氧化性主要与朊蛋白序列中的八肽重复区有关．但是迄今为止它的抗氧化机制仍旧不清楚．我们构建表达了野生型朊蛋白（PrP-PG5）和它的不同八肽重复区突变体0（PrP-PG0）,9（PrP-PG9）和12（PrP-PGl2）．各种原核表达突变体蛋白在H202氧化后出现分子量的增加,并可导致羰基产生．MTT和细胞计数实验显示表达各种突变体的细胞存活率明显低于表达野生型朊蛋白（PrP—PG5）的细胞．细胞内ROS检测发现表达各种突变体的细胞内ROS水平明显高于表达野生型朊蛋白（PrP-PG5）的细胞．此外,谷胱甘肽过氧化物酶检测显示表达野生型朊蛋白（PrP-PG5）的细胞内谷胱甘肽过氧化物酶水平明显高于表达各种突变体的细胞．H2O2处理细胞后,转染突变体的细胞总的羰基产物数量明显高于转染野生型朊蛋白（PrP-PG5）的细胞,而表达突变体细胞及转染空载体的细胞较表达野生型朊蛋白（PrP-PG5）的细胞对氧化物质的抵抗性明显减弱．这些结果提示,具有正确八肽重复区数目对于朊蛋白（PrP）的抗氧化作用起关键作用,PrP的抗氧功能的丢失可能参与家族性朊病毒病的病理过程．     

野菊花总黄酮增强细胞自噬促进β淀粉样蛋白清除

目的探讨野菊花总黄酮(total fl avonoids of Chrysanthemu m,TFC)清除β淀粉样蛋白的作用和分子机制。方法采用CCK-8法观察不同浓度TFC对SH-SY5Y神经细胞株增殖的影响,ELISA法检测TFC对转染过表达淀粉样前体蛋白(APP)和淀粉蛋白前β位分解酶1(BACE1)的CHO细胞培养上清液中Aβ水平的影响,MDC染色检测TFC处理后的神经元内自噬小体的形成,Western blot检测自噬蛋白LC3表达水平。结果 CCK-8法分析显示,TFC对SH-SY5Y神经元活力无明显影响;ELISA检测显示,TFC处理使过表达APP和BACE1的CHO细胞培养上清液中Aβ水平呈浓度依赖性降低;荧光倒置显微镜下观察和Western blot检测发现,TFC处理后SH-SY5Y细胞自噬增加,LC3/LC3自噬标志物蛋白表达增强。结论 TFC可能通过增强细胞自噬而促进Aβ的清除,减少Aβ细胞毒性。     

神经胶质瘤细胞ATF5表达水平对HCMV复制的影响

人巨细胞病毒(human cytomegalovirus,HCMV)在神经胶质瘤细胞中的复制水平不一,其机制尚不清楚。本研究通过下调转录激活因子5(ATF5)在神经胶质瘤细胞中的表达,检测HCMV感染神经胶质瘤细胞后病毒复制水平的变化。首先用HCMV AD169(MOI=5)分别感染U87、SY5Y及A172细胞,观察细胞形态变化,分别在24、48、72、96、120 h取各时间点上清液检测病毒滴度;Real-time PCR检测HCMV即刻早期基因(IE2)、早期基因(UL44)、晚期基因(UL99)及ATF5的表达情况;Western-blot检测病毒基因编码蛋白及ATF5表达的情况。结果显示HCMV在U87、SY5Y细胞中复制水平与病毒在A172细胞中复制水平相比,U87、SY5Y细胞组明显高于A172细胞组(P0.05),ATF5表达在U87、SY5Y细胞组与A172细胞组相比,U87、SY5Y细胞组ATF5表达明显高于A172组(P0.05);利用慢病毒介导的RNA干扰技术下调ATF5在U87、SY5Y细胞的表达,用HCMV感染细胞检测病毒基因及蛋白的表达,结果ATF5表达下调可抑制HCMV的复制(P0.05)。以上结果表明,在胶质瘤细胞中下调ATF5表达水平可以抑制HCMV的复制水平。     

