神经干细胞分离培养方法的介绍

在成体的许多组织中发现了多能干细胞,这些干细胞可以进行自我复制,参与组织的正常修复。神经干细胞在体外能分化为神经元、星形胶质细胞和少突胶质细胞,并具有多向分化潜能。成体神经干细胞和胚胎干细胞都能分化成成体神经系统中的各种神经细胞。神经干细胞具有自我更新能力,因此神经干细胞可以应用于神经损伤或者神经疾病的修复。本文概述了神经干细胞体外分离培养的方法及其生长影响因子。     

神经营养因子与神经干细胞

生长因子在神经干细胞的增殖,分化和存活过程中有重要作用。神经营养因子是其中的一类,它包括神经生长因子（NGF）家族,胶质源性神经营养因子（GDNF）家族和其它神经营养因子。NGF家族包括NGF,BDNF,NT－3,NT－4／5和NT－6。这一家族可促进epidermic growth facter(EGF)反应 海马及前脑室管膜下区神经干细胞的存活和分化。GDNF家族包括GDNF,NTN,PSP和ART。GDNF家族促神经发育的作用主要在外周,它促进肠神经嵴前体细胞的存活和增殖,且对外周感觉神经的发育至关重要。其它生长因子如bFGF和EGF,它们能促进神经干细胞增殖和存活;CNTF和LIF等在神经干细胞的分化中也有重要作用。     

大鼠胚胎神经干细胞纹状体内移植后特性

观察大鼠胚胎神经干细胞移植入成年大鼠纹状体后的存活、迁移和分化状况。自14天胎鼠脑室下区分离获得神经干细胞,利用无血清培养基培养扩增并进行鉴定。经4～5代的扩增后,以BrdU标记的神经干细胞通过脑立体定位注射移植入成年大鼠纹状体内,然后分别于移植后2周、4周、6周和8周时做脑冰冻切片,通过免疫组织化学和免疫荧光方法检测移植细胞的数量、定位和分化情况。8周后移植细胞的检出率约16%;移植细胞向周围宿主组织有广泛的迁移表现,尤以沿着白质束向头尾方向的迁移最为显著,最远向后侧达到内囊;纹状体中移植细胞主要分化为神经元和星形胶质细胞。星形胶质细胞数量最多,主要位于移植区与宿主组织临界部位,而神经元处于移植区中央。培养的大鼠胚胎神经干细胞可以作为移植替代治疗神经退行性疾病研究的供体细胞源,而移植中的迁移现象值得注意。     

神经干细胞研究进展

神经干细胞研究是当今生命科学研究的热点之一。神经干细胞是神经系统发育过程中保留下来的具有自我更新和多分化潜能的原始细胞。随着对神经干细胞认识的不断深入,其临床应用前景与价值得到了越来越多研究者的肯定。从神经干细胞的生物学特征、来源、培养鉴定、分化及应用等几个方面对目前的研究做一概述。     

神经干细胞和祖细胞

近年来利用细胞培养技术分离的神经干细胞和祖细胞为研究神经发育提供了一条新的途径,研究神经干细胞和祖细胞的增殖、迁移和分化将为阐明神经发育机制提供有力的证据。同时,神经干细胞还为治疗中枢退行性疾病带来了希望。无论是作为脑组织移植的供体,还是作为在体诱导的靶细胞,神经干细胞都为中枢神经系统功能重建和神经再生提供了新的思路。     

神经干细胞迁移浅识

神经干细胞的迁移是近年来神经科学领域研究的热点之一.神经干细胞的增殖、迁移、分化和网络重建的特性为研究中枢神经系统退行性疾病及损伤后功能的恢复奠定了基础,其中神经干细胞的迁移发挥了重要作用.目前已经有大量研究探索神经干细胞的迁移,本文将分别从神经干细胞的迁移现象、神经干细胞迁移的影响因素及其应用意义等方面做一综述.     

乳鼠海马神经干细胞的体外分离、扩增和分化研究

探讨海马神经干细胞（neuralstemcells,NSCs）在体外分离扩增和诱导分化的可行性。无菌条件下分离新生（24h）SD大鼠海马神经干细胞,采用无血清培养和胎牛血清诱导分化。免疫荧光染色技术分别检测诱导前细胞巢蛋白（Nestin）的表达,以及分化细胞的神经元特异性烯醇化酶（neuron specific enolase,NSE）、胶质纤维酸性蛋白（glialfibrillaryacidicprotein,GFAP）的表达,以鉴定细胞类型。流式细胞仪检测神经干细胞分化前后增殖能力的变化。结果显示：从乳鼠海马分离培养的细胞生长状态良好,具有克隆增殖能力,并呈Nestin表达阳性,分化后可出现NSE及GFAP表达阳性的细胞。流式细胞仪检测显示：诱导前,细胞增殖活跃,S＋G2／M期细胞为（36．27±1．99）％,而分化各阶段（3,7,10d）S＋G2／M期细胞比例与诱导前（Ctrl）相比则明显下降（尸〈0．05）,分别为（26．39±1．10）％、（26．33±1．33）％和（24．54±1．12）％。这些结果表明乳鼠海马存在神经干细胞,并具有自我更新和多向分化的潜能,可用于基础和临床的相关研究。     

脊髓神经干细胞的研究进展

近十多年来,人们对神经干细胞的研究进展迅速。本文从生长因子EGF和bFGF的反应性、分化特点、临床应用前景三个方面来综述脊髓神经干细胞的研究进展。为深入开展脊髓神经干细胞的基础研究及临床应用研究提供科学依据。     

神经干细胞研究进展

神经干细胞(neural stem cells, NSCs)是中枢神经系统中保持分裂和分化潜能的细胞,对它的研究和应用已成为近年来脑科学研究的一个重要领域.神经干细胞体外培养技术的建立提供了对其进行研究的有力手段.目前的研究主要集中于神经干细胞在脑中的起源、分布及在中枢神经系统疾病治疗中的应用等方面.     

