神经干细胞研究方法

神经干细胞(neural stem cells，NSCs)具有自我更新能力、高增生潜能以及多分化潜能，可分化成神经元、少突胶质细胞和星形胶质细胞等。NSCs对中枢神经系统的发育、维持和修复有十分重要的意义，利用NSCs进行细胞替代治疗和基因治疗有很好的临床应用前景。目前，NSCs的研究已成为近年来医学生物学研究中的一项重大课题，对它的研究主要集中在NSCs的分离、体外纯化培养、鉴定和移植应用等方面，现就其研究方法进行论述。     

胚胎大鼠神经干细胞体外分化的激光共聚焦显微镜观察

目的:采用激光扫描共聚焦显微镜观察体外培养胎鼠大脑皮质神经干细胞(neural stem cells,NSCs)分化情况。方法:利用无血清培养方法,分离培养胚胎大鼠大脑NSCs,进行体外扩增培养、传代;采用溴脱氧尿嘧啶核苷(bromodeoxyuridine,BrdU)掺入、双重免疫荧光细胞化学标记方法和激光扫描共聚焦显微镜,用神经细胞的特异性抗体(神经元β-微管蛋白、胶质纤维酸性蛋白),鉴定NSCs向神经元与星形胶质细胞分化的情况。结果:从胎鼠大脑皮质及皮质下分离的组织,经原代和传代培养均可形成细胞克隆,并表达神经上皮干细胞蛋白(Nestin)抗原。在血清诱导下,分化后的细胞表达神经元、星形胶质细胞2种神经细胞的特异性抗原,NSCs分化为星形胶质细胞神经元的比例分别为(43.70±8.55)%和(23.00±3.69)%。结论:从胎鼠大脑皮质分离出的细胞可获得呈集落样生长的神经干细胞团,并能表达NSCs的特异性抗原;激光扫描共聚焦显微镜可清晰地观察到培养细胞具有分化为神经元和星形胶质细胞的潜能。     

SRC-1基因在小鼠神经干细胞分化过程中的变化

目的观察类固醇受体辅助活化因子-1(SRC-1)基因在小鼠神经干细胞(NSCs)体外培养分化过程中表达的变化。方法用机械分离和酶消化法从1~3 d小鼠大脑皮质获取NSCs原代细胞,体外培养获神经球,传代并消化,经血清诱导神经球细胞分化。形态学、细胞免疫荧光实验观察神经球形态、Nestin表达鉴定NSCs;抗体分别标记神经元、星形胶质细胞,检测细胞分化d 3、9的细胞类型。RT-qPCR和免疫印迹法检测细胞分化d 0、3、9 SRC-1基因mRNA和蛋白的表达。结果新生小鼠大脑皮质获取的细胞,体外培养能扩增形成神经球,Nestin阳性表达。NSCs分化d 3主要为神经元,d 9主要为星形胶质细胞。与NSCs分化d 0相比,SRC-1表达在分化d 3明显升高,在分化d 9明显降低。 结论 成功分离并获取新生小鼠皮质NSCs。在NSCs分化中,SRC-1表达量有明显变化,分布依次为神经元>NSCs>星形胶质细胞。     

胎鼠脑神经干细胞的分离、培养和鉴定

目的 研究胎鼠脑皮质神经干细胞(NSCs)的分离、培养及鉴定方法。方法 从孕15d胎鼠的大脑皮层和海马区脑组织中获取NSCs,在含有B27、表皮生长因子(EGF)和碱性成纤维生长因子(bFGF)的DMEM/F12无血清培养液中培养;传代后用5%胎牛血清培养液诱导NSCs分化。结果 体外分离培养的NSCs在无血清培养液中形成大量的神经球。经3-5代传代的细胞生长稳定。经巢蛋白染色鉴定,大部分为阳性细胞,神经细胞球经胎牛血清培养液贴壁培养后可分化为神经元特异烯醇化酶、胶质纤维酸性蛋白和半乳糖脑苷脂表达阳性的细胞。结论 从孕15d胎鼠大脑皮质和海马组织分离出的NSCs具有自我更新能力和多向分化潜能,其在5%胎牛血清培养液中具有向神经元和神经胶质细胞分化的潜能。     

