神经干细胞向神经元定向诱导分化方法的研究进展

神经干细胞(NSCs)是一类具有自我更新能力和多种分化潜能的细胞,它来源于神经组织,在适当条件下可分化成神经元、少突胶质细胞、星形胶质细胞和小胶质细胞。NSCs体外诱导分化的成体细胞可以用于中枢神经损伤疾病及退行性神经系统疾病的治疗。因此,找到一系列体外诱导NSCs定向分化的模式,诱导其分化为特定的神经细胞,达到神经修复和细胞治疗的目的乃当今神经科学界的研究热点之一。该文主要阐述NSCs向神经元诱导分化方法的研究进展。     

成年小鼠脑室下区神经干细胞培养与鉴定体系的建立

目的 建立系统的成年小鼠脑室下区神经干细胞分离、培养及鉴定体系.方法 用无血清方法分离培养成年小鼠脑室下区来源的神经干细胞;用克隆培养、BrdU整合的方法检验培养细胞的干细胞特性;用免疫荧光细胞化学方法检测Brdu、神经干细胞标记物nestin和SOX2,分化的细胞标记物Tuj1、GFAP、NG2.用Western boltting和RT-PCR方法进一步检测神经干细胞标记物nestin和SOX2的表达.结果 从成年小鼠脑室下区分离培养出具有自我更新、增殖的神经球,构成神经球的细胞nestin和SOX2呈阳性,它们分化后产生Tujl阳性的神经元、GFAP阳性的星型胶质细胞、NG2阳性的少突胶质细胞.结论 建立了简单、稳定的培养成年小鼠脑室下区神经干细胞方法.     

大鼠胎脑神经干细胞的分离培养及电穿孔转染

目的 分离培养大鼠胎脑神经干细胞(NSC),研究电穿孔技术在NSC中的转染效率.方法 从sD胎鼠脑组织中分离、培养和扩增NSC,利用免疫荧光组织化学技术对NSC及其分化细胞进行鉴定.使用电穿孔技术将质粒pEGFP-N1转染入NSC,利用其表达的绿色荧光蛋白(GFP)作为转染成功的报告物或标记物,在荧光显微镜下根据发出绿色荧光的NSC数量,计算出转染效率.结果 从SD胎鼠脑组织中分离的细胞在体外可长时间自我增殖,原代及传代克隆细胞均表达NSC的特异性抗原巢蛋白,且诱导分化后可表达星形胶质细胞的特异性抗原-胶质纤维酸性蛋白(GFAP)和神经元的特异性抗原-神经元特异性烯醇化酶(NSE).用电穿孔技术转染神经干细胞,其效率为17.9%～69.1%,平均30.5%.结论 本实验成功分离出具有自我增殖和多向分化潜能的胎鼠NSC,并证实电穿孔技术可对其进行高效转染,为进一步研究NSC的转基因治疗提供实验基础.     

神经干细胞移植治疗帕金森病的研究进展

神经干细胞(NSCs)是指具有自我更新能力,多向分化潜能力,并具有提供一定量脑组织能力的一组细胞群。近年来,NSCs的研究已经成为了神经生物学研究的一个热点。研究人员试图将NSCs移植治疗包括帕金森病(PD)在内的神经系统临床疾病,并且在动物模型中已经取得了一定的成果。现就NSCs的特点、NSCs移植治疗PD的策略以及移植过程中的常见难题等予以综述。     

全反式视黄酸对新生大鼠纹状体神经干细胞向神经元样细胞分化的作用

目的观察全反式视黄酸（atRA）体外对神经干细胞（NSCs）的诱导分化作用及其可能的分子机制。方法分离培养新生大鼠纹状体NSCs；以免疫组织化学染色观察不同浓度atRA对NSCs分化的影响；RT-PCR分析视黄酸受体表达情况：结果atRA诱导NSCs向神经元方向分化，其作用在一定范围内呈剂量依赖性，诱导剂量以1．0pmol／L较为适宜。NSCs的RARβ基因的表达对atRA存在剂量依赖性和时间依赖性。结论atRA诱导NSCs向神经元方向分化，此作用与atRA上调细胞的RARβ基因转录水平有关。     

Biological properties of neural progenitor cells isolated from the hippocampus of adult cynomolgus monkeys

Background The existence of neurogenesis in the hippocampus of adult nonhuman primates has been confirmed in recent years, however, the biological properties of adult neural stem cells or neural progenitor cells （NPCs） from this region remain to be extensively explored. The present work was to investigate on the expansion of NSCs/NPCs from the hippocampus of adult cynomolgus monkeys and the examination of their characteristics in vitro. Methods NPCs isolated from the hippocampus of adult cynomolgus monkeys were expanded in vitro in serum-free media containing growth factors, and were then allowed to differentiate by removing mitotic factors. The expansion capacity of NPCs and their differentiation potential were assayed by immunohistochemical and immunocytochemical analysis. Results During primary culture, NPCs underwent cell division, proliferation and aggregation to form neurospheres that were growing in suspension. Without mitotic stimulation, most neurospheres adhered to the culture dish and started to differentiate. Eventually, nearly 12% of the differentiated cells expressed neuron specific marker-β Ⅲ-tubulin （Tuj1） and 84% expressed astrocyte specific marker-fibrillary acidic protein （GFAP）. In addition, the expression of a neural stem cell marker, nestin, was found both in NPCs and in the subgranular zone of adult monkey hippocampus, where NPCs were originally derived. Conclusions NPCs from the hippocampus of adult cynomolgus monkeys can be expanded to some extent in vitro and are capable of differentiating into neurons and astrocytes. Further experiments to promote the in vitro proliferation capacity of NPCs will be required before adult NPCs can be used as a useful cell model for studying adult neurogenesis and cell replacement therapy using adult stem cells.     