小鼠胚胎神经干细胞的分离培养及其鉴定

且的探索小鼠胚胎神经干细胞的体外培养方法,并获取高纯度的神经干细胞,为神经干细胞的深入研究提供实验材料。方法无菌条件下分离E15天小鼠胚脑皮质,制成单细胞悬液,在bFGF和B27存在的培养基中培养扩增,通过免疫细胞化学染色鉴定神经干细胞及其子代细胞的分化方向。结果培养的部分细胞在B27和bFGF存在的无血清培养基中可以在体外分裂增殖,同时表达神经干细胞特异性抗原nestin,并在撤出B27和bFGF的有血清培养基中向神经细胞和胶质细胞分化。结论小鼠胚脑皮质存在具有多向分化潜能的神经干细胞,这些细胞可以在体外稳定培养、传代并自然分化,为细胞替代治疗提供了理想的细胞来源。     

Pluripotent stem cell-derived neural stem cells: From basic research to applications

Basic research on pluripotent stem cells is designed to enhance understanding of embryogenesis, whereas applied research is designed to develop novel therapies and prevent diseases. Attainment of these goals has been enhanced by the establishment of embryonic stem cell lines, the technological development of genomic reprogramming to generate induced-pluripotent stem cells, and improvements in vitro techniques to manipulate stem cells. This review summarizes the techniques required to generate neural cells from pluripotent stem cells. In particular, this review describes current research applications of a simple neural differentiation method, the neural stem sphere method, which we developed.     

干细胞的研究进展及临床应用前景

干细胞是一类具有自我复制和分化潜能的影响,它包括胚胎干细胞和成体干细胞。干细胞的发育受多种内在机制和微环境因素的影响。目前人类胚胎干细胞已可在体外培养。最新研究发现,成体干细胞可以横向分化为其他类型的细胞,为干细胞的广泛应用提供了基础。     

室管膜下区神经干细胞与神经发生的研究进展

室管膜下区(subventricular zone,SVZ)存在着神经干细胞(nueral stem cells,NSCs),是成年哺乳动物脑内重要的神经发生区域。神经发生过程极为复杂,包括一系列的生物学事件。在病理状态下,SVZ区的细胞增殖,新生的神经细胞迁移到病灶处,取代或修复受损的细胞,起到保护脑组织的作用。该文就SVZ区的神经干细胞、神经发生过程及病理状态下神经发生的相关研究做一综述。     

Proteomics of neural stem cells

The isolation of neural stem cells from fetal and adult mammalian CNS and the demonstration of functional neurogenesis in adult CNS have offered perspectives for treatment of many devastating hereditary and acquired neurological diseases. Due to this enormous potential, neural stem cells are a subject of extensive molecular profiling studies with a search for new markers and regulatory pathways governing their self-renewal as opposed to differentiation. Several in-depth proteomic studies have been conducted on primary or immortalized cultures of neural stem cells and neural progenitor cells, and yet more remains to be done. Additionally, neurons and glial cells have been obtained from embryonic stem cells and mesenchymal stem cells, and proteins associated with the differentiation process have been characterized to a certain degree with a view to further investigations. This review summarizes recent findings relevant to the proteomics of neural stem cells and discusses major proteins significantly regulated during neural stem cell differentiation with a view to their future use in cell-based regenerative and reparative therapy.     

Counting human neural stem cells

Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as plating cells for immunocytochemistry or for cell transfections. This protocol describes a straightforward and fast method for differentiating between live and dead cells and quantifying the cell concentration and total cell number using a hemacytometer. This procedure first requires detaching cells from a growth surface and resuspending them in media. Next, the cells are diluted in a solution of Trypan blue (ideally to a concentration that will give 20-50 cells per quadrant) and placed in the hemacytometer. Finally, averaging the counts of viable cells in several randomly selected quadrants, dividing the average by the volume of one 1 mm(2) quadrant (0.1 microl) and multiplying by the dilution factor gives the number of cells per l. Multiplying this cell concentration by the total volume in microl gives the total cell number. This protocol describes counting human neural stem/precursor cells (hNSPCs), but can also be used for many other cell types.     

Passaging human neural stem cells

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for therapeutic purposes as well as to explore many fundamental processes of human neural development and pathology. This protocol presents a simple method of culturing and passaging hNSPCs in hopes of standardizing this technique and increasing reproducibility of human stem cell research. The hNSPCs we use were isolated from cadaveric postnatal brain cortices by the National Human Neural Stem Cell Resource and grown as adherent cultures on flasks coated with fibronectin (Palmer et al., 2001; Schwartz et al., 2003). We culture our hNSPCs in a DMEM:F12 serum-free media supplemented with EGF, FGF, and PDGF and passage them 1:2 approximately every seven days. Using these conditions, the majority of the cells in the culture maintain a bipolar morphology and express markers of undifferentiated neural stem cells (such as nestin and sox2).