神经干细胞的研究进展

神经干细胞(NSCs)在临床的相关研究始于上世纪90年代,神经干细胞目前证实可分化成其他多种神经组织细胞,其中最多见的为少突胶质细胞、神经元、星形胶质细胞等。本次的研究从NSCs的培养鉴定方面,生物学特性及其在缺血性损伤、肿瘤、中枢神经系统的退行性疾病治疗等各方面行探讨研究,分析及临床进展,患者在自身的治疗中取体内NSCs应用最为理想。目前自体移植NSCs对于中枢的神经系统损伤的治疗动物实验已得到较多证明,随着医疗科技的进步,NSCs在临床疾病的治疗中将发挥更大的作用。     

脐血源性神经干细胞移植对缺血缺氧性脑损伤新生大鼠的治疗作用

目的 探讨人脐血源性神经干细胞(NSCs)移植治疗缺血缺氧性脑损伤(HIBD)新生大鼠的可行性.方法 7d龄大鼠随机分成NSCs移植(A)组、HIBD模型对照(B)组和正常对照(C)组,每组40只.从人脐血中分离出单个核细胞(UBC-MNCs),定向诱导分化出NSCs并行溴脱氧核苷尿嘧啶(BrdU)标记,经尾静脉移植到HIBD新生大鼠体内.免疫组化法检测移植后1、7、14、21 d各组动物脑组织易损区室管膜前下区(SVZa)神经巢蛋白(Nestin)、神经元特异性烯醇酶(NSE)、胶质纤维酸性蛋白(GFAP)、BrdU染色阳性细胞表达情况及移植细胞在脑内的存活、迁移和分化情况.结果 UBC-MNCs可体外培养并定向诱导分化为NSCs,A组SVZa区NSE、BrdU阳性细胞表达量较B组均增高(P<0.01);移植早期Nestin增高,移植后14 d达峰,以后逐渐下降(P(0.01);A组GFAP表达较B组降低,大鼠损伤区面积显著减小(P<0.01).结论 移植的NSCs可在HIBD新生大鼠SVZa存活、增殖及分化,促进组织结构恢复和细胞功能重建,NSCs移植是治疗HIBD的一种新途径.     

骨髓基质细胞和神经干细胞体外共培养的实验研究

目的:研究神经干细胞(NSCs)和骨髓基质细胞(MSCs)共培养时对各自分化的影响。方法:将2种细胞按接种数目分为5组,A组为NSCs∶MSCs=105∶0;B组为NSCs∶MSCs=105∶104;C组为NSCs∶MSCs=105∶105;D组为NSCs∶MSCs=104∶105;E组为NSCs∶MSCs=0∶105。培养7d后分别进行5-溴-2-脱氧尿嘧啶(BrdU)和神经元特异性烯醇化酶(NSE)、胶质细胞纤维酸性蛋白(GFAP)、微管相关蛋白2(MAP2)、半乳糖神经酰胺(GalC)免疫细胞化学双染色。结果:随着MSCs比例的提高,各组BrdU和NSE或MAP2的双阳性细胞表达差异有统计学意义(P<0.05),且B、C及D组分别与A组比较差异亦有统计学意义(均P<0.01);随NSCs比例的增高,B、C、D组NSE、MAP2和GFAP双阳性MSCs表达逐渐增高(均P<0.01)。结论:MSCs可以促进NSCs分化为神经元,而且在NSCs诱导下部分MSCs可以转化为神经元和星形细胞。     

