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1.
骨髓基质细胞源性神经干细胞体外分化及电生理特性的研究   总被引:15,自引:4,他引:11  
目的探索骨髓基质细胞(BMSCs)诱导分化为神经干细胞(NSCs),比较血清、维甲酸(RA)、胶质细胞源神经营养因子(GDNF)、脑源性神经营养因子(BDNF)及2-巯基乙醇(2-ME)等不同浓度诱导条件下BMSCs分化情况,以及分化细胞的电生理特性。方法以恒河猴骨髓中分离出的BMSCs为实验对象,利用神经干细胞培养基和RA、GDNF、BDNF、2-ME等生长因子在不同血清浓度下进行培养增殖和诱导分化。Nestin、CD133抗体免疫细胞化学染色鉴定NSCs,NSE、β-tublin鉴定神经元、GFAP鉴定神经胶质细胞。膜片钳检测细胞的电生理特性。结果低浓度血清(2.5%)+RA(0.3mg/L)+GDNF(20μg/L)诱导分化效果较好,且分化的神经元样细胞较未分化细胞的膜特性[静息膜电位(RMP)、膜电容(Cm)、串联电阻值(Rs)]有了显著改变(P﹤0.01)。部分形态成熟的神经元样细胞表现出TTX敏感的快速激活、快速失活的电压依赖性的Na+通道,而未分化细胞却未记录到内向电流;两类细胞均可记录到外向的K+电流,但神经元样细胞的电流峰值强度要显著高于未分化细胞,并且包括两种电流成分:瞬时外向K+电流和延迟整流型的K+电流。结论RA+GDNF及配合使用低浓度血清能够有效诱导骨髓源神经干细胞向成熟神经系细胞分化,且分化的神经元样细胞具有快速激活、快速失活的电压依赖性Na+通道,类似神经细胞的电生理特性。  相似文献   

2.
海马干细胞分化的神经元电生理特征的初步探讨   总被引:12,自引:1,他引:11  
目的探讨大鼠海马干细胞分化的神经元的电生理特征。方法无血清方法分离、培养新生大鼠海马干细胞,诱导其分化为神经元,采用全细胞膜片钳技术记录分化的神经元的静息膜电位、动作电位及离子单通道等电生理指标。结果海马干细胞分化的神经元具有一定的电生理特征,记录到静息膜电位及动作电位,其50%和90%复极化动作电位时程分别为69.75±4.57ms及79.75±6.45ms,并同时记录到三种外向钾电流。结论在现有培养条件下海马干细胞可分化为具有一定电生理特征的神经元。  相似文献   

3.
目的探索大鼠嗅鞘细胞对神经干细胞(NSC)分化的影响,以及分化后神经元电生理特性。方法取新生鼠大脑皮质,原代培养大鼠NSC。NSC分为实验组和对照组,实验组将无血清培养的NSC中加入嗅鞘细胞条件培养液,对照组单纯无血清培养NSC。光镜下观察细胞分化情况,免疫组化法分别检测巢蛋白(nestin)、神经生长因子受体(NGFRp75)、神经丝蛋白(NF200)和胶质纤维酸性蛋白(GFAP)的表达,膜片钳检测神经元电生理特性。结果实验组嗅鞘细胞主要诱导NSC分化为神经元,少量分化为胶质细胞。对照组NSC逐渐萎缩,最终死亡。分化后的神经元记录到快速激活、快速失活能被河豚毒素特异阻断的钠电流,以及慢激活、慢失活能被四乙铵特异阻断的延迟整流性钾电流。结论嗅鞘细胞能诱导NSC分化成神经元,分化后的神经元具有活跃的电生理特性。  相似文献   

