Necdin蛋白与神经元的生长分化

在神经系统,Necdin只在成熟神经元的细胞核中表达,可能与成熟神经元分裂静止状态的保持有关.近年的研究表明,Necdin是一种生长抑制蛋白,能与多种因子如SV40大T抗原,腺病毒E1A,转录因子E2F1以及肿瘤抑制蛋白p53等结合,在功能上类似于成视网膜瘤蛋白Rb.necdin基因缺陷时,会引起脑内,特别是下丘脑神经元分化障碍.人类necdin基因位于PWS综合征的基因缺失区,可能与PWS的一些症状有关.本文从Necdin蛋白的基本概况,生物功能以及Necdin与疾病三个方面进行了综述.     

Necdin与神经系统退行性改变

Necdin是黑素瘤相关抗原家族Ⅱ成员之一,主要表达于成熟神经元的细胞核。Necdin作为一种生长抑制蛋白,通过与多种胞核及胞浆中的细胞因子结合,调控细胞周期、细胞分化及细胞凋亡过程。人类Necdin基因位于Prede—Willi综合征（PWS）的基因缺失区,可能与PWS的一些症状有关。Necdin缺失的小鼠表现出运动缺陷,可能参与了神经退行性改变。本文就Necdin的对神经系统的影响做一综述。     

脂肪干细胞向神经元诱导分化的研究

目的研究脂肪干细胞向神经元分化的可行性,为神经系统疾病的替代治疗寻求行之有效的可种植细胞。方法用β-2-巯基乙醇和丁酸酯羟基茴香醚诱导脂肪干细胞向神经元分化,对干预后细胞进行形态学观察和细胞免疫组化鉴定。结果人类腹部脂肪来源的基质细胞进行原代和传代培养后细胞形态类似于成纤维细胞,经过诱导分化后细胞形态表现为典型的神经元形态。神经元特异性烯醇化酶鉴定绝大部分有阳性染色,但在不到一周的时间内死亡。结论脂肪组织中存在能分化为神经元的干细胞．有巨大的研究价值和临床应用前景。     

中脑多巴胺能神经元发育分化的基因调控机制

中脑多巴胺能神经系统由于涉及帕金森病、精神分裂症、药物成瘾等多种严重神经精神障碍的病理过程而受到重视。多巴胺神经元的发育是一包括多种转录因子和生长因子参与的严格控制过程。在胚胎发育早期，是由控制中脑发育的多种同源域蛋白调控多巴胺前体细胞的增殖和迁移；在特异性转录因子Nurrl和Ptx3表达后，多巴胺神经元开始表达特定的表型并合成DA；最终在与纹状体靶细胞的正确联系建立的前提下分化成熟；同时这一过程要受到来自周围细胞信号的相互影响。     

大鼠骨髓间充质干细胞向神经元样细胞诱导分化及神经蛋白的动态表达

背景：现阶段骨髓间充质干细胞定向诱导分化为神经元样细胞的方法并不统一。目的：探讨大鼠骨髓间充质干细胞体外向神经样细胞诱导分化的条件,及其定向分化过程中神经蛋白的动态表达。设计、时间及地点：细胞形态学观察及蛋白分子水平检测,于2006-09/2007-01在苏州大学生命科学学院完成。材料：清洁级SD大鼠2只。胎牛血清为杭州四季青产品,成纤维生长因子、丁羟基茴香醚为Sigma公司产品,二甲基亚砜为Amresco产品。 方法：Percoll法体外分离培养、扩增纯化大鼠骨髓间充质干细胞,调整细胞密度为5×107 L-1。设立2组,诱导组用含10%胎牛血清和10 μg/L成纤维生长因子的L-DMEM培养基预诱导24 h后,再以含2%二甲基亚砜和200 μmol/L丁羟基茴香醚的L-DMEM无血清培养基诱导1,3,5 h。对照组始终用含10%胎牛血清的L-DMEM进行培养。主要观察指标：细胞形态学变化,免疫荧光染色鉴定神经细胞表型。结果：预诱导24 h,胞体由原来的长梭形变成多角形或不规则形,伸出多个短棒状突起,可见2~3个核仁,细胞间漩涡状生长趋势消失,排列无规律;诱导1~3 h,细胞逐渐变圆,胞质收缩明显,折光性增强,双级或多极的突起互相连接呈网状;诱导5 h,突起出现一、二级分支。诱导1 h后部分细胞表达神经前体细胞表面抗原巢蛋白;3 h后巢蛋白达高峰,同时表达成熟神经元标志物神经元特异性烯醇化酶和微管蛋白;5 h后巢蛋白表达下降,神经元特异性烯醇化酶和微管蛋白达高峰。对照组细胞形态无明显变化,巢蛋白表达为阴性。结论：在添加化学诱导剂之前先使用成纤维生长因子进行预诱导,能促进骨髓间充质干细胞向神经前体细胞和神经元转化,且在诱导分化过程中神经蛋白分子的表达顺序与神经细胞发育过程一致。     