β淀粉样蛋白膜内片段对APP表达的影响

MTT法和荧光分光光度计检测发现β淀粉样蛋白膜内片段(intramembranous fragments of amyloid-β,IF-Aβ)可以抑制β淀粉样蛋白42(amyloid-β,Aβ42)对体外培养神经元的毒性.通过体外检测IF-Aβ对淀粉样前体蛋白(amyloid precursor protein,APP)表达的影响,探索IF-Aβ对Aβ42的神经毒性抑制机制.分别从mRNA水平和蛋白质水平用RT-PCR方法和Western-blot方法检测IF-Aβ对体外培养神经元APP表达的影响.发现IF-Aβ加入原代培养的神经元后,APP从mRNA水平和蛋白质水平均表达下降,说明IF-Aβ通过抑制APP的表达,减少了Aβ生成,达到神经保护的作用.     

Puerarin Protects Human Neuroblastoma SH‐SY5Y Cells against Glutamate‐Induced Oxidative Stress and Mitochondrial Dysfunction

Glutamate, the principal excitatory neurotransmitter, plays a central role in brain metabolism; however, aberrant neurotransmission of glutamate has been linked to neurodegenerative diseases. Therefore, the effective agents that target at glutamate‐induced cell injury will be useful for prevention and treatment of neurodegenerative diseases. In this study, the neuroprotective effect of puerarin, an active isoflavone extracted from the Chinese herb Radix puerariae, against glutamate‐induced cell injury in human neuroblastoma SH‐SY5Y cells was evaluated for the first time. The results showed that the pretreatment of puerarin could attenuate glutamate‐induced cell injury in a dose‐dependent manner. This protective effect was mediated through inhibiting reactive oxygen species generation, attenuating the upregulation of Bax and downregulation of Bcl‐2, preserving mitochondrial membrane potential (MMP), preventing cytochrome c release, and reducing caspase activity. These findings may significantly contribute to a better understanding of the neuroprotective effect of puerarin and provide new insights into its application toward neurodegenerative diseases in the future.     

Prevention of paraquat-induced apoptosis in human neuronal SH-SY5Y cells by lipocalin-type prostaglandin D synthase

Paraquat is a widely used herbicide that is structurally similar to the known dopaminergic neurotoxicant 1-methyl-4-phenyl-pyridine and acts as a potential etiologic factor for the development of Parkinson's disease. In this study, we investigated the protective roles of lipocalin-type prostaglandin (PG) D synthase (L-PGDS) against paraquat-mediated apoptosis of human neuronal SH-SY5Y cells. The treatment of SH-SY5Y cells with paraquat decreased the intracellular GSH level, and enhanced the cell death with elevation of the caspase activities. L-PGDS was expressed in SH-SY5Y cells, and its expression was enhanced with the peak at 2?h after the initiation of the treatment with paraquat. Inhibition of PGD? synthesis and exogenously added PGs showed no effects regarding the paraquat-mediated apoptosis. SiRNA-mediated suppression of L-PGDS expression in the paraquat-treated cells increased the cell death and caspase activities. Moreover, over-expression of L-PGDS suppressed the cell death and caspase activities in the paraquat-treated cells. The results of a promoter-luciferase assay demonstrated that paraquat-mediated elevation of L-PGDS gene expression occurred through the NF-κB element in the proximal promoter region of the L-PGDS gene in SH-SY5Y cells. These results indicate that L-PGDS protected against the apoptosis in the paraquat-treated SH-SY5Y cells through the up-regulation of L-PGDS expression via the NF-κB element. Thus, L-PGDS might potentially serve as an agent for prevention of human neurodegenerative diseases caused by oxidative stress and apoptosis.     