神经干细胞中Notch和生长因子/细胞因子信号通路间的相互作用

多能神经干细胞增殖和分化的精确控制对神经系统的正常发育是至关重要的。在哺乳动物中枢神经系统发育过程中，3种主要神经细胞来源于神经干细胞（NSCs）：神经元、星形胶质细胞和少突胶质细胞。成纤维细胞生长因子（FGF2）和表皮生长因子（EGF）参与NSCs的调控。在晚期发育中这些生长因子，特别是骨形成蛋白（BMPs）和白介素-6家族细胞因子也调节NSCs对神经胶质原信号的应答。通过Notch跨膜受体的信号则是NSCs另一个重要的调节机制。     

EGB对大鼠神经干细胞分化为星形胶质样细胞的影响

目的：观察银杏叶提取物（EGB）对大鼠海马神经干细胞（NSCs）增殖及分化为星形胶质样细胞的影响。方法：取新生24h内的Wistar大鼠海马组织的细胞进行体外培养，1周后将其接种在培养板中．观察不同浓度的EGB对细胞的生长作用．并检测胶质原纤维酸性蛋白（GFAP）的表达。结果：各实验组NSCs的生长明显好于对照组．免疫细胞化学显示：同一时间内，诱导NSCs分化为星形胶质样细胞的百分率增高；同一浓度的EGB，诱导神经干细胞分化为星形胶质样细胞的百分率随着时间的延长呈上升的趋势。结论：EGB能促进神经干细胞的存活、增殖及向星型胶质样细胞的方向分化。     

NT-3高表达促进神经干细胞向胆碱能神经元分化

目的体外研究神经营养因子3(neurotrophin-3,NT-3)基因转染神经干细胞(neural stem cells,NSCs)后对其向胆碱能神经元分化的影响,并探讨其机制。方法体外分离培养新生小鼠脑源NSCs,免疫荧光细胞化学法对其进行鉴定;将NSCs分为NSCs组(不作任何处理的NSCs)、GFP-NSCs组(转染GFP的NSCs)、NT-3-NSCs组(转染NT-3的NSCs),免疫荧光细胞化学法和ELISA法检测各组NSCs中NT-3的表达;免疫荧光细胞化学法和RT-PCR法检测各组NSCs向胆碱能神经元分化的能力;乙酰胆碱检测试剂盒检测乙酰胆碱分泌情况;RT-PCR法检测Notch信号通路相关靶基因Hes1、Mash 1和Neurogenin 1(Ngn1)表达情况。结果免疫荧光细胞化学法结果显示,NSCs表达其特异性标志蛋白Nestin和Sox2,与NSCs组和GFP-NSCs组相比,NT-3-NSCs组能够分化为更多的胆碱能神经元(P<0.01),分化的胆碱能神经元可分泌乙酰胆碱(P<0.01),且能够减少Notch通路靶基因Hes 1 mRNA的表达,增加Mash1、Ngn 1 mRNA的表达(P<0.05)。结论 NT-3高表达可促进NSCs分化为更多的胆碱能神经元,其机制可能与抑制Notch信号通路有关。     

Endogenous Neural Stem Cells in the Adult Brain

Despite progress in our understanding molecular mechanisms of neuronal cell death in many central nervous system (CNS) diseases, widely effective treatments remain elusive. Recent studies have shown that neural stem cells (NSCs) are present in the subventricular zone (SVZ) lining the lateral ventricles and the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG) in adult mouse, rat, nonhuman primate, and human brain. Newly generated cells in the SGZ can differentiate into mature, functional neurons and integrate into the DG as granule cells, which are involved in memory formation. In addition, many CNS diseases can stimulate the proliferation of neuronal stem/progenitor cells located in the SVZ and SGZ of the adult rodent brain, and the resulting newborn cells migrate into damaged brain regions, where they express mature neuronal markers. Therefore, it might be possible for damaged cells to be replaced from endogenous neural stem cell pools. However, the capacity of self-repair is obviously not enough. Proliferation, migration, and neuronal differentiation of endogenous NSCs could be manipulated by pharmaceutical tools to reach the adequate benefits for the treatment of CNS diseases. This work is supported in part by National Institute of Health (NIH) grant AG21980 (K.J.).     