4.
NSCs在OECs诱导下定向分化及神经元电生理特性研究   总被引:1,自引:0,他引:1  
目的探索大鼠嗅鞘细胞(OECs)对神经干细胞(NSCs)分化的影响,并记录分化后神经元电生理特性。方法在无血清改良Eagle培养基(DMEM/F12)培养的NSCs中,加入OECs条件培养基,观察分化情况,巢蛋白(nestin)鉴定NSCs、神经丝蛋白200(NF200)鉴定神经元、膜片钳检测神经元电生理特性。结果OECs能促进NSCs分化为神经元,分化后的神经元记录到快速激活快速失活、能被河豚毒素(TTX)特异阻断的钠电流及慢激活慢失活、能被四乙基氯化铵(TEA)特异阻断的延迟整流性钾电流。结论OECs能诱导NSCs分化,分化后的神经元具有活跃的电生理特性,具有替代凋亡、坏死神经元的潜能。  相似文献   

5.
背景:以往研究证实,骨髓间充质干细胞经体外诱导先分化为神经干细胞,然后分化为神经样细胞,但是对于分化的神经样细胞是否具有电生理特性尚不明确。 目的:观察骨髓间充质干细胞诱导分化为神经样细胞的离子通道是否具有电生理特性? 设计、时间及地点:观察性实验,于2005-03在华中科技大学同济医学院附属同济医院神经内科实验室完成。 材料:4周龄Wistar大鼠37只,雌雄不拘,体质量150 g左右。 方法:取Wistar大鼠股骨和胫骨,进行骨髓间充质干细胞原代培养,传至第15~20代融合状态的间充质干细胞置于预诱导培养基中培养24 h后,换用诱导培养基。在倒置显微镜下连续观察间充质干细胞的形态变化,分别采用免疫组织化学和Western Blot对未经诱导分化的间充质干细胞和诱导后第3天的间充质干细胞进行检测。利用电生理膜片箝技术检测50个骨髓间充质干细胞诱导分化后的神经样细胞。 主要观察指标:记录到的钾电流、GABA电流。 结果:①诱导分化前呈梭形细胞,在加入预诱导培养基后未出现明显的形态变化,换用诱导培养基1 h,胞体开始收缩,出现少数较小的卵圆形或纺锤形细胞,诱导24 h后,约60%细胞变成双极形、多极形和锥形,出现类似神经元细胞的形态。②免疫细胞化学检测未经诱导分化的间充质干细胞无NeuN、Nestin、GFAP表达,诱导分化后第3天的间充质干细胞NeuN表达明显增强,有Nestin表达,无GFAP表达。③Western Blot检测在未诱导分化时和预诱导24 h,无NeuN、Nestin表达,Nestin表达在诱导分化后6 h呈强阳性,在诱导分化24 h和48 h逐渐减弱。NeuN在诱导分化后6 h有表达,在诱导分化后24 h和48 h表达增强。④在检测的50个细胞中,共有25个细胞检测到ATP电流(50%)、GABA电流13个(26%)、总钾电流46(92%)、复极化钾电流46(92%)。 结论:在诱导分化的神经样细胞上存在功能性离子通道,具有电生理特性。  相似文献   

6.
背景:人类胚胎干细胞可以在饲养细胞依赖性培养体系和化学限定性培养体系下维持未分化状态,能够在体内外诱导分化成三胚层来源的细胞类型。 目的:比较饲养细胞和化学限定性培养体系对人类胚胎干细胞特性的影响。 方法:将在饲养细胞培养体系下培养27代的人类胚胎干细胞转入到化学限定性培养基体系中培养56代,然后再将其转回到饲养细胞培养体系中,将3种培养条件下的人类胚胎干细胞(饲养细胞培养体系培养70代、化学限定性培养体系培养56代、化学限定性培养体系下培养70代后转回饲养细胞培养体系下培养13代和20代)进行多能性分子标记SSEA4流式分析等检测分析,同时对3种培养条件下人类胚胎干细胞经拟胚体诱导分化后分别检测多能性基因和三胚层分化基因的表达。 结果与结论:人类胚胎干细胞在饲养细胞和化学限定性培养体系下表现出不同的诱导分化倾向,在化学限定性培养体系下表现出向神经诱导分化抑制,这种不同的诱导分化倾向可发生可逆性转换,当人类胚胎干细胞由化学限定性培养体系转回到饲养细胞培养体系时,诱导分化倾向表现出与其在饲养细胞下诱导分化一致的模式。在拟胚体分化中,多能性基因Nanog高可能对诱导分化倾向起着重要作用。与此同时,人类胚胎干细胞SSEA4细胞亚群发生相应的变化,人类胚胎干细胞在饲养细胞和化学限定下培养体系下表现的分化倾向与人类胚胎干细胞亚群所占的比例存在关联。  相似文献   

