多巴胺诱导SH-SY5Y多巴胺能细胞凋亡

目的 动态研究多巴胺对多巴胺能神经细胞的细胞毒作用.方法 用不同浓度的多巴胺处理SH-SYSY细胞,在一定时程内的不同时间点观察细胞活性,各种氧化应激指标.结果 研究发现多巴胺作用于多巴胺能细胞系SH-SY5Y,可导致细胞存活率明显减少,细胞存活率的下降呈时间与浓度依赖;多巴胺处理后,细胞的氧化应激指标OH-自由基、超氧化物岐化酶(SOD)、线粒体膜电位(△ψM)、活性氧(ROS)均有不同程度的改变.结论 本次实验结果提示多巴胺对多巴胺能细胞的毒性是通过氧化应激途径起作用的.     

MPP^＋诱导SHSY5Y细胞凋亡的可能机制

探讨MPP 诱导SHSY5Y细胞凋亡的可能机制。采用流式细胞仪测定SHSY5Y细胞的凋亡率、Bcl 2 /Bax蛋白的表达率、活性氧的生成量 ,免疫细胞化学染色法观察活化型CPP32的表达。结果显示 ,MPP 诱导SHSY5Y细胞凋亡 ,两者间呈明显的量效和时效关系。在凋亡过程中Bcl 2蛋白表达显著降低 ,Bax蛋白表达随之增高。随着MPP 作用浓度的增加和作用时间的延长 ,ROS的生成逐渐增加 ,活化型Caspase 3阳性细胞的百分比也逐渐增加 ,均有明显的量效和时效关系。提示Bcl 2 /Bax蛋白是MPP 诱导SHSY5Y细胞凋亡的重要调节蛋白 ,ROS和CPP32在凋亡过程中可能具有重要作用。     

突变型Notch3(R90C)蛋白调控自噬诱导VSMC细胞凋亡的分子机制

目的探讨Notch 3(R90C)突变蛋白诱导VSMC细胞凋亡机制。方法采用真核细胞转染技术将Flag-Notch 3(WT)及Flag-Notch 3(R90C)质粒转入大鼠主动脉血管平滑肌细胞A10,通过Western blot检测Beclin 1及LC3Ⅱ/LC3Ⅰ;单丹磺酰尸胺(monodansylcadaverin,MDC)染色检测细胞自噬空泡的变化;PI/Annexin-V-FITC检测细胞凋亡率。结果与转染Flag-Notch 3(WT)组相比,过表达Flag-Notch 3(R90C)下调细胞内源性Beclin 1蛋白的表达,LC3Ⅱ/LC3Ⅰ的比值降低。与转染Flag-Notch 3(WT)组相比,过表达Flag-Notch 3(R90C)诱导A10细胞自噬空泡聚集减少。与转染Flag-Notch 3(WT)组相比,过表达Flag-Notch 3(R90C)使A10细胞凋亡率增加。结论 Notch 3(R90C)突变蛋白可通过抑制细胞巨自噬水平诱导血管平滑肌细胞凋亡。     

氧化应激是MPP+诱导SHSY5Y细胞凋亡的可能机制

探讨MPP+诱导SHSY5Y细胞凋亡的可能机制.采用流式细胞仪测定SHSY5Y细胞的凋l亡率、Bcl-2/Bax蛋白的表达率、活性氧的生成量,免疫细胞化学染色法观察活化型CPP32的表达.结果显示,MPP+诱导SHSY5Y细胞凋亡,两者间呈明显的量效和时效关系.在凋亡过程中Bcl-2蛋白表达显著降低,Bax蛋白表达随之增高.随着MPP+作用浓度的增加和作用时间的延长,ROS的生成逐渐增加,活化型Caspase-3阳性细胞的百分比也逐渐增加,均有明显的量效和时效关系.提示Bcl-2/Bax蛋白是MPP+诱导SHSY5Y细胞凋亡的重要调节蛋白,ROS和CPP32在凋亡过程中可能具有重要作用.     