Adult neural stem cell therapy: expansion in vitro, tracking in vivo and clinical transplantation

Neural stem cells (NSCs) are present not only in the developing nervous systems, but also in the adult human central nervous system (CNS). It is long thought that the subventricular zone of the lateral ventricles and the dentate gyrus of the hippocampus are the main sources of human adult NSCs, which are considered to be a reservoir of new neural cells. Recently adult NSCs with potential neural capacity have been isolated from white matter and inferior prefrontal subcortex in the human brain. Rapid advances in the stem cell biology have raised appealing possibilities of replacing damaged or lost neural cells by transplantation of in vitro-expanded stem cells and/or their neuronal progeny. However, sources of stem cells, large scale expansion, control of the differentiations, and tracking in vivo represent formidable challenges. In this paper we review the characteristics of the adult human NSCs, their potentiality in terms of proliferation and differentiation capabilities, as well as their large scale expansion for clinical needs. This review focuses on the major advances in brain stem cell-based therapy from the clinical perspective, and summarizes our work in clinical phase I-II trials with autologuous transplantation of adult NSCs for patients with open brain trauma. It also describes multiple approaches to monitor adult human NSCs labeled superparamagnetic nanoparticles after transplantation and explores the intriguing possibility of stem cell transplantation.     

Neurogenic drugs and compounds to treat CNS diseases and disorders

Neurological diseases and related conditions affect an estimated 1 billion of individuals worldwide [1]. There is still no cure for neurological diseases and disorders, barely a few treatments more or less efficient. This mandates the design and development of novel paradigms and strategies, to discover and develop new treatments and cures for these diseases. Neurogenesis occurs in the adult brain of mammals primarily in two regions, the dentate gyrus (DG) of the hippocampus and the subventricular zone, in various species including in humans. Neural progenitor and stem cells have been isolated, propagated and characterized in vitro from the adult brain of mammals, including humans. The confirmation that neurogenesis occurs in the adult brain and neural stem cells (NSCs) reside in the adult central nervous system (CNS) of mammals, opens new avenues and opportunities for treating neurological diseases and injuries [2].     

Group I Metabotropic Glutamate Receptors: A Potential Target for Regulation of Proliferation and Differentiation of an Immortalized Human Neural Stem Cell Line

Human neural stem cells (NSCs) from the developing embryo or the subventricular zone of the adult brain can potentially elicit brain repair after injury or disease, either via endogenous cell proliferation or by cell transplantation. Profound knowledge of the diverse signals affecting these cells is, however, needed to realize their therapeutic potential. Glutamate and group I metabotropic glutamate receptors (mGluRs) affect proliferation and survival of rodent NSCs both during embryonic and post‐natal development. To investigate the role of group I mGluRs (mGluR1 and mGluR5) on human NSCs, we differentiated an immortalized, forebrain‐derived stem cell line in the presence or absence of glutamate and with addition of either the group I mGluR agonist DHPG or the selective antagonists, MPEP (mGluR5) and LY367385 (mGluR1). Characterization of differentiated cells revealed that both mGluR1 and mGluR5 were present on the cells. Addition of glutamate to the growth medium significantly increased cell proliferation and reduced cell death, resulting in increased cell numbers. In the presence of glutamate, selective activation of group I mGluRs reduced gliogenesis, whereas selective inhibition of group I mGluRs reduced neurogenesis. Our results substantiate the importance of glutamate signalling in the regulation of human NSCs and may as such be applied to promote proliferation and neuronal differentiation.     

