Parkin基因过表达质粒和细胞模型的构建

目的:构建Parkin基因过表达质粒并转染SH-SY5Y细胞,为进一步研究帕金森病的发病机制及中药的作用环节奠定基础。方法:首先构建Parkin基因过表达质粒,采用脂质体转导技术,将Parkin过表达质粒应用Lipo3000转染SH-SY5Y细胞。荧光显微镜观察细胞绿色荧光蛋白的表达;RT-PCR检测其Parkin mRNA的表达;Western Blot技术检测其Parkin蛋白的表达。结果:转染组可观测到较多的绿色荧光蛋白表达;Parkin过表达细胞Parkin mRNA和蛋白的表达均显著提高(P0.01)。结论:通过脂质体转导技术,应用Lipo3000可将Parkin过表达质粒成功转染入SH-SY5Y细胞,转染的基因和蛋白表达均较高,提示此法可成功构建Parkin基因过表达的细胞模型。     

Beclin-1 shRNA对低氧诱导的SH-SY5Y细胞自噬的影响

目的：构建Beclin-1基因短发夹干扰RNA（shRNA）慢病毒载体,感染人SH-SY5Y细胞,观察沉默Beclin-1基因后低氧对SH-SY5Y细胞自噬的影响。方法：构建特异性靶向Beclin-1基因的shRNA慢病毒表达载体和阴性对照序列慢病毒载体;再将载体转染入SH-SY5Y细胞;RT-PCR检测Beclin-1的mRNA表达;Western blot检测Beclin-1蛋白表达;CCK-8法测定Beclin-1 shRNA对SH-SY5Y细胞活力的影响。再将空白对照、阴性对照、转染型三种细胞分别以21%常氧及5%低氧培养,Western blot检测各组细胞LC3蛋白表达;电镜观察自噬小体。结果：Beclin-1 shRNA能明显抑制SH-SY5Y细胞Beclin-1的mRNA及蛋白的表达;沉默Beclin-1基因后,Beclin-1 shRNA组细胞存活率与阴性对照组相比无差异;成功建立了稳定表达Beclin-1 shRNA的SH-SY5Y细胞。5%低氧处理后,与阴性对照组相比较,Beclin-1 shRNA组细胞中LC3Ⅱ/LC3Ⅰ比值下调,细胞内自噬小体数量减少。结论：慢病毒介导的Beclin-1shRNA对SH-SY5Y细胞的活力无影响,但可以抑制低氧诱导的自噬。     

Neuroligin对SH-SY5Y细胞增殖和凋亡的影响

目的:探讨Neuroligin(NLG)对人神经母细胞瘤SH-SY5Y增殖和凋亡的影响。方法:体外培养SH-SY5Y细胞24 h后,分别用浓度为50,100,200μmol/L的NLG siRNA转染SH-SY5Y细胞并共孵育24 h,然后采用噻唑蓝(MTT)法检测不同浓度NLG siRNA对SHSY5Y细胞增殖率的影响,以及Real-time PCR法检测SH-SY5Y细胞中凋亡基因Bax、Bcl-2、Bcl-x L和caspase-9基因表达水平的变化。结果:与空白对照组及siRNA阴性对照组相比,转染NLG siRNA后SH-SY5Y细胞增殖率显著下降,细胞凋亡相关基因被激活,导致细胞凋亡。结论:NLG对神经元细胞具有保护作用,抑制神经细胞凋亡。     