7.
人胚脊髓神经干细胞的分离培养和鉴定   总被引:1,自引:0,他引:1  
目的:探讨人胚脊髓神经干细胞的体外培养和分化的方法,观察其增殖和分化特点。方法:利用无血清培养和单细胞克隆技术从人胚脊髓组织中分离培养出神经干细胞并用血清诱导其分化,应用免疫荧光细胞化学技术对培养细胞及其分化细胞进行鉴定。结果:从人胚脊髓组织分离的细胞在EGF单独存在时无法形成神经球,在bFGF单独存在时只形成少量神经球,在EGF和bFGF共同存在时形成大量具有连续增殖能力的神经球,表达神经干细胞的标志物Nestin,经血清诱导后分化为神经元、星形胶质细胞和少突胶质细胞并表达特异性抗原NSE、GFAP和CNP。结论:在体外培养条件下可从人胚脊髓组织中培养出神经干细胞,它可为神经干细胞的基础研究和临床应用提供材料。  相似文献   

8.
目的:检测人胚胎干细胞源TH阳性细胞的神经元性电生理特性。方法:采用我们实验室改良后的“无血清四步法经拟胚体培养体系”的方法,体外诱导人胚胎干细胞源性TH阳性细胞,在对其细胞核型及特异性标志物进行检测的基础上,运用全细胞膜片钳记录的方法,检测其细胞膜上电压门控性离子通道的电生理特性。结果:分化前后细胞核型保持正常;诱导得到的细胞形态一致,大多数细胞(>90%)表达多巴胺能神经元的标志β-tubulion和TH,并仍表达神经前体细胞的标志nestin;膜片钳检测显示诱导分化的TH阳性细胞具有神经元性电压门控钠、钾离子通道。结论:人胚胎干细胞经体外定向诱导分化为TH阳性细胞后具有一定的DA能神经元特性,特别是神经元性电生理特性。  相似文献   

9.
目的探讨猫骨髓分离培养、诱导分化神经干细胞的可行性。方法无菌条件下行骨穿,梯度密度离心获取猫骨髓基质细胞,以“神经干细胞培养基”培养,用分化诱导因子进行体外培养和诱导分化。结果猫骨髓基质细胞在相应培养条件下能在体外培养中增殖、分化,克隆形成细胞球(或称“神经球”),这些细胞球能表达神经干细胞特异性抗原nestin,而且能进一步诱导分化出胶质样细胞和神经元样细胞,免疫细胞化学检测可见有胶质源性纤维酸性蛋白抗体(GFAP)和神经元特异性烯醇化酶(NSE)抗原表达。结论猫骨髓基质细胞在一定条件诱导下可分化成神经胶质样和神经元样细胞。  相似文献   

