Notch1受体在脑缺血损伤中表达变化及作用的体外研究

目的观察Notch1受体在脑缺血损伤中的表达变化及其配体Jagged1对缺血损伤的影响,探讨Notch信号通路可能参与脑缺血损伤的机制。方法建立PC12诱导的神经元样细胞氧糖剥夺(OGD)体外脑缺血模型,应用Real-time PCR及Western-blot技术,检测OGD 3h、6h、9h、12h、16h、24h Notch1表达变化;MTT法检测外源性配体Jagged1对细胞缺血损伤的影响。结果与对照组相比,OGD后不同时间Notch1 mRNA及蛋白的表达量均有显著升高,3h时达最高峰,其后随OGD时间的延长逐渐下降,OGD 24h达最低点(P<0.05)。分别与对照组比较,外源性Jagged1干预可使正常及缺血神经元存活率均下降(P<0.05)。结论 Notch信号转导通路可能通过内源性的Notch1受体表达上调参与脑缺血性损伤过程。     

RA诱导P19细胞神经元分化过程中nestin基因的表达变化

目的 观察nestin基因在RA诱导P19细胞向神经分化过程中表达水平的变化,探讨nestin基因在神经分化过程中的特点.方法 P19细胞经1×10-6M视黄酸(RA)诱导悬浮培养4d形成细胞聚集体(aggregation),将聚集体用生长培养基黏附培养至12d,观察神经细胞分化情况,用神经元标记分子NF160对分化第10天的P19细胞进行免疫染色,观察其产生神经元情况,并采用RT-PCR技术对P19细胞、RA连续诱导培养的细胞聚集体以及细胞分化成熟的不同时段检测P19细胞中nestin基因的表达水平.结果 经RA成团诱导4d后,P19细胞向神经方向分化,随着分化时间延长逐渐形成神经网络;NF160对分化第10天的P19细胞检测呈阳性;nestin基因在P19细胞RA诱导前后皆有表达,在RA诱导P19细胞向神经元方向分化时表达水平逐渐上调,各时间点之间差异显著(P<0.05),而在随后P19细胞的分化过程中,随着P19神经元的成熟nestin基因的表达逐渐下降,直至接近于消失.结论 nestin基因在P19细胞向神经元诱导过程中表达逐渐上调,而在P19细胞神经元分化后表达明显下调,它参与神经元的分化和发育过程.     

PINK1介导缺氧损伤时星形胶质细胞对神经元的保护作用

目的探究星形胶质细胞内PTEN诱导假定激酶1(PINK1)缺失对缺血时神经保护作用的影响及其作用机制。方法离体培养原代星形胶质细胞,使用小干扰RNA(si RNA)沉默PINK1表达,氧糖剥夺(OGD)建立细胞缺氧模型,分为4组:PINK1沉默组(si RNA+转染剂)、空质粒组(空质粒+转染剂)、转染剂组(只加转染剂)和对照组(星形胶质细胞),各组均与神经元共培养;另设立神经元单独培养组。免疫荧光染色观察神经元凋亡情况。定量PCR及ELISA检测星形胶质细胞促红细胞生成素(EPO)及血管内皮生长因子(VEGF)表达量;Western blot检测星形胶质细胞内缺血诱导因子(HIF)及核因子κB(NF-κB)通路相关蛋白水平。结果 OGD损伤后神经元凋亡率较高,与星形胶质细胞共培养后神经元凋亡率显著降低(P0.05)。PINK1基因沉默后共培养神经元凋亡增加,星形胶质细胞EPO及VEGF分泌量减少、胞内EPO及VEGF转录水平降低(P0.05);HIF-1、HIF-2与NF-κB通路活化水平均显著降低(P0.05)。结论星形胶质细胞对OGD损伤神经元有保护作用,其作用通过EPO及VEGF实现;PINK1基因沉默后星形胶质细胞对缺血神经元保护作用减弱,可能与NF-κB通路活化水平降低、HIF激活受损进而下调EPO和VEGF表达量有关。     