AGEs对SH-SY5Y细胞增殖、凋亡以及阿尔茨海默病相关mRNA的影响

目的 研究晚期糖基化终产物(AGEs)对人神经母细胞瘤SH-SY5Y细胞株增殖、凋亡以及AD相关mRNA的影响。 方法 利用小牛血清白蛋白(BSA)和葡萄糖体外制备BSA-AGEs;将SH-SY5Y细胞与不同浓度BSA-AGEs保温后,MTT法测定SH-SY5Y细胞的增殖率,流式细胞仪测定细胞凋亡和细胞周期的变化,RT-PCR检测细胞中AD相关mRNA的表达水平。 结果 BSA-AGEs对SH-SY5Y细胞增殖有明显抑制作用,明显促进神经元的凋亡,细胞周期被阻滞于G1/G0期,呈药物浓度依赖性。RT-PCR结果表明,经过BSA-AGEs刺激后,IL-6、高迁移率族蛋白B1(HMGB1)、淀粉先质蛋白(APLP1) mRNA表达水平均明显升高。 结论 BSA-AGEs能有效抑制SH-SY5Y细胞的增殖,促进炎症细胞因子产生,诱导细胞凋亡,提示AGEs在AD的发生与发展过程中具有促进作用。     

缺血后处理对SH-SY5Y细胞缺血再灌注模型的保护作用

目的研究缺血后处理对SH-SY5Y细胞缺血再灌注损伤模型的保护作用,并初步探讨其作用机制。方法将SH-SY5Y细胞分为三组：正常组、缺血再灌注组、缺血后处理组,缺血再灌注组予以糖氧剥夺12 h后正常培养,缺血后处理组经糖氧剥夺12 h后予以3个循环的正常培养10 min/糖氧剥夺10 min,正常培养12 h后通过光镜、荧光显微镜以及细胞计数法、流式细胞术、SOD活力检测SH-SY5Y细胞的改变。结果缺血后处理组的SH-SY5Y细胞存活率较缺血再灌注组明显增加而凋亡率明显下降（P〈0.01）,经缺血后处理的SH-SY5Y细胞内SOD的活力较缺血再灌注组增加32.53%（P〈0.05）。结论缺血后处理在细胞离体状态下可以发挥保护作用,能够降低SH-SY5Y细胞缺血再灌注损伤引起的细胞凋亡,提高细胞内SOD的活力。     

Nogo-A在维甲酸诱导SH-SY5Y细胞分化后的表达分布

目的研究Nogo-A在维甲酸(RA)诱导SH-SY5Y细胞分化后的表达及分布。方法采用免疫荧光单标记法和免疫荧光双标记法染色,显微镜观察。结果SH-SY5Y细胞经RA诱导后,细胞生长出较长的突起,具有典型的神经元形态。Nogo-A在未分化的SH-SY5Y细胞中主要分布于胞浆中。经RA诱导后,Nogo-A分布于胞浆及突起中,尤以突起末梢生长锥处免疫阳性强度高。结论Nogo-A在RA诱导SH-SY5Y细胞分化后的轴丘和生长锥处的表达提示其在神经元突起生长过程中可能起到重要作用。     

外源性Bax基因过表达对神经母细胞瘤SH-SY5Y细胞株Smac蛋白表达的影响

目的观察Bcl-2相关X蛋白（Bax）基因过表达对人神经母细胞瘤SH-SY5Y细胞第二个线粒体来源的半胱氨酸酶的激活剂（Smac）蛋白表达的影响。方法采用脂质体Lipofectamine 2000将携带人Bax基因的过表达质粒转入SH-SY5Y细胞（转染组）,同时设空载体组和未转染组。通过G-418筛选后获得转入目的基因的细胞克隆。Hoechst33258染色观察凋亡细胞,免疫印迹法法检测细胞中Bax、Smac蛋白表达,免疫荧光染色检测Bax、Smac蛋白在细胞中的分布。结果转染后用G-418持续筛选2周可获得G418抗性的细胞克隆。Smac主要分布于胞质,Bax主要位于细胞核。转染组SH-SY5Y细胞Bax蛋白表达水平（1.067±0.036）较空载体组（0.436±0.035）和未转染组（0.444±0.046）显著增高（P〈0.05）;转染组Smac蛋白表达水平（1.408±0.044）相比空载体组（0.708±0.021）和未转染组（0.748±0.041）显著增高（P〈0.05）;空载体和未转染组细胞Bax和Snac蛋白表达均无显著差异（P〉0.05）。转染组较未转染组细胞凋亡率显著增加（P〈0.05）。结论外源性Bax基因转入人神经母细胞瘤细胞中并稳定过表达,能够上调Smac蛋白表达,促进细胞凋亡。     