10.
目的探讨新生小鼠端脑组织神经干细胞是否能够分化成胆碱能神经元。方法取新生小鼠端脑组织.用无血清方法分离培养神经干细胞;用克隆培养的方法检验培养细胞的干细胞特性;用免疫荧光细胞化学的方法检测神经干细胞标志巢蛋白(nestin)及干细胞诱导分化后神经元标志微管相关蛋白2(MAP2)、星形胶质细胞标志胶质纤维酸性蛋白(GFAP)、胆碱能标志胆碱乙酰转移酶(CHAT);比较不同的诱导分化条件(5%胎牛血清、5%胎牛血清+碱性成纤维细胞生长因子)对胆碱能神经元分化的影响。结果从新生小鼠端脑组织分离培养出具有自我更新、扩增能力的神经球;各培养基中神经球均为nestin阳性。诱导分化后均能够产生MAP2阳性神经元、GFAP阳性星形胶质细胞以及ChAT阳性的胆碱能神经元。分化培养中加入碱性成纤维细胞生长因子能够提高胆碱能神经元分化的比例。结论新生小鼠端脑组织神经干细胞能够分化成胆碱能神经元。  相似文献   

11.
Ma K  Fox L  Shi G  Shen J  Liu Q  Pappas JD  Cheng J  Qu T 《Neurological research》2011,33(10):1083-1093
Under appropriate culture conditions, bone marrow (BM)-derived mesenchymal stem cells are capable of differentiating into diverse cell types unrelated to their phenotypical embryonic origin, including neural cells. Here, we report the successful generation of neural stem cell (NSC)-like cells from BM-derived human mesenchymal stem cells (hMSCs). Initially, hMSCs were cultivated in a conditioned medium of human neural stem cells. In this culture system, hMSCs were induced to become NSC-like cells, which proliferate in neurosphere-like structures and express early NSC markers. Like central nervous system-derived NSCs, these BM-derived NSC-like cells were able to differentiate into cells expressing neural markers for neurons, astrocytes, and oligodendrocytes. Whole-cell patch clamp recording revealed that neuron-like cells, differentiated from NSC-like cells, exhibited electrophysiological properties of neurons, including action potentials. Transplantation of NSC-like cells into mouse brain confirmed that these NSC-like cells retained their capability to differentiate into neuronal and glial cells in vivo. Our data show that multipotent NSC-like cells can be efficiently produced from BM-derived hMSCs in culture and that these cells may serve as a useful alternative to human neural stem cells for potential clinical applications such as autologous neuroreplacement therapies.  相似文献   

12.
Self-renewing and multipotent neural stem cells are present in the adult human brain. We successfully harvested neural stem cells from mice and humans using misexpressed EGFP proteins under the control of the nestin second intron enhancer. High-level EGFP expressors derived from mouse embryos included a distinct subpopulation of cells that were self-renewable and multipotent. Further, we obtained that neural progenitor cells from rat fetal spinal cords using a neurosphere technique, and demonstrated their ability to divide and differentiate into neurons in vivo, where they were integrated into the host tissue in the injured rat spinal cord with resultant behavioral improvement of the recipient rat. We also harvested tyrosine hydroxylase-positive neurons from a transgenic mouse expressing GFP under the control of the tyrosine hydroxylase promoter, and successfully transplanted them into the striatum of rats with parkinsonism with marked improvement of the neurological symptoms. Since neural stem cells can adapt well in the host CNS, studies should focus on their application as a vector in gene therapy and on the introduction in vivo or ex vivo of genes to control their proliferation and differentiation. Neural stem cells are a potential, useful source for developing new therapy for CNS disorders.  相似文献   

13.
Multipotent precursors similar to stem cells of the embryonic neural crest (NC) have been identified in several postnatal tissues, and are potentially useful for research and therapeutic purposes. However, their neurogenic potential, including their ability to produce electrophysiologically active neurons, is largely unexplored. We investigated this issue with regard to skin-derived precursors (SKPs), multipotent NC-related precursors isolated from the dermis of skin. SKP cultures follow an appropriate pattern and time-course of neuronal differentiation, with proliferating nestin-expressing SKPs generating post-mitotic neuronal cells that co-express pan-neuronal and peripheral autonomic lineage markers. These SKP-derived neuron-like cells survive and maintain their peripheral phenotype for at least 5 weeks when transplanted into the CNS environment of normal or kainate-injured hippocampal slices. Undifferentiated SKPs retain key neural precursor properties after multi-passage expansion, including growth factor dependence, nestin expression, neurogenic potential, and responsiveness to embryonic neural crest fate determinants. Despite undergoing an apparently appropriate neurogenic process, however, SKP-derived neuron-like cells possess an immature electrophysiological profile. These findings indicate that SKPs retain latent neurogenic properties after residing in a non-neural tissue, but that additional measures will be necessary to promote their differentiation into electrophysiologically active neurons.  相似文献   