神经干细胞向多巴胺能神经元分化的条件

帕金森氏病 (ＰＤ)病理改变的最大特点是病变只侵犯单一类型的神经元[1] 。因此 ,ＰＤ就成为细胞替代治疗战略最有代表性的疾病。但无论是自体肾上腺髓质还是胚胎中脑以及经过基因修饰的胶质细胞、成纤维细胞、成肌细胞 ,在移植到宿主体内以后很快就失去了功能 ,说明这些细胞可能还不是理想的移植材料[2 ] 。存在于哺乳动物胚胎期及成年期的神经干细胞 (ｎｅｕｒａｌｓｔｅｍｃｅｌｌｓＮＳＣｓ) [3 ,4] 不但具有自我更新的能力 ,而且具有多向分化的潜能 ,在移植到宿主体内以后还可以与宿主的局部细胞相整合。这些优点都使ＮＳＣｓ成为细胞…     

神经干细胞原代培养及GABA能神经元的诱导分化

目的探讨神经干细胞的原代培养及γ-氨基丁酸(GABA)能神经元的诱导分化.方法取孕16 dWistar大鼠的胚鼠全脑,进行体外培养,并对培养的神经干细胞以及诱导分化的GABA能神经元进行鉴定.结果培养24h后,出现2～4个细胞的细胞球.分化2d后,神经球贴壁后伸出细长突起,并可和周边神经球伸出的突起连接.神经球周边可见大量散在贴壁的双极或多极细胞.免疫荧光染色可见神经球均有nestin、NF200、GFAP阳性细胞.取第3代神经球行GABA能神经元定向分化.分化24h后,实验组细胞球贴壁.3d后,实验组细胞球周边有大量散在分布细胞贴壁生长,胞体圆形较大,有1～2个细长突起.免疫荧光显示,实验组周边散在的贴壁细胞多为GAD65阳性细胞.GAD65阳性细胞分化率实验组(85.97±2.78)%、对照组(18.16±2.29)%,P<0.01.结论本实验利用寡核苷酸序列特异性阻断了bHLH基因家族的调控因子之一Hes1,解除了其对bHLH的抑制,促进了神经干细胞向神经元的分化.实验还发现,阻断Hes1后大大提高了神经干细胞向GABA神经元分化的比率.     

脑提取液诱导骨髓基质细胞向神经元分化的研究

近年来,研究发现骨髓基质细胞(bone marrow stromal cells, BMSCs)能够分化为多种组织细胞(神经元、心肌细胞等)[1～4] ,使其成为生命科学领域研究的热点之一.     

音速波状蛋白促进骨髓间充质干细胞向多巴胺能神经元分化

目的探讨音速波状蛋白（Shh）促进人骨髓间充质干细胞（MSCs）体外定向分化为多巴胺能神经元样细胞的作用。方法体外分离、扩增和鉴定人骨髓MSCs。采用不同诱导方案诱导MSCs向神经元和多巴胺能神经元样细胞定向转化后,进行抗神经巢蛋白（Nestin）、神经元特异烯醇化酶（NSE）、神经胶质纤维酸性蛋白（GFAP）、酪氨酸羟化酶（TH）和多巴胺转运体（DAT）等免疫细胞化学染色,并计算阳性细胞百分率。结果实验组诱导后MSCs能分化为具有典型神经元形态的细胞,可见NSE、Nestin、GFAP、TH和DAT等神经细胞标志表达;对照组MSCs细胞形态无明显变化,上述特异性标志物表达均为阴性。实验2组（诱导方案含Shh）与1组（诱导方案不含Shh）的NSE、Nestin、GFAP阳性细胞百分率的差异无统计学意义,但实验2组TH和DAT阳性细胞百分率明显高于实验1组,差异具有统计学意义（P〈0.05）。结论Shh可促进MSCs分化为多巴胺能神经元样细胞。     