Human TAO kinase 1 induces apoptosis in SH-SY5Y cells

The human TAO kinase 1 (hTAOK1) is a member of the Ste20 group of kinases with the kinase domain located at the N-terminus. The rat homologue, originally named TAO1, has been demonstrated to be highly expressed in brain. In this study, the human TAO kinase 1 was transfected into human neuroblastoma SH-SY5Y cells and its biological effects on the cell morphology were observed by co-expressing the enhanced green fluorescent protein (EGFP). It was found that after 16 h of transfection the cells had shrunk, and finally became rounded when transfected with wild-type or mutant K57A genes encoding either the kinase domain (residues 1-376) or the full-length molecule (residues 1-1001). Thirty-four hours after transfection, cells floated and apoptotic bodies were observed after nuclear staining with DAPI. On the other hand, the cells that were transfected with the gene encoding the C-terminal regulatory region (residues 377-1001) of hTAOK1, appeared to remain unchanged. In order to know the signaling events involved in the above biological phenomena, caspase-3-like activities of the transfected cells were measured in the absence or presence of JNK inhibitor SP600125, in which caspase-3 and JNK (C-jun-N-terminal kinase) are both known to be critical components of the neuronal apoptosis. The results showed that the apoptotic cells exhibited elevated caspase-3-like activity, which could be reduced by SP600125 to some extent. It is concluded that human TAO kinase 1 induces apoptosis in SH-SY5Y cells and the kinase domain is essential, but its catalytic activity seems to be dispensable in this case.     

Flavonoids from Potentilla parvifolia Fisch. and Their Neuroprotective Effects in Human Neuroblastoma SH‐SY5Y Cells in vitro

Potentilla parvifolia Fisch . (Rosaceae) is a traditional medicinal plant in P. R. China. In this study, seven flavonoids, ayanin ( 1 ), tricin ( 2 ), quercetin ( 3 ), tiliroside ( 4 ), miquelianin ( 5 ), isoquercitrin ( 6 ), and astragalin ( 7 ), were separated and purified from ethyl acetate extractive fractions from ethanol extracts of P. parvifolia using a combination of sevaral chromatographic methods. The human neuroblastoma SH‐SY5Y cells were differentiated with all trans‐retinoic acid and treated with okadaic acid to induce tau protein phosphorylation and synaptic atrophy, which could establish an Alzheimer's disease cell model. The neuroprotective effects of these flavonoids in cellular were evaluated in vitro by this cell model. Results from the Western blot and morphology analysis suggested that compounds 3 and 4 had the better neuroprotective effects.     

β‐Methylphenylalanine exerts neuroprotective effects in a Parkinson's disease model by protecting against tyrosine hydroxylase depletion

We evaluated the neuroprotective effects of β‐methylphenylalanine in an experimental model of rotenone‐induced Parkinson's disease (PD) in SH‐SY5Y cells and rats. Cells were pre‐treated with rotenone (2.5 µg/mL) for 24 hours followed by β‐methylphenylalanine (1, 10 and 100 mg/L) for 72 hours. Cell viability, reactive oxygen species (ROS) levels, mitochondrial membrane potential (MMP), mitochondrial fragmentation, apoptosis, and mRNA and protein levels of tyrosine hydroxylase were determined. In a rat model of PD, dopamine (DA) and 3,4‐dihydroxyphenylacetic acid (DOPAC) levels, bradykinesia and tyrosine hydroxylase expression were determined. In rotenone–pre‐treated cells, β‐methylphenylalanine significantly increased cell viability and MMP, whereas ROS levels, apoptosis and fragmented mitochondria were reduced. β‐Methylphenylalanine significantly increased the mRNA and protein levels of tyrosine hydroxylase in SH‐SY5Y cells. In the rotenone‐induced rat model of PD, oral administration of β‐methylphenylalanine recovered DA and DOPAC levels and bradykinesia. β‐Methylphenylalanine significantly increased the protein expression of tyrosine hydroxylase in the striatum and substantia nigra of rats. In addition, in silico molecular docking confirmed binding between tyrosine hydroxylase and β‐methylphenylalanine. Our experimental results show neuroprotective effects of β‐methylphenylalanine via the recovery of mitochondrial damage and protection against the depletion of tyrosine hydroxylase. We propose that β‐methylphenylalanine may be useful in the treatment of PD.     