14.
The electrophysiological properties of potassium ion channels are regarded as a basic index for determining the functional differentiation of neural stem cells. In this study, neural stem cells from th...  相似文献   

15.
We investigated the neurogenic potential of full-term human umbilical cord blood (hUCB)-derived multipotent mesenchymal stem cells (MSCs) in response to neural induction media or coculture with rat neural cells. Phenotypic and functional changes were assessed by immunocytochemistry, RT-PCR, and whole-cell patch-clamp recordings. Naive MSCs expressed both mesodermal and ectodermal markers prior to neural induction. Exposure to retinoic acid, basic fibroblast growth factor, or cyclic adenosine monophosphate (cAMP) did not stimulate neural morphology, whereas exposure to dibutyryl cAMP and 3-isobutyl-1-methylxanthine stimulated a neuron-like morphology but also appeared to be cytotoxic. All protocols stimulated increases in expression of the neural precursor marker nestin, but expression of mature neuronal or glial markers MAP2 and GFAP was not observed. Nestin expression increases were serum level dependent. Electrophysiological properties of MSCs were studied with whole-cell patch-clamp recordings. The MSCs possessed no ionic currents typical of neurons before or after neural induction protocols. Coculture of hUCB-derived MSCs and rat neural cells induced some MSCs to adopt an astrocyte-like morphology and express GFAP protein and mRNA. Our data suggest hUCB-derived MSCs do not transdifferentiate into mature functioning neurons in response to the above neurogenic protocols; however, coculture with rat neural cells led to a minority adopting an astrocyte-like phenotype.  相似文献   

16.
人神经干细胞的分离、克隆和动物脑内移植及转基因表达   总被引:16,自引:3,他引:13  
目的分离和克隆人神经干细胞,并在体外和体内分析其生物学特征.方法我们联合采用四步法从人胚胎前脑分离制备多潜能神经干细胞,并使用重组腺病毒相关病毒载体(rAAV)将LacZ基因和胶质细胞起源的神经营养因子(GDNF)基因转移到神经干细胞.结果二株人神经干细胞被成功建立.这些克隆化后的神经干细胞在细胞培养中和移植到新生小鼠脑内后能发育分化成神经元、少枝胶质细胞和星形胶质细胞.在rAAV转导基因后,神经干细胞可在体外和体内表达转基因产物.结论这种具有转基因表达能力的神经干细胞为神经系统疾病的进一步治疗研究提供了有潜在价值的细胞资源.  相似文献   

17.
Recent evidence indicates that neural stem cell properties can be found among a mammalian skin-derived multipotent population. A major barrier in the further characterization of the human skin-derived neural progenitors is the inability to isolate this population based on expression of cell surface markers. Our work has been devoted to purified human skin-derived stem cells that are capable of neural differentiation, based on the presence or absence of the AC133 cell surface marker. The enriched skin-derived AC133(+) cells express the CD34 and Thy-1 antigens. These cells cultured in a growth medium containing epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) proliferate, forming spheres, and differentiate in vitro into neurons, astrocytes, and rarely into oligodendrocytes. Single cells from sphere cultures initiated from human purified AC133(+) cells were replated as single cells and were able to generate new spheres, demonstrating the self-renewing ability of these stem cell populations. Brain engraftment of cells obtained from human purified AC133(+)-derived spheres generated different neural phenotypes: immature neurons and a most abundant population of well differentiated astrocytes. The AC133-derived astrocytes assumed perivascular locations in the frontal cortex. No donor-derived oligodendrocytes were found in the transplanted mouse brains. Several donor small, rounded cells that expressed endothelial markers were found close to the host vessel and near the subventricular zone. Thus, mammalian skin AC133-derived cells behave as a multipotent population with the capacity to differentiate into neural lineages in vitro and, prevalently, endothelium and astrocytes in vivo, demonstrating the great plasticity of these cells and suggesting potential clinical application.  相似文献   