丹参诱导鼠骨髓间充质细胞向神经元分化

目的：研究丹参对鼠骨髓间充质细胞的分化作用。方法：丹参注射液诱导鼠骨髓间充质细胞向神经元方向分化，采用免疫细胞化学方法对分化的和未分化的细胞进行鉴定。结果：丹参可诱导鼠骨髓间充质细胞向神经细胞分化，分化的细胞早期表达巢蛋白和Musashi1蛋白，后期则表达神经元的标志物神经元特异性醇化酶和神经微丝M，在最适合的诱导条件下约50％-60％的细胞表达这两种神经元的标志物。结论：骨髓组织中存在能分化为神经元的干细胞，丹参能够诱导这种干细胞向神经元分化，这种细胞可能成为中枢神经系统自体细胞移植的另一个干细胞的来源。     

Effect of aucubin on neural precursor cell survival during neuronal differentiation

Purpose of the study: Aucubin (ACB) is an iridoid glycoside with various biological activities. Previously, it is reported that ACB reduces cell survival and proliferation in many human tumors, whereas it facilitates cell survival and neuroprotection in damaged neuronal cells and disease models. However, its effects on cell survival in the non-proliferating or differentiated neurons are not known.

Materials and methods: We examined whether ACB facilitated cell survival in differentiating neural precursor cells, HiB5, compared with the proliferating HiB5 cells at various concentrations.

Results: The cell viabilities were evaluated by measuring MTT values, cell numbers, amounts of neurotransmittersD1 and protein amounts of neuronal markers. Here, we showed that ACB promotes cell survival in differentiated neurons (10–200 μg/mL), but reduces it in proliferating NPCs (200–400 μg/mL). Protein amounts of neurofilament proteins, NF-H, NF-M, PSD-95 in post-synaptic density, GAP-43 in growing neurites and NeuN in differentiated neurons were upregulated by addition of ACB, indicating that cell survival increased in differentiated neurons, shown by immunoblot analysis. Especially, when PDGF was added into N2 media to facilitate neuronal differentiation of HiB5 cells, the viability of differentiated HiB5 cells was significantly elevated following the increase of ACB concentration. Furthermore, ACB promoted cell survival of specific neuron types, such as GABAergic neurons and glutamatergic neurons. When differentiated neurons were immunostained with markers for specific neurons, neuronal subtypes producing GABA and GAD 65/67 were immunostained more than subtypes producing glutamate and vGluT1.

Conclusion: These results indicate that ACB improves neuronal cell survival in differentiated cells, suggesting it may be a therapeutic compound for neurodegenerative disorders.     

effects of NGF and dibutyryl cAMP on neuronal differentiation of embryonal carcinoma cells

The P19S18O1A1 embryonal carcinoma cell line is capable of neuronal differentiation and is therefore useful in studying neuronal development and the influence of growth modulators on neuronal differentiation. We report here on the effects of nerve growth factor (NGF) and dibutyryl cyclic adenosine monophosphate (db cAMP), individually and combined, on differentiation of P19S18O1A1 cells. NGF alone did not induce any significant neuron-like changes in cultures exposed to NGF for as long as 12 days. Treatment with db cAMP resulted in changes in a significant population of the cells, including development of a neuron-like morphology, seen at both the light and electron microscopic level, loss of stage-specific embryonic antigen expression and the appearance of two neuronal markers, neurofilament protein and neuron-specific enolase. These changes were similar to changes seen when embryonal carcinoma (EC) cells are treated with retinoic acid. NGF in combination with dibutyryl cyclic adenosine monophosphate brought about similar changes as dibutyryl cyclic adenosine monophosphate alone, and was therefore not synergistic for induction of neuronal properties. In retinoic acid-treated cultures, the neuron-like cells had ultrastructural features very similar to neurons in non-tumorous, normal tissue, with typical organelles, such as one nucleolus, neurotubules and neurofilaments, while db cAMP-treated EC cells showed similar findings at the electronmicroscopic level. The results suggest that db cAMP can induce the neuronal phenotype in EC cells alone without pre-treatment with retinoic acid.     