APPswe基因慢病毒表达质粒的构建及对其SH-SY5Y细胞生长和凋亡的影响

为考察APPswe基因的表达对细胞生长和凋亡作用的影响,本研究采用gateway分子重组技术构建表达APPswe基因的慢病毒质粒,并将该质粒转染至SH-SY5Y细胞中.用RT-PCR和Western blot技术分别测定APPswe基因的mRNA转录和蛋白翻译水平.活细胞计数法和细胞外乳酸脱氢酶法分别测定细胞存活力和细胞膜损伤程度.Annexin V-FITC/PI染色流式细胞术测定细胞凋亡水平,Hoescht 33342染色观察细胞核形态变化,DCFH-DA染色法测定胞内活性氧水平.转录和翻译水平的验证表明,APPswe基因能够在SH-SY5Y细胞中相对稳定地表达.表达APPswe基因后,细胞的存活能力降低,损伤程度增加,细胞内活性氧水平上升,细胞核浓缩聚集,细胞发生凋亡反应.试验结果表明,APPswe基因的表达对SH-SY5Y细胞生长产生抑制作用,造成细胞损伤和凋亡,表达APPswe基因的SH-SY5Y细胞系可应用于体外AD病理发生机制和药物作用的研究.     

血管外膜肌成纤维细胞分化的基因表达谱

我们以往的研究表明,TGF-β1可以诱导血管外膜成纤维细胞（adventitial fibroblasts,AFs）向肌成纤维细胞（myofibroblasts,MFs）分化。为寻找可能涉及MF分化的基因,本实验采用寡核苷酸芯片技术动态检测细胞表型转化过程中基因表达的变化,实时定量RT-PCR验证芯片结果。在芯片上的15866条总探针组中,2121个探针组在TGF-β1刺激后至少一个时间点的表达发生2倍以上变化,其中l318个基因表达上调,761个基因表达下调,还有少数基因（42个）在不同的时间点既有上调又有下调表达。在1231个已知功能基因中,分泌磷蛋白l（secreted phosphoprotein1．APP1）、Rhoassociated coiled-coil forming kinase2（ROCK2）的表达趋势与标志基因α-平滑肌肌动蛋白（α-SM-actin）的表达趋势相同,TGF-D1诱导MF分化过程中上调了电压门控性钾通道Shal家族成员2（potassium voltage-gated channel,Shal-related family and member2,KCND2）的表达,这些基因参与了MF的分化;此外,还发现内皮素1（endothelin 1,EDN1）、补体成分、NADPH氧化酶4（NADPH oxidase 4,NOX4）和NAD（P）H dehydrogenase,quinone 1 （NQO1）可能参与了MF分化。本实验用寡核苷酸芯片技术验证了通过其它技术证实的同MF分化相关的基因,并发现了新的涉及该过程的基因,基因表达谱研究有利于鉴定参与细胞分化的基因和通路。     

TIMP-1基因缄默削弱SH-SY5Y细胞的增殖潜能并诱导其凋亡增强

前期研究发现,人基质金属蛋白酶组织抑制剂-1(tissue inhibitors of metalloproteinases-1,TIMP-1)在唐氏综合征（Down’s syndrome ,DS)胎儿脑组织内表达下调．为了探讨TIMP-1表达下调参与DS脑病变发生的可能机制,本研究以人神经母细胞瘤细胞（SH-SY5Y）为模型,观察TIMP-1基因沉默后对其增殖和凋亡的影响．应用LipofectaminTM2000将TIMP-1特异性短发卡 RNA( short hairpin RNA,shRNA)导入SH-SY5Y细胞,经嘌呤霉素筛选获得稳定表达TIMP-1-shRNA细胞株;应用RT-PCR、real-time PCR和Western 印迹对干扰效率进行鉴定：与SH-SY5Y细胞相比,无论在mRNA水平还是蛋白水平,SH-SY5Y-TIMP-1-shRNA细胞中TIMP-1的表达显著下调（下调率接近100%）．结果显示,已成功构建了TIMP-1基因沉默的SH-SY5Y细胞模型．在此基础上,通过MTT检测发现,TIMP-1基因沉默后SH-SY5Y细胞增殖减慢;流式细胞仪和荧光显微镜凋亡检测显示,TIMP-1基因沉默后SH-SY5Y细胞凋亡明显增加．这些研究结果表明,TIMP-1基因沉默能削弱SH-SY5Y细胞的增殖能力并增强SH-SY5Y的凋亡效应,提示TIMP-1可能是通过影响神经细胞的增殖和凋亡参与DS智力低下的发病过程．     