锂剂对SH-SY5Y细胞氧糖剥夺模型的保护作用及其对AIF凋亡通路的影响

目的 研究不同浓度的锂剂对SH-SY5Y细胞氧糖剥夺模型的保护效果,确定有效的浓度,并探讨其对AIF凋亡通路的作用,分析其神经保护作用的机制.方法 以不同浓度的锂剂对制备氧糖剥夺模型的SH-SY5Y细胞进行预处理,观察其对细胞的保护作用.MTT法描绘细胞生长曲线.Annexin V和PI双染,流式细胞仪检测各组细胞凋亡情况.免疫荧光检测凋亡诱导因子(AIF)蛋白的变化.结果 MTT显示不同浓度的锂剂对SH-SY5Y细胞氧糖剥夺模型有较强的细胞保护作用,1mmol/L氯化锂为细胞保护的有效剂量.Annexin V和PI双染,流式细胞仪检测可见锂剂处理组凋亡细胞明显减少.免疫荧光染色可见锂剂处理组AIF蛋白发生核移位减少.结论 锂剂对氧糖剥夺模型的SH-SY5Y细胞具有较强的保护作用,1mmol/L氯化锂为细胞保护的有效剂量,其作用机制之一可能是对AIF凋亡通路的抑制.     

SH-SY5Y细胞的神经分化与神经退行性疾病

SH-SY5Y细胞来源于人骨髓,是神经母细胞瘤细胞系的亚克隆,其形态、生理、生化功能与人体细胞相似,可转化为神经元样细胞,表现出一系列具有高度指导意义的神经生物学特性,且具有可简单快速地大规模扩增、可重复性、低成本维持、与原代神经元的培养相比没有伦理问题等优势,被广泛运用于神经系统疾病的体外实验研究中。SH-SY5Y细胞可以制备成未分化和分化两种形式,通过多种分化方案可使细胞分化为更接近于人体内成熟的神经元样细胞。不同的分化方案可使SH-SY5Y细胞表达不同的神经元表型,且表现出应用于不同疾病病理生理机制研究的优点和局限性,使其更适合研究神经退行性疾病,例如帕金森病、阿尔茨海默病等。该文就SH-SY5Y细胞的神经分化方法及其在神经退行性疾病实验研究中的应用进行综述。     

Biochanin A protects SH-SY5Y cells against isoflurane-induced neurotoxicity by suppressing oxidative stress and apoptosis

Biochanin A (BCA) is a natural organic O-methylated isoflavone with a variety of pharmacological effects, and has been reported to have neuroprotective properties. Here, we explored whether BCA protects neurocytes against isoflurane-induced neurotoxicity and investigated the underlying mechanism. Cell viability was tested by cell counting kit-8 and lactate dehydrogenase release assays. Apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and caspase-3/7 activity assays. Superoxide dismutase (SOD) and catalase (CAT) activities and levels of glutathione (GSH) and malondialdehyde (MDA) were measured to assess oxidative stress. Expression of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase (NQO1) was determined by western blotting. Treatment with BCA significantly attenuated the reduction of cell viability induced by isoflurane in SH-SY5Y cells. In addition, BCA treatment reversed isoflurane-induced SOD and CAT activity reduction, GSH level decline and MDA level increase. Isoflurane-induced apoptosis was also attenuated by treatment with BCA. The increase in nuclear Nrf2, HO-1 and NQO1 expression induced by isoflurane was amplified by treatment with BCA. These inhibitory effects of BCA on isoflurane-induced oxidative stress, viability reduction and cell apoptosis were attenuated in Nrf2 knockdown SH-SY5Y cells. Our findings indicate that BCA protects SH-SY5Y cells against isoflurane-induced neurotoxicity via inducing the Nrf2/ARE pathway.     