18.
Discrimination of neural stem cells from other progenitors in the developing mammalian brain has been hampered by the lack of specific markers. Identifying the progenitor pools and signalling pathways that guide mammalian neurogenesis are central to understanding the complex mechanisms that govern development of the nervous system. Notch signalling plays a pivotal role in the development of the mammalian nervous system by maintaining multipotent neural stem cells and regulating their fate. In order to identify putative neural stem cells in situ, we generated transgenic mice that express Green Fluorescent Protein (GFP) and report Notch signalling activity in the developing CNS. Here we show the subdivision of progenitors within the neural tube of these mice. We purify progenitors from the neural tube and show that cells with the highest levels of Notch-reporter activity have self-renewal capability and multipotency, whereas those lacking Hes5 expression do not form neurospheres in vitro. Using marker protein co-expression and cell sorting, we show that both neuroepithelial cells as well as some radial glia at all axial levels of the embryonic neural tube display active Notch signalling. However, Tbr2-positive basal progenitors of the developing telencephalon and differentiating Islet1/2- and Lim1-positive motor neurons outside the ventricular zone do not express Hes5-GFP. Quantitative analysis showed that Hes5 expression correlates better with neural stem cell potential than expression of the related gene Hes1. Thus, Notch activity through Hes5 identifies multipotent progenitors with stem cell properties and subdivides the different progenitors into defined pools.  相似文献   

19.
Existence of multipotent neural stem cells (NSC) has been known in developing or adult mammalian CNS, including humans. NSC have the capacity to grow indefinitely and have multipotent potential to differentiate into three major cell types of CNS, neurons, astrocytes and oligodendrocytes. Stable clonal lines of human NSC have recently been generated from the human fetal telencephalon using a retroviral vector encoding v‐myc. One of the NSC lines, HB1.F3, carries normal human karyotype of 46XX and has the ability to self‐renew, differentiate into cells of neuronal and glial lineages, and integrate into the damaged CNS loci upon transplantation into the brain of animal models of Parkinson disease, HD, stroke and mucopolysaccharidosis. F3 human NSC were genetically engineered to produce L‐dihydroxyphenylalanine (L‐DOPA) by double transfection with cDNA for tyrosine hydroxylase and guanosine triphosphate cylohydrolase‐1, and transplantation of these cells in the brain of Parkinson disease model rats led to L‐DOPA production and functional recovery. Proactively transplanted F3 human NSC in rat striatum, supported the survival of host striatal neurons against neuronal injury caused by 3‐nitropro‐pionic acid in rat model of HD. Intravenously introduced through the tail vein, F3 human NSC were found to migrate into ischemic lesion sites, differentiate into neurons and glial cells, and improve functional deficits in rat stroke models. These results indicate that human NSC should be an ideal vehicle for cell replacement and gene transfer therapy for patients with neurological diseases. In addition to immortalized human NSC, immortalized human bone marrow mesenchymal stem cell lines have been generated from human embryonic bone marrow tissues with retroviral vectors encording v‐myc or teromerase gene. These immortalized cell lines of human bone marrow mesenchymal stem cells differentiated into neurons/glial cells, bone, cartilage and adipose tissue when they were grown in selective inducing media. There is further need for investigation into the neurogenic potential of the human bone marrow stem cell lines and their utility in animal models of neurological diseases.  相似文献   

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