应用两种方法诱导SH-SY5Y 细胞分化的研究

目的:观察全反式维甲酸(ATRA)处理和ATRA与十四烷酰佛波醇乙酸酯(TPA)序贯处理(ATRA/TPA)对人类神经母细胞瘤细胞系SH-SY5Y细胞增殖抑制和形态分化的影响。方法:应用10μM ATRA处理6天和10μM ATRA处理3天继以80 nMTPA处理3天这两种方法使SH-SY5Y细胞分化;用倒置光学显微镜动态观察SH-SY5Y细胞形态学变化;并用MTT比色法比较两种分化方法对SH-SY5Y细胞的体外抗增殖作用。结果:ATRA处理和ATRA与TPA序贯处理对SH-SY5Y细胞都有抗增值和诱导细胞分化作用,细胞形态发生明显的变化,分化成神经元表型,前者主要表现为两端带有长突起的纺锤体样细胞形态,而后者主要是由细胞体延伸出多个突起的多边形的细胞。ATRA分化6天的细胞的存活率下降为78.7%±2.0%。当去除ATRA后,继续培养1天的细胞存活率上升为89%±0.2%,而继续培养2天的细胞存活率为86.3%±1.4%;ATRA与TPA序贯分化6天细胞存活率下降为75.9±0.4%。当去除TPA后,继续培养一天的细胞存活率为75.5±0.7%,继续培养2天的细胞存活率为74.9±1.0%。结论:维甲酸(ATRA)处理和ATRA与十四烷酰佛波醇乙酸酯(TPA)序贯处理(ATRA/TPA)均能明显诱导SH-SY5Y细胞分化。这两种分化细胞为神经科学的研究提供了优良的体外培养模型细胞,尤其是ATRA与TPA序贯处理能获得分化完全而稳定的神经元样细胞。     

阿尔茨海默病中miR-29c对APP表达调控的初步研究

目的探讨microRNA29c（miR-29c）在阿尔茨海默病中的作用。方法采用了microarray芯片检测3月龄、6月龄APPswe/PSΔE9双转基因阿尔茨海默病小鼠大脑中microRNA表达情况并利用实时定量PCR验证结果可靠性,通过microRNA靶基因数据库预测选出与阿尔茨海默病相关的靶基因,构建miR-29c表达载体,将其转染SH-SY5Y及HEK-293T细胞,在高表达miR-29c的SH-SY5Y及HEK-293T细胞系中验证miR-29c对靶基因的调控作用。将靶基因APP mRNA的野生及突变3’UTR序列克隆到双荧光素酶报告基因载体,用双荧光素酶报告检测系统检测miR-29c与靶基因的结合位点。结果根据microRNA芯片结果筛选出在3、6月龄APPSWE/PS1ΔE9双转基因小鼠大脑表达均有差异的miR-29c,通过实时定量PCR证实miR-29c在3月、6月、9月龄小鼠中表达明显升高。通过microRNA靶基因数据库预测miR-29c可以调控阿尔茨海默病的靶基因APP,利用Western blot检测到高表达miR-29c的SH-SY5Y细胞及HEK-293T细胞中APP蛋白表达减少。将miR-29c表达载体与带有野生及突变APPmRNA的3’UTR的双荧光素酶报告载体共转染HEK-293T细胞,通过双荧光素酶检测系统检测未找到miR-29c与APP mRNA 3’UTR的结合位点。结论 miR-29c对APP表达的具有负向调控作用,但其调控位点可能不位于其3’UTR区域。     