局灶性脑缺血再灌注大鼠海马CA1区神经元凋亡研究

目的 观察大鼠局灶性脑缺血再灌注(ischemia reperfusion,I/R)损伤后海马CA1区神经元凋亡、TUNEL阳性细胞变化,以及凋亡相关蛋白Bcl-2与Bax蛋白的表达情况.方法 将健康雄性SD (Sprague-Dawley)大鼠随机分为假手术组和I/R组,每组再分为缺血再灌注后3、6、12、24、48、72 h亚组.应用免疫组化方法检测再灌注后不同时间点大鼠海马CA1区神经元凋亡基因Bcl-2和Bax蛋白的表达及Bcl-2/Bax比值变化,采用原位细胞凋亡检测(TUNEL)技术检测凋亡阳性细胞数.结果 各组非缺血侧相应区域神经元胞质中Bcl-2均有微弱表达.I/R组缺血侧海马CA1区于再灌注3 h开始出现Bcl-2和Bax蛋白微弱表达,随再灌注时间延长神经元内Bcl-2表达逐渐增强,再灌注24 h后Bcl-2表达达高峰,假手术组与I/R组比较差异有统计学意义(P<0.05).结论 I/R损伤后海马CA1区神经元不仅存在变性坏死,还存在明显的细胞凋亡且细胞凋亡在大鼠I/R损伤中发挥重要作用;I/R可诱导海马CA1区细胞凋亡基因Bcl-2和Bax蛋白表达,且其表达呈一定规律.     

神经元机械性损伤后Homer蛋白表达及意义

目的研究体外培养神经元机械损伤后不同亚型Homer蛋白表达的变化规律,进一步阐明Homer与神经元损伤的关系.方法胎鼠脑皮质神经元体外培养7 d,以微量移液器塑料滴头划割培养的神经元,横、竖各划8道,划伤宽度约1 mm,造成机械性损伤.在伤后不同时间(10、30 min,1、3、6、12、24、72 h),采用链酶亲合素-过氧化物酶复合物法(SABC法)行免疫组化染色.对照组不进行机械性划割,其它处理同损伤组.结果对照组神经元在各时间点Homer 1a免疫组化染色呈弱阳性.机械性损伤后从伤后10 min持续至24 h Homer 1a表达量增加,免疫组化染色呈阳性.阳性染色呈颗粒状分布于胞浆、胞膜及突起.Homer 1b/c,Homer 2a/b和Homer 3在对照组神经元中即有一定程度表达,机械性损伤后,其表达量无明显变化.结论神经元机械性损伤后Homer 1a表达明显升高,而Homer 1b/c、2a/b和3表达无明显变化. Homer 1a对第1组代谢型谷氨酸受体(mGluR)的功能具有负反馈作用,Homer 1b/c、2a/b和3则调节mGluR在细胞表面的分布,提高其稳定性.据此推测,增加Homer 1a表达或减少Homer 1b/c、2a/b和3的表达可能改变mGluR受体信号的传递效率,对神经元具有保护作用.     

心肌营养素-1对超氧化损伤神经元的保护作用

目的在体外培养神经元凋亡损伤模型,观察重组腺病毒-心肌营养素1(Adv-CT1)对损伤神经元存活的保护作用,了解CT-1对神经元的作用和机制,为神经损伤提供新的治疗措施。方法诱导大鼠神经干细胞(NSCs)分化为神经元,建立超氧化诱导神经元凋亡损伤模型,以Adv-CT1转染神经元,应用免疫组化、流式细胞仪凋亡检测等技术,观察CT-1对神经元生长存活的作用以及CT-1和caspase-3基因在损伤神经元表达的变化。结果分离培养NSCs,应用无血清小剂量碱性成纤维细胞生长因子(bFGF)神经元培养基诱导NSCs定向分化培养神经元;在神经元凋亡模型中转染Adv-CT1,免疫组化显示神经元中CT-1表达增高(P<0.05,或P<0.01),caspase-3表达降低(P<0.05);流式细胞检测显示CT-1可减少损伤神经元凋亡比例(P<0.01,或P<0.05),促进细胞存活。结论Adv-CT1转染到凋亡神经元后,CT-1表达增加,caspase-3表达降低,提示Adv-CT1对损伤神经元有保护作用,是CT-1通过减少神经元凋亡基因caspase-3表达,抑制凋亡发生,从而促进神经元存活。     

刺五加多糖对H_2O_2损伤的海马神经元表达c-fos和p53基因的影响

目的研究刺五加多糖(ASPS)对氧化应激损伤的海马神经元表达c-fos和p53基因的影响,进一步探讨ASPS对氧化应激损伤海马神经元的保护机制。方法从新生大鼠分离出海马,其神经元经培养后分为阴性对照组、损伤模型组(1 mmol/LH_2O_2诱导)和ASPS干预组(该组按ASPS 10、5、2.5μg/ml的终浓度又分为三个亚组)。采用H_2O_2建立海马神经元氧化应激损伤模型。用甲基噻唑基四唑(MTT)法检测各组海马神经元活性,免疫组化法检测其c-fos和p53的表达。结果 ASPS干预组神经元活性显著高于损伤模型组(P0.05)。c-fos和p53在ASPS干预组神经元中的表达水平较损伤模型组明显降低(P0.05)。结论 ASPS能够减少受损海马神经元c-fos和p53基因的表达,增强其活性,对海马神经元有明显的保护作用。     