Novel strategies for discovering inhibitors of Dengue and Zika fever

Neurogenesis occurs in discrete regions of the adult brain, particularly the hippocampus. It is enhanced in the hippocampus of animal models and patients with neurological diseases and disorders, such as Alzheimer's disease (AD) and epilepsy. Adult hippocampal neurogenesis is modulated by drugs used for treating AD and depression, particularly galantamine, memantine and fluoxetine. This reveals that adult neurogenesis and newly generated neuronal cells of the adult hippocampus are involved in neurological diseases and disorders and that adult neurogenesis and neural stem cells (NSCs) of the adult hippocampus are the target of drugs used for treating AD and depression. Hence, adult neurogenesis and NSCs open new opportunities for our understanding of the pathology of the nervous system and new avenues to discover and develop novel drugs for treating neurogical diseases and disorders; drugs that would target specifically the NSCs of the neurogenic regions in the adult brain, or neurogenic drugs, and that would reverse or compensate deficits and impairments associated with neurological diseases and disorders, particularly those associated with the hippocampus. Adult NSCs represent a model to discover and develop novel drugs for treating neurological diseases and disorders. These drugs may also have potential for regenerative medicine and the treatment of brain tumors.     

Adult neurogenesis and neural stem cells as a model for the discovery and development of novel drugs

Neurogenesis occurs in discrete regions of the adult brain, particularly the hippocampus. It is enhanced in the hippocampus of animal models and patients with neurological diseases and disorders, such as Alzheimer's disease (AD) and epilepsy. Adult hippocampal neurogenesis is modulated by drugs used for treating AD and depression, particularly galantamine, memantine and fluoxetine. This reveals that adult neurogenesis and newly generated neuronal cells of the adult hippocampus are involved in neurological diseases and disorders and that adult neurogenesis and neural stem cells (NSCs) of the adult hippocampus are the target of drugs used for treating AD and depression. Hence, adult neurogenesis and NSCs open new opportunities for our understanding of the pathology of the nervous system and new avenues to discover and develop novel drugs for treating neurogical diseases and disorders; drugs that would target specifically the NSCs of the neurogenic regions in the adult brain, or neurogenic drugs, and that would reverse or compensate deficits and impairments associated with neurological diseases and disorders, particularly those associated with the hippocampus. Adult NSCs represent a model to discover and develop novel drugs for treating neurological diseases and disorders. These drugs may also have potential for regenerative medicine and the treatment of brain tumors.     

Untangling the functional potential of PSA-NCAM-expressing cells in CNS development and brain repair strategies

Central nervous system (CNS) neural stem cells (NSCs), which are mostly defined by their ability to self-renew and to generate the three main cell lineages of the CNS, were isolated from discrete regions of the adult mammalian CNS including the subventricular zone (SVZ) of the lateral ventricle and the dentate gyrus in the hippocampus. At early stages of CNS cell fate determination, NSCs give rise to progenitors that express the polysialylated form of the neural cell adhesion molecule (PSA-NCAM). PSA-NCAM(+) cells persist in adult brain regions where neuronal plasticity and sustained formation of new neurons occur. PSA-NCAM has been shown to be involved in the regulation of CNS myelination as well as in changes of cell morphology that are necessary for motility, axonal guidance, synapse formation, and functional plasticity in the CNS. Although being preferentially committed to a restricted either glial or neuronal fate, cultured PSA-NCAM(plus) progenitors do preserve a relative degree of multipotentiality. Considering that PSA-NCAM(+) cells can be neatly used for brain repair purposes, there is much interest for studying signaling factors regulating their development. With this regard, it is noteworthy that neurotransmitters, which belong to the micro-environment of neural cells in vivo, regulate morphogenetic events preceding synaptogenesis such as cell proliferation, migration, differentiation and death. Consistently, several ionotropic but also G-protein-coupled neurotransmitter receptors were found to be expressed in CNS embryonic and postnatal progenitors. In the present review, we outlined the ins and outs of PSA-NCAM(plus) cells addressing to what extent our understanding of extrinsic and in particular neurotransmitter-mediated signaling in these CNS precursor cells might represent a new leading track to develop alternative strategies to stimulate brain repair.     