The involvement of autophagy and cytoskeletal regulation in TDCIPP-induced SH-SY5Y cell differentiation

Exposure and toxicity to organophosphate-based flame retardants are an increasing health concern. Neurons appear to be particularly vulnerable to the effects of these chemicals. For example, in vitro studies have shown that tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) induces apoptosis and autophagy in neural cells. In the present study, we investigated the cell biological mechanisms of TDCIPP-induced neurotoxicity using undifferentiated human SH-SY5Y neuroblastoma cells as a model. Interestingly, TDCIPP treatment promoted differentiation in SH-SY5Y cells, which displayed various alterations including neurite elongation, an expansion of the numbers of neurite-bearing cells, and an increase in expression of cytoskeletal components normally enriched in neurons. Furthermore, the upregulation of microtubule-associated protein light chain 3, the degradation of p62/sequestosome 1, and the formation of autophagosomes occurred in treated cells, suggesting that TDCIPP exposure induces autophagy. However, pretreatment with the autophagy inhibitor 3-methyladenine suppressed TDCIPP-induced autophagy and reduced expression of the aforementioned cytoskeletal components. This correlated with a reduction in neurite outgrowth and numbers of neurite-bearing cells. Taken together, these results indicate that autophagy might promote TDCIPP-induced SH-SY5Y cell differentiation, which leads to an increase in expression of cytoskeletal components and neurite outgrowth. This study offers key insights into the mechanisms of neurotoxicity associated with this commonly used organophosphate.     

SH-SY5Y神经细胞APP高表达对胆碱酯酶活性影响

目的研究类阿尔茨海默病(AD)神经细胞模型中APP异常代谢下胆碱酯酶活性变化,探讨AD的发病机制。方法采用脂质体转染法将含有瑞典家族性AD双突变的淀粉样蛋白前体蛋白APP670/671基因(APPSWE)转染至神经母细胞瘤细胞(SH-SY5Y)中;利用实时荧光定量PCR方法和Western blotting的方法测定APPmRNA、可溶性APP(αAPPs)和总APP蛋白表达水平;采用改进的Ellman比色法测定细胞培养基中乙酰胆碱酯酶(Acetylcholinesterase,AChE)及丁酰胆碱酯酶(Butyrylcholinesterase,BuChE)活性。结果转染APPSWE质粒的SH-SY5Y细胞APP总mRNA和蛋白表达水平显著升高,αAPPs分泌量下降。转染组SH-SY5Y细胞与对照组相比,AChE和BuChE活性都升高。结论体外神经细胞实验证明APP蛋白过度表达能升高AChE和BuChE的活性,其机制可能与APP蛋白代谢紊乱使Aβ升高及下调αAPPs分泌有关。     

Potent blocking action of lead on voltage-activated calcium channels in human neuroblastoma cells SH-SY5Y

Human neuroblastoma cells (SH-SY5Y) have two types of voltage-activated calcium channels, which are equivalent to the N and L types. Both types of calcium channels were equally blocked by lead in a concentration-dependent and reversible manner, with Ki congruent to 1 microM. This lead concentration is in the same order of magnitude as that found in the blood of children exhibiting neuropsychological disorders. Sodium and potassium channel currents were not significantly affected by lead at a concentration of 10 microM.     

A link between monoamine oxidase-A and apoptosis in serum deprived human SH-SY5Y neuroblastoma cells

Summary Increased monoamine oxidase (MAO) activity was recently shown to accompany apoptotic cell death of various neuronal cells following growth factor deprivation. Here we show that in serum deprived SH-SY5Y cells, MAO-A mRNA levels and catalytic activities are increased, linked with activation of the apoptotic executioner caspase-3. Importantly, specific inhibition of MAO-A activity resulted in loss of apoptotic cell morphology. Our study indicates that MAO catalytic activity is involved in apoptotic signalling in response to serum withdrawal in neuronal cells.     