内质网应激在乙型脑炎病毒诱导神经元细胞凋亡中的作用及机制研究

内质网应激(Endoplasmic reticulum stress,ERS)的激活与创伤、缺血再灌注等病理刺激引起的神经元凋亡有关,流行性乙型脑炎病毒(JEV)感染能够促进神经元凋亡、激活ERS,但ERS在JEV诱导神经元细胞凋亡中的作用尚不清楚.为了研究ERS在JEV诱导神经元细胞凋亡中的作用及机制,本研究以神经细胞株SH-SY5Y为对象,感染JEV并加用ERS激动剂、ERS抑制剂或转染阴性对照(NC) siRNA、蛋白激酶R样内质网激酶(PERK)siRNA,检测细胞存活率、凋亡率、ERS蛋白PERK、肌醇必需酶-1α(IRE1α)、活化转录因子6(ATF6)及凋亡蛋白C/EBP同源蛋白(CHOP)、含半胱氨酸的天冬氨酸蛋白水解酶12(Caspase-12)、Bcl-2相关X蛋白(Bax)的表达.结果 显示:JEV组SH-SY5Y细胞的凋亡率及PERK、CHOP、Caspase-12、Bax的表达水平高于对照组,存活率低于对照组(P＜0.05),IRE1α、ATF6的表达水平与对照组比较无显著差异(P＞0.05).与JEV组比较,激动剂组SH-SY5Y细胞的凋亡率及PERK、CHOP、Caspase-12、Bax的表达水平显著增加,存活率显著降低(P＜0.05);抑制剂组SH-SY5Y细胞的凋亡率及PERK、CHOP、Caspase-12、Bax的表达水平显著降低,存活率显著增加(P＜0.05);与si-NC+JEV组比较,si-PERK+JEV组SH-SY5Y细胞的凋亡率及PERK、CHOP、Caspase-12、Bax的表达水平P显著降低,存活率显著增加(P＜0.05).以上结果表明ERS的PERK通路激活与JEV诱导神经元凋亡有关.     

红花黄色素B对冈田酸致SH-SY5Y神经元损伤的保护作用

本研究目的是考察红花黄色素B(SYB)对冈田酸(OA)致SH-SY5Y神经元损伤的保护作用。采用全反式维甲酸(ATRA)诱导SH-SY5Y细胞分化为成熟神经元,OA诱导神经元损伤,建立Tau蛋白过度磷酸化的神经元突触萎缩模型;Giemsa染色法观察SH-SY5Y细胞形态学变化;Western Blot检测Tau蛋白262位点磷酸化水平;流式细胞术检测细胞总活性氧(ROS)和线粒体源ROS水平,以及线粒体膜电位的变化。结果表明,ATRA可诱导SH-SY5Y细胞分化为成熟神经元;OA可致神经元突触萎缩和Tau蛋白在262位点过度磷酸化;SYB能够改善OA所致成熟神经元损伤,降低Tau蛋白在262位点的磷酸化水平,其保护作用机制可能与减少胞内及线粒体源ROS产生,提高线粒体膜电位有关。     

A PKCbeta isoform mediates phorbol ester-induced activation of Erk1/2 and expression of neuronal differentiation genes in neuroblastoma cells.

Protein kinase C (PKC) activation induces neuronal differentiation of SH-SY5Y neuroblastoma cells. This study examines the role of PKCbeta isoforms in this process. The PKCbeta-specific inhibitor LY379196 had no effect on 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced neurite outgrowth from SH-SY5Y neuroblastoma cells. On the other hand, PKCbeta inhibition suppressed the TPA-stimulated increase in neuropeptide Y mRNA, activation of neuropeptide Y gene promoter elements, and phosphorylation of Erk1/2. The TPA-induced increase in neuropeptide Y expression was also inhibited by the MEK inhibitor PD98059. These data indicate that activation of a PKCbeta isoform, through a pathway involving Erk1/2, leads to increased expression of neuronal differentiation genes in neuroblastoma cells.     

