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.     

神经干细胞向神经元分化调控的研究进展

神经干细胞是一类具有分裂潜能和自更新能力的母细胞,它可以通过对称分裂和不对称分裂方式产生神经组织的各类细胞,包括神经元、星形胶质细胞和少突胶质细胞。中枢神经系统受到损伤后,神经元和胶质细胞的损伤导致了临床症状,内源性神经干细胞的修复作用不大,原因是干细胞的数量有限,微环境的不允许。移植的神经干细胞进入体内后,由于受到多种因素的影响,常保持未分化状态或大部分分化为胶质细胞。神经干细胞向神经元分化的调控机制及其影响因素直接决定神经干细胞源性神经元的比例和神经元之间功能性突触的数量。现就其研究进展做一综述。     

脐带血来源干细胞神经分化的研究进展

中枢神经系统损伤后的自身修复能力有限,因而研究者致力于寻找一种合适的细胞进行移植以代替受损的神经细胞修复神经损伤。近年来的研究表明,脐带血干细胞能够在体外诱导条件下向神经样细胞分化,并在动物体内实验中促进神经损伤的恢复,有可能作为一种有效的细胞资源,应用于人类中枢神经系统疾病的细胞替代治疗以及神经保护与支持。     

神经干细胞移植在神经退行性疾病中的研究进展

神经退行性疾病是一类可导致感觉丧失、运动功能丧失和记忆衰竭等症状的难治性疾病,传统治疗方法虽能延缓疾病进展,但局限性明显。而神经干细胞移植作为一种潜在的新型治疗方式能够有效促进神经细胞的功能恢复及组织再生,在神经退行性疾病的治疗应用方面前景广阔。因此,本文通过对神经干细胞的现有来源及其在神经退行性疾病治疗中的研究进展进行综述,以期为神经干细胞移植在神经退行性疾病治疗中的应用提供新的思路。     

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。     

神经干细胞研究介绍

神经干细胞研究是近年脑科学研究的热点,本文综述了神经干细胞的分离培养方法、脑内迁移路线、发育分化的影响因素以及可能的应用前景。     

小分子化合物在干细胞神经分化中的研究进展

神经系统疾病是发生于中枢、周围、自主神经系统的以感觉、运动、意识、自主神经功能障碍为主要表现的疾病,是目前对人类危害最大的疾病之一。随着干细胞生物学的发展,人类在诱导干细胞神经分化方面取得了显著进展,并可能会解决神经修复与再生的关键问题。其中,小分子化合物因其在使用的便捷性、可控性和功能多样性等方面的显著优势,正越来越多地被用于干预和研究干细胞的增殖、分化和重编程等生物学行为。针对小分子化合物诱导干细胞神经分化的研究现状作一概述。     

诱导间充质干细胞定向神经分化的研究进展

间充质干细胞(mesenchymal stem cells,MSCs)具有自我更新和多向分化的潜能,但分化方向缺乏引导性,受基因、细胞因子、内环境等多种因素的影响。干细胞移植是神经性疾病治疗的常用手段,本文就MSCs定向神经分化的诱导方法如神经营养因子、化学诱导剂、光生物调节、中药诱导等及其可能机制进行阐述。     

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。     

Large-scale generation of human iPSC-derived neural stem cells/early neural progenitor cells and their neuronal differentiation

Induced pluripotent stem cell (iPSC)-based technologies offer an unprecedented opportunity to perform high-throughput screening of novel drugs for neurological and neurodegenerative diseases. Such screenings require a robust and scalable method for generating large numbers of mature, differentiated neuronal cells. Currently available methods based on differentiation of embryoid bodies (EBs) or directed differentiation of adherent culture systems are either expensive or are not scalable. We developed a protocol for large-scale generation of neuronal stem cells (NSCs)/early neural progenitor cells (eNPCs) and their differentiation into neurons. Our scalable protocol allows robust and cost-effective generation of NSCs/eNPCs from iPSCs. Following culture in neurobasal medium supplemented with B27 and BDNF, NSCs/eNPCs differentiate predominantly into vesicular glutamate transporter 1 (VGLUT1) positive neurons. Targeted mass spectrometry analysis demonstrates that iPSC-derived neurons express ligand-gated channels and other synaptic proteins and whole-cell patch-clamp experiments indicate that these channels are functional. The robust and cost-effective differentiation protocol described here for large-scale generation of NSCs/eNPCs and their differentiation into neurons paves the way for automated high-throughput screening of drugs for neurological and neurodegenerative diseases.     