Enhancement of Arsenic Trioxide (As2O3)- Mediated Apoptosis Using Berberine in Human Neuroblastoma SH-SY5Y Cells

Objective

Arsenic trioxide (As₂O₃) has been used as an anticancer agent in traditional Chinese medicine for thousand years and berberine is an isoquinoline alkaloid present that has indicated significant antimicrobial activity. We have examined the combined anticancer effects of As₂O₃ and berberine against the human neuroblastoma (HNB) SH-SY5Y cells in vitro, and to elucidate underlying molecular mechanism.

Methods

HNB SH-SY5Y cells were treated with 2 µM As₂O₃ and 75 µg/ml berberine, and their survival, cell death mechanism as well as synergistic cytotoxic effects were estimated by using MTT assay, DAPI staining, agarose gel electrophoresis, flow cytometric analysis, and western blot analysis.

Results

The combined treatment of two drugs also markedly decreased cell viability. The cytotoxic effects of two drugs were revealed as apoptosis characterized by chromatin condensation, DNA fragmentation, and the loss of mitochondrial membrane potential. The apoptotic cytotoxicity was accompanied by activation of caspase-3 protease as well as decreased the expression of Bcl-2, Bid, and Bcl-x/L. In addition, the cells treated with combination of two drugs also showed significantly increased intracellular reactive oxygen species levels and lipid peroxidation compared to cells As₂O₃ or berberine only.

Conclusion

Combined treatment of As₂O₃ with berberine induced activation of apoptotic signaling pathways in HNB SH-SY5Y cells. These results suggest that the possibility of the combined treatment of two chemotherapeutic agents with low concentration improving cytotoxic effect for cancer cells with minimal side effects.     

6-羟基多巴胺诱导SH-SY5Y细胞帕金森病模型中Peroxiredoxin6表达的研究

目的 应用蛋白质组学技术研究6-羟基多巴胺(6-OHDA)诱导SH-SY5Y细胞帕金森病(PD)模型中Peroxiredoxin6(Prx6)的表达变化.方法 在建立6-OHDA诱导SH-SY5Y细胞帕金森病模型的基础上,分别提取6-OHDA实验组和对照组孵育24h的细胞总蛋白,应用荧光差异双向凝胶电泳(DIGE)技术获得蛋白点的差异表达信息,运用基质辅助激光解吸/电离飞行时间质谱仪(MALDI-TOF MS)鉴定出差异蛋白质.结果 DIGE分析发现实验组有一个表达明显上调的差异蛋白质点(P<0.01),经质谱分析鉴定确认为Prx6.结论 6-OHDA诱导SH-SY5Y细胞的帕金森病模型中Prx6表达上调,提示PD中有氧化应激存在,Prx6上调及氧化应激可能与PD的发病机制有关.     

Netrin-1通过下调氧糖剥夺诱导的人神经母细胞瘤自噬促进细胞存活

目的通过建立体外人神经母细胞瘤(SH-SY5Y)氧糖剥夺(oxygen and glucose deprication,OGD)模型模拟脑梗死后神经元缺血缺氧环境(OGD),探索神经导向因子Netrin-1是否影响OGD后细胞的生存,以及自噬在其中的可能作用。方法体外培养SH-SY5Y细胞,检测Netrin-1对细胞活性及自噬的影响。使用自噬诱导剂雷帕霉素、自噬抑制剂3-甲基腺嘌呤干预自噬水平,同时构建过表达Netrin-1受体UNC5H2-HA基因的HEK293T细胞;CCK-8试剂盒检测细胞活性,免疫印迹检测自噬相关标志物(Beclin 1、LC3、p62)的表达及免疫荧光检测LC3斑点的形成。结果与OGD对照组相比,Netrin-1及3-甲基腺嘌呤的干预均显著提高细胞活性,伴有LC3-Ⅱ斑点形成减少;相反,雷帕霉素增加LC3-Ⅱ斑点的同时抑制细胞活性,并减弱了Netrin-1提高细胞活性的作用。过表达UNC5H2-HA细胞较对照质粒组相比,LC3-Ⅱ表达增加;同时进行Netrin-1干预后仅过表达组出现LC3-Ⅱ表达下降,p62表达上升。结论 Netrin-1可能通过受体UNC5H2下调OGD损伤的人神经母细胞瘤自噬而提高细胞活性。