体外培养原代神经元损伤模型的建立及损伤后脑红蛋白的表达

目的建立原代神经元机械性损伤的体外细胞模型并探讨神经元损伤后脑红蛋白（NGB）的表达变化。方法取出生12h内Wistar鼠的大脑皮质，培养10-12d后，采用所设计的标准模板，直视下进行损伤，采用免疫组化方法和图像分析技术观测损伤前、后不同时间点神经元的NGB免疫组化反应及其阳性信号的灰度值。结果皮质神经元损伤2h后，NGB阳性信号强度即开始增加，16h后达高峰，随后逐渐下降，128h后恢复至损伤前水平。结论皮质神经元体外损伤模型可用于在体外观察神经元受到机械性损伤后细胞内蛋白表达变化的一些分子事件，简单实用。机械性损伤可以诱导神经元NGB在损伤后一定时间段内表达上调。     

高糖诱导大鼠海马神经元细胞模型建立及细胞凋亡的影响

目的通过不同葡萄糖浓度和不同作用时间诱导SD大鼠海马神经元,建立稳定的糖尿病脑病细胞模型,探讨高糖对海马神经元凋亡情况的影响。方法 (1)海马神经元的原代培养及纯度鉴定。(2)最适高糖浓度探索:CCK-8法检测海马神经元各组细胞活性。(3)最适浓度下高糖作用时间的探索:Western blot检测海马神经元细胞Bcl-2、Bax的表达情况。(4)最适浓度和作用时间下,流式细胞术检测细胞的凋亡率。结果 (1)成功培养海马神经元,神经元细胞经鉴定纯度达85%以上。(2) 45 mmol/L组海马神经元存活率均 80%。(3) 45 mmol/L-48 h为最适作用时间组,细胞内Bcl-2表达下降、Bax表达升高(P 0. 01)。(4)最适高糖组细胞凋亡率显著升高(P 0. 01)。结论高糖浓度为45 mmol/L、作用时间48 h为理想的糖尿病脑病细胞模型,且高糖通过影响Bcl-2、Bax的表达导致海马神经元凋亡率升高。     

15d-PGJ_2对神经元氧糖剥夺/复氧损伤的影响

目的观察过氧化物酶体增殖物激活受体γ(PPAR-γ)激动剂15d-PGJ_2对神经元氧糖剥夺/复氧(OGD/R)损伤的影响,并探讨其可能的作用机制。方法采用原代培养的小鼠胎鼠大脑皮质神经元,建立OGD/R损伤模型。将培养的神经元细胞进行分组,Western blot检测各组神经元内LC3-1、LC3-Ⅱ、Bcl-2、Beclin1、AMPK、m TOR及p70S6K等蛋白的表达;MTT法检测细胞活性,乳酸脱氢酶(LDH)漏出率测定细胞毒性。结果 OGD/R损伤6 h、12 h及24 h后神经元LC3-Ⅱ、Beclin 1表达明显增高,而p62表达持续下降,神经元的生存率明显下降,LDH漏出率增加。15d-PGJ_2可显著降低LC3-Ⅱ和Beclin 1表达水平,改善神经元的生存率及降低LDH的漏出率。OGD/R后Bcl-2的蛋白表达水平均明显减少,而Beclin 1表达则显着增加。15d-PGJ_2预处理显著增加OGD/R 24 h的Bcl-2表达量。利用Bcl-2 siRNA或scRNA转染神经元细胞发现,Bcl-2 siRNA可消除15d-PGJ_2对OGD/R后各时间点的抑制效应。结论 15d-PGJ_2能够有效地对神经元OGD/R损伤起到保护作用,其机制可能是通过上调Bcl-2的表达进而抑制自噬的发生实现的。     