Differentiation Induces Dramatic Changes in miRNA Profile,Where Loss of Dicer Diverts Differentiating SH-SY5Y Cells Toward Senescence

MicroRNAs (miRNAs) are generated by endonuclease activity of Dicer, which also helps in loading of miRNAs to their target sequences. SH-SY5Y, a human neuroblastoma and a cellular model of neurodevelopment, consistently expresses genes related to neurodegenerative disorders at different biological levels (DNA, RNA, and proteins). Using SH-SY5Y cells, we have studied the role of Dicer and miRNAs in neuronal differentiation and explored involvement of P53, a master regulator of gene expression in differentiation-induced induction of miRNAs. Knocking down Dicer gene induced senescence in differentiating SH-SY5Y cells, which indicate the essential role of Dicer in brain development. Differentiation of SH-SY5Y cells by retinoic acid (RA) or RA + brain-derived neurotrophic factor (BDNF) induced dramatic changes in global miRNA expression. Fully differentiated SH-SY5Y cells (5-day RA followed by 3-day BDNF) significantly (p < 0.05 and atleast >3-fold change) upregulated and downregulated the expression of 77 and 17 miRNAs, respectively. Maximum increase was observed in the expression of miR-193-5p, miR-199a-5p, miR-192, miR-145, miR-28-5p, miR-29b, and miR-222 after RA exposure and miR-193-5p, miR-146a, miR-21, miR-199a-5p, miR-153, miR-29b, and miR-222 after RA + BDNF exposure in SH-SY5Y cells. Exploring the role of P53 in differentiating SH-SY5Y cells, we have observed that induction of miR-222, miR-192, and miR-145 is P53 dependent and expression of miR-193a-5p, miR-199a-5p, miR-146a, miR-21, miR-153, and miR-29b is P53 independent. In conclusion, decreased Dicer level enforces differentiating cells to senescence, and differentiating SH-SY5Y cells needs increased expression of P53 to cope up with changes in protein levels of mature neurons.     

Morphological Differentiation Towards Neuronal Phenotype of SH-SY5Y Neuroblastoma Cells by Estradiol,Retinoic Acid and Cholesterol

Human SH-SY5Y neuroblastoma cells maintain their potential for differentiation and regression in culture conditions. The induction of differentiation could serve as a strategy to inhibit cell proliferation and tumor growth. Previous studies have shown that differentiation of SH-SY5Y cells can be induced by all-trans-retinoic-acid (RA) and cholesterol (CHOL). However, signaling pathways that lead to terminal differentiation of SH-SY5Y cells are still largely unknown. The goal of this study was to examine in the RA and CHOL treated SH-SY5Y cells the additive impacts of estradiol (E₂) and brain-derived neurotrophic factor (BDNF) on cell morphology, cell population growth, synaptic vesicle recycling and presence of neurofilaments. The above features indicate a higher level of neuronal differentiation. Our data show that treatment for 10 days in vitro (DIV) with RA alone or when combined with E₂ (RE) or CHOL (RC), but not when combined with BDNF (RB), significantly (p < 0.01) inhibited the cell population growth. Synaptic vesicle recycling, induced by high-K⁺ depolarization, was significantly increased in all treatments where RA was included (RE, RC, RB, RCB), and when all agents were added together (RCBE). Specifically, our results show for the first time that E₂ treatment can alone increase synaptic vesicle recycling in SH-SY5Y cells. This work contributes to the understanding of the ways to improve suppression of neuroblastoma cells’ population growth by inducing maturation and differentiation.     

Differences in early and late responses between neurotrophin-stimulated trkA- and trkC-transfected SH-SY5Y neuroblastoma cells.