Neural stem cells: the basic biology and prospects for brain repair]

Neural stem cells (NSCs) are multipotential progenitor cells that have self-renewal activities. A single NSC is capable of generating various kinds of cells within the CNS, including neurons, astrocytes, and oligodendrocytes. Because of these characteristics, there is an increasing interest in NSCs and neural progenitor cells from the aspects of both basic developmental biology and therapeutic applications for damaged brain. By understanding the nature of NSCs present in the CNS, extracellular factors and signal transduction cascades involved in the differentiation and maintenance of NSCs, population dynamics and localization of NSCs in embryonic and adult brains, prospective identification and isolation of NSCs, and induction of NSCs into particular neuronal phenotypes, it would be possible to develop a feasible strategy to manipulate cells in situ to treat damaged brain.     

A comparative study of neural and mesenchymal stem cell-based carriers for oncolytic adenovirus in a model of malignant glioma

Glioblastoma multiforme is a primary malignancy of the central nervous system that is universally fatal due to its disseminated nature. Recent investigations have focused on the unique tumor-tropic properties of stem cells as a novel platform for targeted delivery of anticancer agents to the brain. Neural stem cells (NSCs) and mesenchymal stem cells (MSCs) both have the potential to function as cell carriers for targeted delivery of a glioma restricted oncolytic virus to disseminated tumor due to their reported tumor tropism. In this study, we evaluated NSCs and MSCs as cellular delivery vehicles for an oncolytic adenovirus in the context of human glioma. We report the first preclinical comparison of the two cell lines and show that, while both stem cell lines are able to support therapeutic adenoviral replication intracellularly, the amount of virus released from NSCs was a log higher than the MSC (p < 0.001). Moreover, only virus loaded NSCs that were administered intracranially in an orthotopic glioma model significantly prolonged the survival of tumor bearing animals (median survival for NSCs 68.5 days vs 44 days for MSCs, p < 0.002). Loading oncolytic adenovirus into NSCs and MSCs also led to expression of both pro- and anti-inflammatory genes and decreased vector-mediated neuroinflammation. Our results indicate that, despite possessing a comparable migratory capacity, NSCs display superior therapeutic efficacy in the context of intracranial tumors. Taken together, these findings argue in favor of NSCs as an effective cell carrier for antiglioma oncolytic virotherapy.     

磁标记神经干细胞移植治疗脑梗死大鼠MRI示踪及形态学研究

目的:利用磁共振成像(MRI)技术追踪移植的超顺磁性氧化铁标记神经干细胞(NSCs),无创、动态地监测NSCs在活体脑内的存活和迁移。方法:大鼠32只制备右侧大脑中动脉瘤局灶脑缺血/再灌注(MCAO)模型,BrdU标记NSCs,立体定向磁标记NSCs脑内移植,MRI下活体动态追踪脑内移植的磁标记NSCs,实验动物于磁标记NSCs脑内移植第1、5和14天达到观察时相点后,取脑组织做石蜡切片及HE染色。结果:24只大鼠出现明显的神经功能缺失症状。MRI追踪示:磁标记NSCs脑内移植1d后,针道和侧脑室部位有低信号物质存在;磁标记NSCs脑内移植5d后,低信号物质沿胼胝体腹侧迁移;磁标记NSCs脑内移植2周,右侧(病灶侧)侧脑室部位低信号物质向缺血区迁移,其前端已达到缺血区的边缘。脑组织病理学检测:BrdU阳性磁标记NSCs沿胼胝体腹侧向缺血/再灌注区迁移。随着脑内磁标记移植时间的推移,侧脑室区的NSCs向缺血/再灌注区迁移数量逐渐增加。结论:超顺磁性氧化铁颗粒和BrdU双标记的神经干细胞移植至脑梗死大鼠脑内后可迁移到病灶区;MRI成像能够在活体内连续示踪观察神经干细胞的迁移及分布情况。