VIP and PACAP induce selective neuronal differentiation of mouse embryonic stem cells

The capacity of embryonic stem cells (ES cells) to differentiate into neuronal cells represents a potential source for neuronal replacement and a model for studying factors controlling early stages of neuronal differentiation. Various molecules have been used to induce such differentiation but so far neuropeptides acting via functional G-protein-coupled receptors (GPCRs) have not been investigated. Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are neuropeptides expressed in early development which affect neuronal precursor proliferation and neuronal differentiation. VIP and PACAP share two common receptors (VPAC1 and VPAC2 receptors) while only PACAP binds with high affinity to PAC1 receptors. The aim of the study was to determine whether VIP and PACAP could produce functional neuronal differentiation of ES cells. Mouse ES cells were allowed to aggregate in embryoid bodies (EBs) in the presence or not of VIP and PACAP for 1 week. VIP and PACAP potently increased the proportion of EB-derived cells expressing specifically a neuronal phenotype shown by immunocytochemistry and neurite outgrowth without altering glial cell number. Binding and RT-PCR analyses demonstrated the presence of VPAC2 and PAC1 receptors on ES cells. Accordingly, both peptides increased cyclic AMP and intracellular calcium. In contrast, EB-derived cells only expressed a functional PAC1 receptor, suggesting a switch in GPCR phenotype during ES cell differentiation. These original data demonstrate that functional GPCRs for VIP and PACAP are present on ES cells and that these neuropeptides may induce their differentiation into a neuronal phenotype. It opens an exciting new field for neuropeptide regulation of tissue ontogenesis.     

Ultrastructural study of synapses at the time of neuronal migration and early differentiation in the tangential vestibular nucleus of the chick embryo in vivo

The chick tangential nucleus is a primary vestibular nucleus whose two main neuron types migrate and begin to differentiate between 5 and 8 days in the embryo (gestation takes 20-21 days). Based on rapid Golgi impregnations of developing tangential neurons and growing fibers, we have identified ultrastructural counterparts and characterized interactions in the nucleus from 5 to 8 days. Developing tangential neurons received the earliest synapses at 5 days on their primitive processes and subsequently on their cell bodies by longitudinal fibers of unknown origins. In contrast, the primary vestibular afferents did not form identified synapses on the developing tangential neurons until 7 1/2 days. In conclusion, the earliest synapses in the tangential nucleus are formed by longitudinal fibers, which are probably not primary vestibular afferents. Since a specific class of fibers forms particular synapses on the tangential neuron precursors at predictable times prior to and during neuronal migration and also at the onset of differentiation, the role of these synapses in developmental events should be explored.     

Actin-Binding protein,drebrin, accumulates in submembranous regions in parallel with neuronal differentiation

Drebrins are developmentally regulated actin-binding proteins. In this study, we analyzed subcellular distribution of drebrin E in neuroblastoma cells (SHSY5Y) in culture, especially in terms of its relationship to actin filaments. In undifferentiated cells, drebrin E was scattered as flocculus small dots along the stress fibers and also accumulated at adhesion plaques. In parallel with the neuronal differentiation following retinoic acid treatment, drebrin E was accumulated, accompanying filamentous (F) actin, in the submembranous cortical cytoplasm. Similar submembranous localization of drebrins was observed in primary cultured neurons. In the presence of drebrin E F-actin was more stable against cytochalasin D than F-actin lacking drebrin E. These results suggest that drebrin E plays a role in neuronal morphological differentiation by changing its subcellular localization with stabilized F-actin. © 1994 Wiley-Liss, Inc.     