Despite their sympathetic neuroblast origin, highly malignant neuroblastoma tumors and derived cell lines have no or low expression of the neurotrophin receptor genes, trkA and trkC. Expression of exogenous trkA in neuroblastoma cells restores their ability to differentiate in response to nerve growth factor (NGF). Here we show that stable expression of trkC in SH-SY5Y neuroblastoma cells resulted in morphological and biochemical differentiation upon treatment with neurotrophin-3 (NT-3). To some extent, trkA- and trkC-transfected SH-SY5Y (SH-SY5Y/trkA and SH-SY5Y/trkC) cells resembled one another in terms of early signaling events and neuronal marker gene expression, but important differences were observed. Although induced Erk 1/2 and Akt/PKB phosphorylation was stronger in NT-3-stimulated SH-Y5Y/trkC cells, activation of the immediate-early genes tested was more prominent in NGF-treated SH-SY5Y/ trkA cells. In particular, c-fos was not induced in the SH-SY5Y/trkC cells. There were also phenotypic differences. The concentrations of norepinephrine, the major sympathetic neurotransmitter, and growth cone-located synaptophysin, a neurosecretory granule protein, were increased in NGF-treated SH-SY5Y/trkA but not in NT-3-treated SH-SY5Y/trkC cells. Our data suggest that NT-3/p145trkC and NGF/p140trkA signaling differ in some aspects in neuroblasoma cells, and that this may explain the phenotypic differences seen in the long-term neurotrophin-treated cells.     

Retinoid signalling and gene expression in neuroblastoma cells: RXR agonist and antagonist effects on CRABP-II and RARbeta expression

9-cis Retinoic acid (RA) induces gene expression in neuroblastoma cells more effectively and with different kinetics than other RA isomers, and could be acting in part through Retinoid X Receptors (RXRs). The aim of this study was to characterise the effects of an RXR agonist and RXR homodimer antagonist on the induction of cellular RA binding protein II (CRABP-II) and RA receptor-beta (RARbeta) in neuroblastoma cells in response to different retinoids. The RXR agonist, LDG1069, was as effective as all-trans RA in inducing gene expression, but less effective than 9-cis RA. The RXR-homodimer antagonist, LG100754, inhibited the induction of CRABP-II mRNA in SH-SY5Y neuroblastoma cells by 9-cis RA or the RXR-specific agonist LGD1069, but had no effect when used with all-trans RA. Conversely, LG100754 did not inhibit induction of RARbeta mRNA by 9-cis or all-trans RA, or by LGD1069. RAR- and RXR-specific ligands used together induced CRABP-II and RARbeta as effectively as 9-cis RA. These results demonstrate the value of combining RXR- and RAR-specific ligands to regulate RA-inducible gene expression. The possibility that RXR-homodimers mediate, in part, the induction of CRABP-II by 9-cis RA and RXR-specific ligands is discussed.     

Reverse Engineering the Neuroblastoma Regulatory Network Uncovers MAX as One of the Master Regulators of Tumor Progression

Neuroblastoma is the most common extracranial tumor and a major cause of infant cancer mortality worldwide. Despite its importance, little is known about its molecular mechanisms. A striking feature of this tumor is its clinical heterogeneity. Possible outcomes range from aggressive invasion to other tissues, causing patient death, to spontaneous disease regression or differentiation into benign ganglioneuromas. Several efforts have been made in order to find tumor progression markers. In this work, we have reconstructed the neuroblastoma regulatory network using an information-theoretic approach in order to find genes involved in tumor progression and that could be used as outcome predictors or as therapeutic targets. We have queried the reconstructed neuroblastoma regulatory network using an aggressive neuroblastoma metastasis gene signature in order to find its master regulators (MRs). MRs expression profiles were then investigated in other neuroblastoma datasets so as to detect possible clinical significance. Our analysis pointed MAX as one of the MRs of neuroblastoma progression. We have found that higher MAX expression correlated with favorable patient outcomes. We have also found that MAX expression and protein levels were increased during neuroblastoma SH-SY5Y cells differentiation. We propose that MAX is involved in neuroblastoma progression, possibly increasing cell differentiation by means of regulating the availability of MYC:MAX heterodimers. This mechanism is consistent with the results found in our SH-SY5Y differentiation protocol, suggesting that MAX has a more central role in these cells differentiation than previously reported. Overexpression of MAX has been identified as anti-tumorigenic in other works, but, to our knowledge, this is the first time that the link between the expression of this gene and malignancy was verified under physiological conditions.