Notch配体刺激白血病细胞内Cleaved Notch1蛋白及基因的表达*☆

背景：Notch信号系统对干细胞的自身复制与分化有调节的作用。 目的：监测Notch配体刺激下白血病细胞蛋白与基因表达的变化,解析Notch信号系统在细胞内的作用。 方法：应用被Notch配体包被的培养皿,培养TMD7和THP-1细胞株,进行短期细胞增殖效果的评估。并且在刺激不同时间段后,应用免疫印迹法对Notch蛋白的活性进行检测,应用定量RT-PCR法测定各种基因表达的变化。 结果与结论：Notch配体刺激对TMD7细胞株的增殖有促进作用,对THP-1细胞株的增殖有抑制作用。配体刺激下,免疫印记结果显示2种不同相对分子质量的活性化Notch1蛋白片段。配体刺激后,各种基因的发现量的变化是不同的。而同一细胞株对3种Notch配体刺激的反应基本类似。可见Notch信号系统比现在所知道的更为复杂,可能存在着两种正向和负向的影响。     

Notch1 and Numb genes are inversely expressed as oligodendrocytes differentiate

The Notch1 pathway plays a fundamental role during the establishment of cell fates in the central nervous system (CNS) by regulating neural cell differentiation. In oligodendrocytes (OLs), Notch1 activity prevents these cells from becoming terminally mature, thereby influencing CNS myelination. Little is known of how OLs regulate the expression of this receptor at the gene level or if OLs have mechanisms to control the level of intracellular activity of the Notch1 pathway. In this study, we have found that Notch1 gene expression was higher in proliferative OL progenitor cells (OPCs) and was reduced when cells were forced to withdraw from the cell cycle and became mature, indicating that Notch1 gene expression is developmentally regulated in OLs. We observed that the blockade of terminal differentiation of OPCs by incubation with Delta1, an activator of Notch1, was a dominant process and OL-differentiating signals such as thyroid hormone could not overcome this inhibition in culture. This suggests that a downregulation of the Notch1 pathway might be required to allow OPCs to enter terminal differentiation. We also provide evidence that OPCs and OLs express the Numb gene, a known negative regulator of Notch1 activity. In vivo, Numb was found in postnatal OLs from cerebellar and cerebral white matter. In vitro, Numb expression showed to be inversely correlated to that of Notch1, with higher levels of Numb proteins in mature OLs, in association with myelin-like membranes.     

Notch1 signaling influences v2 interneuron and motor neuron development in the spinal cord

The Notch signaling pathway plays a variety of roles in cell fate decisions during development. Previous studies have shown that reduced Notch signaling results in premature differentiation of neural progenitor cells, while increased Notch activities promote apoptotic death of neural progenitor cells in the developing brain. Whether Notch signaling is involved in the specification of neuronal subtypes is unclear. Here we examine the role of Notch1 in the development of neuronal subtypes in the spinal cord using conditional knockout (cKO) mice lacking Notch1 specifically in neural progenitor cells. Notch1 inactivation results in accelerated neuronal differentiation in the ventral spinal cord and gradual disappearance of the ventral central canal. These changes are accompanied by reduced expression of Hes1 and Hes5 and increased expression of Mash1 and Neurogenin 1 and 2. Using markers (Nkx2.2, Nkx6.1, Olig2, Pax6 and Dbx1) for one or multiple progenitor cell types, we found reductions of all subtypes of progenitor cells in the ventral spinal cord of Notch1 cKO mice. Similarly, using markers (Islet1/2, Lim3, Sim1, Chox10, En1 and Evx1/2) specific for motor neurons and distinct classes of interneurons, we found increases in the number of V0-2 interneurons in the ventral spinal cord of Notch1 cKO mice. Specifically, the number of Lim3+/Chox10+ V2 interneurons is markedly increased while the number of Lim3+/Islet+motor neurons is decreased in the Notch1 cKO spinal cord, suggesting that V2 interneurons are generated at the expense of motor neurons in the absence of Notch1. These results provide support for a role of Notch1 in neuronal subtype specification in the ventral spinal cord.     

Progenitor cell maintenance and neurogenesis in sympathetic ganglia involves Notch signaling

Differentiation of noradrenergic neurons from neural crest-derived precursors results in the formation of primary sympathetic ganglia. As sympathetic neurons continue to divide after the acquisition of adrenergic and neuronal properties it was unclear, whether the increase in neuron number during neurogenesis is due to neuron proliferation rather than differentiation of progenitor cells. Here, we demonstrate Sox10-positive neural crest progenitor cells and continuous sympathetic neuron generation from Phox2b-positive autonomic progenitors during early chick sympathetic ganglion development. In vivo activation of Notch signaling resulted in a decreased neuronal population, whereas expression of the Notch signaling inhibitor Su(H)(DBM) increased the proportion of Scg10-positive neurons. Similar results were obtained for sensory dorsal root ganglia (DRG). The effects of Notch gain- and loss-of-function experiments support the notion that progenitor maintenance and neuron differentiation from progenitor cells are essential for neurogenesis also during early sympathetic ganglion development.     