Comparative analysis of CNS populations in knockout mice with altered growth hormone responsiveness

Recently we have shown that growth hormone (GH) inhibits neuronal differentiation and that this process is blocked by suppressor of cytokine signalling-2 (SOCS2). Here we examine several cortical and subcortical neuronal populations in GH hyper-responsive SOCS2 null (-/-) mice and GH non-responsive GH receptor null (GHR-/-) mice. While SOCS2-/- mice showed a 30% decrease in density of NeuN positive neurons in cortex compared to wildtype, GHR-/- mice showed a 25% increase even though brain size was decreased. Interneuron sub-populations were variably affected, with a slight decrease in cortical parvalbumin expressing interneurons in SOCS2-/- mice and an increase in cortical calbindin and calretinin and striatal cholinergic neuron density in GHR-/- mice. Analysis of glial cell numbers in cresyl violet or glial fibrillary acidic protein (GFAP) stained sections of cortex showed that the neuron : glia ratio was increased in GHR-/- mice and decreased in SOCS2-/- mice. The astrocytes in GHR-/- mice appeared smaller, while they were larger in SOCS2-/- mice. Neuronal soma size also varied in the different genotypes, with smaller striatal cholinergic neurons in GHR-/- mice. While the size of layer 5 pyramidal neurons was not significantly different from wildtype, SOCS2-/- neurons were larger than GHR-/- neurons. In addition, primary dendritic length was similar in all genotypes but dendritic branching of pyramidal neurons in the cortex appeared sparser in GHR-/- and SOCS2-/- mice. These results suggest that GH, possibly regulated by SOCS2, has multiple effects on central nervous system (CNS) development and maturation, regulating the number and size of multiple neuronal and glial cell types.     

Neuronal differentiation of P19 embryonal carcinoma cells in defined media

The P19 embryonal carcinoma cell line is a useful model system for analyzing the factors that regulate neuronal differentiation. In order to analyze the extrinsic factors that are involved in differentiation, it is necessary to carry out experiments in fully defined media. Here we have investigated the neuronal differentiation of P19 cells in two defined media. Cells that are propagated and induced with retinoic acid in standard serum-containing medium are capable of differentiating into neuron-like cells in N2 medium. Dividing fibroblast-like cells also appeared in these cultures. After about 10 days in culture in N2 medium, the great majority of neuron-like cells died. On the other hand, culturing induced cells in N2 medium for 5 days and then switching to a defined medium consisting of Neurobasal medium plus B27 supplement allowed the neuron-like cells to survive for prolonged periods of time. This defined medium thus provides a suitable system for analyzing extrinsic factors that affect the survival and differentiation of P19 neurons. P19 cells induced with retinoic acid and plated in N2 were exposed to bFGF and EGF, which are known to be mitogens for neuronal precursor cells. Both growth factors were mitogenic for a subpopulation of the induced cells. In separate experiments, cells cultured in N2 in the presence of RA were induced to differentiate into neuron-like cells. © 1995 Wiley-Liss, Inc.     

Proliferation, migration and differentiation of neuronal progenitor cells in the adult mouse subventricular zone surgically separated from its olfactory bulb

The subventricular zone of the adult mammalian forebrain contains progenitor cells that, by migrating along a restricted pathway called the ‘rostral migratory stream’ (RMS), add new neurons to the olfactory bulb throughout life. To determine the influence of the olfactory bulb on the development of these progenitor cells, we performed lesions that interrupt this pathway and separate the olfactory bulb from the rest of the forebrain. By labelling cells born at several survival times after the lesions with the thymidine analogue bromodeoxyuridine (BrdU), we found that disconnection from the bulb influences the rate of BrdU incorporation by the progenitor cells. The number of labelled cells in lesioned mice was almost half that found in control mice. In the disconnected migratory pathway, the number of neurons expressing calretinin was increased indicating that neuronal differentiation was enhanced: newly born neurons occurred within and around the RMS, most of them expressed calretinin and left the pathway starting about 2 weeks after the lesion. Thereafter, these neurons preserving their phenotype, spread for long distances, and accumulated ectopically in dorsal regions of the anterior olfactory nucleus and the frontal cortex. Finally, transplantation of adult subventricular cells into the lesioned pathway showed that the lesion neither prevents neuronal migration nor alters its direction. Thus, although the olfactory bulb appears to regulate the pace of the developmental processes, its disconnection does not prevent the proliferation, migration and phenotypic acquisition of newly generated bulbar interneurons that, since they cannot reach their terminal domains, populate some precise regions of the lesioned adult forebrain.