Human neural progenitor cells derived from embryonic stem cells in feeder-free cultures

Derivation of human neural progenitors (hNP) from human embryonic stem (hES) cells in culture has been reported with the use of feeder cells or conditioned media. This introduces undefined components into the system, limiting the ability to precisely investigate the requirement for factors that control the process. Also, the use of feeder cells of non-human origin introduces the potential for zoonotic transmission, limiting its clinical usefulness. Here we report a feeder-free system to produce hNP from hES cells and test the effects of various media components involved in the process. Five protocols using defined media components were compared for efficiency of hNP generation. Based on this analysis, we discuss the role of basic fibroblast growth factor (FGF2), N2 supplement, non-essential amino acids (NEAA), and knock-out serum replacement (KSR) on the process of hNP generation. All protocols led to down-regulation of Oct4/POU5F1 expression (from 90.5% to <3%), and up-regulation of neural progenitor markers to varying degrees. Media with N2 but not KSR and NEAA produced cultures with significantly higher (p<0.05) expression of the neural progenitor marker Musashi 1 (MSI1). Approximately 89% of these cells were Nestin (NES)+ after 3 weeks, but they did not proliferate. In contrast, differentiation media supplemented with KSR and NEAA produced fewer NES+ (75%) cells, but these cells were proliferative, and by five passages the culture consisted of >97% NES+ cells. This suggests that KSR and NEAA supplements did not enhance early differentiation but did promote proliferating of hNP cell cultures. This resulted in an efficient, robust, repeatable differentiation system suitable for generating large populations of hNP cells. This will facilitate further study of molecular and biochemical mechanisms in early human neural differentiation and potentially produce uniform neuronal cells for therapeutic uses without concern of zoonotic transmission from feeder layers.     

神经干细胞移植的临床研究进展

神经系统损伤会导致脑内神经干细胞（neural stem cells,NSCs）的扩增以实现自我修复功能,而通过外源细胞移植的方式来加速这一进程,可能是一种更有效的治疗手段。当前,神经干细胞临床研究所面临的主要问题是如何评价细胞在移植后的行为和功能。该文综述了近几年使用神经干细胞移植治疗几种主要神经系统疾病的临床研究成果,并着重关注了干细胞移植后的示踪研究。     

低氧促进神经干细胞向多巴胺能神经元分化

神经干细胞（neural stem cells,NSCs）作为具有多向分化潜能的神经前体细胞,被广泛应用于细胞移植等研究,而低氧不但调节干细胞的体外增殖,在干细胞分化中也具有重要的作用。本文着重探讨了低氧对NSCs分化的调节作用。采用Wistar孕大鼠（E13．5d）,分离胚胎中脑NSCs,加入无血清DMEM／F12培养液（含20ng／mL EGF、20ng／mL bFGF、1％ N2和B27）,3～5d后传代,细胞培养至第三代进行诱导分化,分别在低氧（3％O2）和常氧（20％O2）条件下诱导分化3d,然后在常氧条件下分化成熟5～7d（DMEM／F12含1％FBS、N2和B27）后进行检测。Nestin、NeuN以及TH免疫组织化学鉴定NSCs;流式细胞术分析测定NSCs向TH阳性神经元方向的分化;高效液相色谱测定细胞培养上清液中多巴胺（dopamine,DA）含量。结果显示,分离培养的NSCs均为nestin阳性细胞;低氧可明显促进NSCs向神经元方向的分化;TH阳性神经元比例在常氧和低氧组分别为（10．25±1．03）％和（19．88±1．44）％。NSCs诱导分化7d后,低氧组细胞培养上清液中DA浓度明显增加,约为常氧组的2倍（P〈0．05,n=8）。上述结果表明,3％低氧可促进NSCs向神经元方向,特别是向DA能神经元方向分化。这为NSCs应用于临床治疗帕金森病提供了基础。     