福建省多层次养老服务供求现状及对策研究

目的 研究氯化锂对移植到胫神经内的神经干细胞的存活、分化的影响;研究神经干细胞移植和氯化锂处理对宿主神经轴突溃变、炎症细胞浸润的影响.方法 结扎成年大鼠右侧胫神经,将培养的神经干细胞注射到结扎部位远端,腹腔注射氯化锂或生理盐水,1周后用免疫荧光染色方法检测神经干细胞的巢蛋白(Nestin)、神经核抗原(NeuN)、胆碱乙酰转移酶(ChAT)、胶质细胞原纤维酸性蛋白质(GFAP)、神经纤丝蛋白200(NF200)、巨噬细胞抗原(ED1)的表达情况.结果 移植到胫神经内的神经干细胞存活良好,并沿神经轴索间隙向周围扩散.移植后的神经干细胞未见Nestin阳性表达,大部分移植细胞呈GFAP阳性表达,无NeuN或ChAT阳性表达.腹腔注射氯化锂能显著减少移植细胞的GFAP阳性表达比例.胫神经结扎后,神经内轴突溃变明显.与未移植神经干细胞组比较,移植组胫神经内NF200阳性表达明显增多,联合应用氯化锂能进一步增加NF200的表达.应用氯化锂能显著减少神经干细胞移植后胫神经内的ED1表达.结论 氯化锂能减少移植后的神经干细胞向胶质细胞方向的分化,神经干细胞移植联合应用氯化锂可以减少宿主神经轴突的溃变程度,同时能抑制神经干细胞移植后的炎症细胞浸润.     

成年大鼠海马神经干细胞移植治疗脑出血的研究

目的:探讨成年大鼠海马神经干细胞(NSCs)对脑出血的移植治疗作用.方法:从成年SD大鼠的海马中分离、培养、鉴定NSCs,制作大鼠脑出血模型,将BrdU标记的NSCs注入到血肿同侧的尾状核内.不同时间处死大鼠,通过免疫组织化学分析NSCs在体内的分化情况.结果:经Nestin染色证实能从成年大鼠的海马中分离出NSCs,通过免疫组织化学证实移植的NSCs能在出血区分化为神经原和神经胶质细胞.结论:移植的NSCs在大鼠脑内血肿周围能够成活.     

Neural stem cells: Brain building blocks and beyond

Abstract

Neural stem cells are the origins of neurons and glia and generate all the differentiated neural cells of the mammalian central nervous system via the formation of intermediate precursors. Although less frequent, neural stem cells persevere in the postnatal brain where they generate neurons and glia. Adult neurogenesis occurs throughout life in a few limited brain regions. Regulation of neural stem cell number during central nervous system development and in adult life is associated with rigorous control. Failure in this regulation may lead to e.g. brain malformation, impaired learning and memory, or tumor development. Signaling pathways that are perturbed in glioma are the same that are important for neural stem cell self-renewal, differentiation, survival, and migration. The heterogeneity of human gliomas has impeded efficient treatment, but detailed molecular characterization together with novel stem cell-like glioma cell models that reflect the original tumor gives opportunities for research into new therapies. The observation that neural stem cells can be isolated and expanded in vitro has opened new avenues for medical research, with the hope that they could be used to compensate the loss of cells that features in several severe neurological diseases. Multipotent neural stem cells can be isolated from the embryonic and adult brain and maintained in culture in a defined medium. In addition, neural stem cells can be derived from embryonic stem cells and induced pluripotent stem cells by in vitro differentiation, thus adding to available models to study stem cells in health and disease.     

Up-regulation of the transient A-type K+ current (IA) in the differentiation of neural stem cells of the early postnatal rat hippocampus

Background Neural stem cells （NSCs） not only are essential to cell replacement therapy and transplantation in clinical settings, but also provide a unique model for the research into neurogenesis and epigenesis. However, little attention has been paid to the electrophysiological characterization of NSC development. This work aimed to identify whether the morphological neuronal differentiation process in NSCs included changes in the electrophysiological properties of transient A-type K^＋ currents （IA）. Methods NSCs were isolated from early postnatal rat hippocampus and were multiplied in basic serum-free medium containing basic fibroblast growth factor. Potassium currents were investigated and compared using whole-cell patch-clamp techniques and one-way analysis of variance （ANOVA）, respectively. Results Compared with NSC-derived neurons, cloned NSCs （cNSCs） had a more positive resting membrane potential, a higher input resistance, and a lower membrane capacitance. Part of cNSCs and NSC-derived neurons possessed both delayed-rectifier K^＋ currents （IDR） and IA, steady-state activation of IA in cNSCs （half-maximal activation at （21.34=L-4.37） mV） occurred at a more positive voltage than in NSC-derived neurons at 1-6 days in vitro （half-maximal activation at （12.85=L-4.19） mV）. Conclusions Our research revealed a developmental up-regulation of the IA component during differentiation of postnatal NSCs. Together with the marked developmental up-regulation of IDR in vitro neuronal differentiation we have previously found, the voltage-gated potassium channels may participate in neuronal maturation process.