SH—SY5Y细胞的钙缓冲研究

目的：研究SH－SY5Y神经杂交瘤细胞的钙缓冲能力。方法：通过膜片钳手段,测量未分化的SH－SY5Y细胞钙离子通道电流;并应用显微荧光测量游离钙离子浓度和高钾去极化的方法,研究胞内Ca^2 浓度上升后浓度恢复的动力学过程。结果：未分化的SH－SY5Y细胞存在钙离子通道电流,在刺激时间间隔较短时（＜150s）,胞内钙浓度的恢复过程会由于缓冲机制的饱和而变慢;而时间间隔＞150s时,缓冲物质则可以基本恢复使得胞内钙的恢复过程基本保持不变。结论：钙缓冲蛋白在细胞内钙浓度的调节中起重要作用。     

Proteomics analysis of methylglyoxal-induced neurotoxic effects in SH-SY5Y cells

Reactive carbonyl compounds contribute to aging, Alzheimer's disease (AD) and other neurodegenerative diseases. Among these compounds, methylglyoxal (MG) can yield advanced glycation end products (AGEs), which are crucial in AD pathogenesis. However, the molecular and biochemical mechanisms of MG neurotoxicity are not completely understood. In the present study, SH-SY5Y cells were treated with MG to induce cell death. 2-D Fluorescence Difference Gel Electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry were employed to determine the changes in protein levels in these cells compared with vehicle-treated cells. Proteomics analysis revealed that 49 proteins were differentially expressed in MG-treated SH-SY5Y cells, of which 16 were upregulated and 33 were downregulated. Among them, eight proteins were identified unambiguously. The significant changes in protein levels of actin, immunoglobulin lambda light chain and protein phosphatase 2 were noteworthy given their functional roles in AD pathogenesis. Taken together, our results suggest that multiple pathways are potentially involved in MG-induced neuron death.     

Tau isoforms which contain the domain encoded by exon 6 and their role in neurite elongation

The regulation of tau protein expression during different stages of cellular differentiation and development as well as its functional role in morphogenesis, neurofibrillary tangle formation, and neurodegeneration have been topics of extensive study but have not been completely clarified yet. Tau undergoes complex regulated splicing in the mammalian nervous system. Our previous study with tau exon 6 demonstrated that it shows a splicing regulation profile which is distinct from that of the other tau exons as well as a unique expression pattern which is spatially and temporally regulated. In this study, we investigated the expression, localization, and effects of tau isoforms which contain exon 6 in neuroblastoma cells which stably overexpress them. We found that expression of one particular combination of tau exons (the longest adult isoform plus the domain of exon 6) significantly inhibits neurite elongation.     

The presence of ascorbate induces expression of brain derived neurotrophic factor in SH-SY5Y neuroblastoma cells after peroxide insult, which is associated with increased survival

Oxidative stress and free radical production have been implicated in Alzheimer's disease, where low levels of the antioxidant vitamin C (ascorbate) have been shown to be associated with the disease. In this study, neuroblastoma SH-SY5Y cells were treated with hydrogen peroxide in the presence of ascorbate in order to elucidate the mechanism(s) of protection against oxidative stress afforded by ascorbate. Protein oxidation, glutathione levels, cell viability and the effects on the proteome and its oxidized counterpart were monitored. SH-SY5Y cells treated with ascorbate prior to co-incubation with peroxide showed increased viability in comparison to cells treated with peroxide alone. This dual treatment also caused an increase in protein carbonyl content and a decrease in glutathione levels within the cells. Proteins, extracted from SH-SY5Y cells that were treated with either ascorbate or peroxide alone or with ascorbate prior to peroxide, were separated by two-dimensional gel electrophoresis and analyzed for oxidation. Co-incubation for 24 hours decreased the number of oxidised proteins (e.g. acyl CoA oxidase 3) and induced brain derived neurotrophic factor (BDNF) expression. Enhanced expression of BDNF may contribute to the protective effects of ascorbate against oxidative stress in neuronal cells.