Prenatal NMDA receptor antagonism impaired proliferation of neuronal progenitor, leading to fewer glutamatergic neurons in the prefrontal cortex

N-methyl-D-aspartate (NMDA) receptor is a glutamate receptor which has an important role on mammalian brain development. We have reported that prenatal treatment with phencyclidine (PCP), a NMDA receptor antagonist, induces long-lasting behavioral deficits and neurochemical changes. However, the mechanism by which the prenatal antagonism of NMDA receptor affects neurodevelopment, resulting in behavioral deficits, has remained unclear. Here, we report that prenatal NMDA receptor antagonism impaired the proliferation of neuronal progenitors, leading to a decrease in the progenitor pool in the ventricular and the subventricular zone. Furthermore, using a PCR array focused on neurogenesis and neuronal stem cells, we evaluated changes in gene expression causing the impairment of neuronal progenitor proliferation and found aberrant gene expression, such as Notch2 and Ntn1, in prenatal PCP-treated mice. Consequently, the density of glutamatergic neurons in the prefrontal cortex was decreased, probably resulting in glutamatergic hypofunction. Prenatal PCP-treated mice displayed behavioral deficits in cognitive memory and sensorimotor gating until adulthood. These findings suggest that NMDA receptors regulate the proliferation and maturation of progenitor cells for glutamatergic neuron during neurodevelopment, probably via the regulation of gene expression.     

A human pluripotent carcinoma stem cell-based model for in vitro developmental neurotoxicity testing: Effects of methylmercury,lead and aluminum evaluated by gene expression studies

The major advantage of the neuronal cell culture models derived from human stem cells is their ability to replicate the crucial stages of neurodevelopment such as the commitment of human stem cells to the neuronal lineage and their subsequent stages of differentiation into neuronal and glial-like cell. In these studies we used mixed neuronal/glial culture derived from the NTERA-2 (NT-2) cell line, which has been established from human pluripotent testicular embryonal carcinoma cells. After characterization of the different stages of cell differentiation into neuronal- and glial-like phenotype toxicity studies were performed to evaluate whether this model would be suitable for developmental neurotoxicity studies. The cells were exposed during the differentiation process to non-cytotoxic concentrations of methylmercury chloride, lead chloride and aluminum nitrate for two weeks. The toxicity was then evaluated by measuring the mRNA levels of cell specific markers (neuronal and glial). The results obtained suggest that lead chloride and aluminum nitrate at low concentrations were toxic primarily to astrocytes and at the higher concentrations it also induced neurotoxicity. In contrast, MetHgCl was toxic for both cell types, neuronal and glial, as mRNA specific for astrocytes and neuronal markers were affected. The results obtained suggest that a neuronal mixed culture derived from human NT2 precursor cells is a suitable model for developmental neurotoxicity studies and gene expression could be used as a sensitive endpoint for initial screening of potential neurotoxic compounds.     

Dicer1 and MiR-9 are required for proper Notch1 signaling and the Bergmann glial phenotype in the developing mouse cerebellum

MicroRNAs (miRNAs) have important roles in the development of the central nervous system (CNS). Several reports indicate that tissue development and cellular differentiation in the developing forebrain are disrupted in the absence of miRNAs. However, the functions of miRNAs during cerebellar development have not been systematically characterized. Here, we conditionally knocked out the Dicer1 gene under the control of the human glial fibrillary acidic protein (hGFAP) promoter to examine the effect of miRNAs in the developing cerebellum. We particularly focused on the phenotype of Bergmann glia (BG). The hGFAP‐Cre activity was detected as early as embryonic day 13.5 (E13.5) at the rhombic lip (RL) in the cerebellar plate, and later in several postnatal cerebellar cell types, including BG. Dicer1 ablation induces a smaller and less developed cerebellum, accompanied by aberrant BG morphology. Notch1 signaling appears to be blocked in Dicer1‐ablated BG, with reduced expression of the Notch1 target gene, brain lipid binding protein (BLBP). Using neuronal co‐culture assays, we showed an intrinsic effect of Dicer1 on BG morphology and Notch1 target gene expression. We further identified miR‐9 as being differentially expressed in BG and showed that miR‐9 is a critical, but not the only, miRNA component of the Notch1 signaling pathway in cultured BG cells. © 2012 Wiley Periodicals, Inc.