Differentiation profile of brain tumor stem cells： a comparative study with neural stem cells

Understanding of the differentiation profile of brain tumor stem cells （BTSCs）, the key ones among tumor cell population, through comparison with neural stem cells （NSCs） would lend insight into the origin of glioma and ultimately yield new approaches to fight this intractable disease. Here, we cultured and purified BTSCs from surgical glioma specimens and NSCs from human fetal brain tissue, and further analyzed their cellular biological behaviors, especially their differentiation property. As expected, NSCs differentiated into mature neural phenotypes. In the same differentiation condition, however, BTSCs exhibited distinguished differences. Morphologically, cells grew flattened and attached for the first week, but gradually aggregated and reformed floating tumor sphere thereafter. During the corresponding period, the expression rate of undifferentiated cell marker CD 133 and nestin in BTSCs kept decreasing, but 1 week later, they regained ascending tendency. Interestingly, the differentiated cell markers GFAP and β-tubulinlII showed an expression change inverse to that of undifferentiated cell markers. Taken together, BTSCs were revealed to possess a capacity to resist differentiation, which actually represents the malignant behaviors of glioma.     

溶血磷脂酸对离体培养的大鼠胚胎神经干细胞向神经胶质细胞分化的影响

本研究用免疫细胞化学荧光双标技术观察了溶血磷脂酸（lysophosphatidic acid,LPA）对大鼠胚胎神经干细胞（neural stem cells,NSCs）分化为少突胶质细胞（galactocerebroside—positive,Gal-C阳性）和星形胶质细胞（grim fibrillary acidic protein-positive,GFAP阳性）的影响,并且用RT-PCR技术对NSCs可能表达的LPA受体进行分析。结果显示：（1）加入不同浓度（0．010．0μmol／L）LPA,第7天进行检测时,少突胶质细胞数量呈明显的剂量依赖性增加,峰值出现在1．0μmol／LLPA组,少突胶质细胞所占百分比从对照组的8．5％增加到32．6％;（2）星形胶质细胞的分化几乎不受LPA的影响,第7天时各LPA处理组星形胶质细胞百分比与对照组相比均无显著性差异;（3）RT-PCR结果显示,大鼠胚胎NSCs的LPA1和LPA3受体表达明显,而LPA3受体表达很弱。以上结果表明,较低浓度的LPA可能作为细胞外信号,通过LPA1和LPA3受体促进大鼠胚胎NSCs向少突胶质细胞分化和生成,但对星形胶质细胞的分化过程无明显影响。     

胚胎干细胞向血液干细胞定向分化的研究进展

骨髓移植是目前治疗恶性白血病以及遗传性血液病最有效的方法之一。但是HLA相匹配的骨髓捐献者严重短缺,骨髓造血干细胞（hematopoietic stem cells,HSCs）体外培养困难,在体外修复患者骨髓造血干细胞技术不成熟,这些都大大限制了骨髓移植在临床上的应用。多能性胚胎干细胞（embryonic stem cells,ESCs）具有自我更新能力,在合适的培养条件下分化形成各种血系细胞,是造血干细胞的另一来源。在过去的二十多年里,血发生的研究是干细胞生物学中最为活跃的领域之一。小鼠及人的胚胎干细胞方面的研究最近取得了重大进展。这篇综述总结了近年来从胚胎干细胞获得造血干细胞的成就,以及在安全和技术上的障碍。胚胎干细胞诱导生成可移植性血干细胞的研究能够使我们更好地了解正常和异常造血发生的机制,同时也为造血干细胞的临床应用提供理论和实验依据。     

肿瘤干细胞研究进展

肿瘤是危害人类健康的重大疾病。肿瘤的起源,即肿瘤的去分化起源和肿瘤的干细胞起源一直是有争议的,而随着干细胞研究的深入,越来越多的实验结果证实肿瘤起源于干细胞的观点。肿瘤干细胞不仅能够从血液系统恶性肿瘤中分离,乳腺癌实体瘤干细胞的成功分离也证实了肿瘤干细胞的存在。针对细胞特异的表面标记,可以靶向消灭肿瘤干细胞,治疗肿瘤。