体外诱导成人骨髓基质细胞分化为神经元样细胞

目的探讨成人骨髓基质细胞(BMSCs)的生物学特性及诱导为神经元样细胞的方法.方法采用Ficoll-Paque液(1.077×103g/L)从成人骨髓中分离BMSC,体外扩增,以碱性成纤维细胞生长因子(bFGF)、表皮生长因子(EGF)、全反式维甲酸(ATRA)、联丁酰基环腺苷酸(dbcAMP)为诱导剂,诱导期间观察细胞形态和数目的变化,并通过免疫细胞化学鉴定神经元烯醇化酶(NSE)、巢蛋白(nestin)、胶质纤维酸性蛋白(GFAP)的表达情况.结果此诱导条件下95%的细胞成为神经元样细胞,免疫组织化学检查显示这些细胞表达NSE、GFAP或nestin.结论bFGF和EGF、ATRA、dbcAMP能诱导成人BMSCs分化为神经元样细胞,而且具有较高的阳性转化率.     

成鼠骨髓基质细胞分化为神经元的体外研究

目的 :通过一定的培养条件 ,使骨髓基质细胞 (BMSC)分化为神经元和胶质细胞。方法 :以含有 EGF、b FGF的培养液培养 BMSC,经传代、换液去除杂质细胞 ,撤掉 EGF、 b FGF并加胶质细胞条件培养液及 BDNF,待细胞分化后进行形态学观察及 NSE、 GFAP染色。结果 :撤掉 EGF、 b FGF并加 BDNF,胶质细胞条件培养液后 2天可见分化细胞 ,NSE阳性细胞占细胞总数的 38.47± 3.2 7% ,GFAP阳性细胞占细胞总数的 5 0 .73± 3.2 6 % ,GFAP阳性细胞占细胞总数的 5 0 .73± 4.6 5 %。结论 :本实验通过 EGF、 b FGF及适宜的培养液成功对骨髓基质细胞进行定向 ,使其转化为神经干细胞并最终诱导其分化为神经元和胶质细胞     

bFGF和N2体外联合定向诱导骨髓间质干细胞向神经元样细胞分化

目的:将成年大鼠骨髓间质干细胞(mesenchymal stem cells，MSCs)体外定向诱导分化为神经元样细胞。方法:成年大鼠骨髓间质干细胞，进行体外扩增、纯化培养，对纯化后的MSCs，使用碱性成纤维生长因子(basic fibroblast growth factor，bFGF)和神经培养添加剂N2进行诱导，使MSCs分化为神经元样细胞，并进行免疫细胞化学方法鉴定。结果：95．6％的MSCs出现形态改变，呈神经元样。免疫细胞化学法鉴定，显示神经元特异性烯醇化酶(NSE)、神经丝蛋白(NF)和神经干细胞标志物巢蛋白(nestin)阳性表达。结论体外MSCs可以分化为神经元样细胞。     

参芪液对成人骨髓问质干细胞的诱导分化作用

目的探讨参芪液体外对成人骨髓间质干细胞(MSC)定向诱导分化为神经元的作用.方法从成人肋骨分离MSC,体外培养扩增,并传代至第5代.含10 mg/mL碱性成纤维细胞生长因子(bFGF)的培养液预诱导24h,再用含不同浓度参芪液的无血清DMEM诱导MSC分化为神经元.免疫组化鉴定神经元烯醇化酶(NSE)、神经丝蛋白(NF)、胶质纤维酸性蛋白(GFAP)的表达.结果成人骨髓间质干细胞在体外扩增5代后,加入参芪液诱导,可见MJSC胞体收缩,突起伸出.免疫组化显示诱导出的神经元样细胞NSE、NF阳性,GFAP阴性.神经分化的定量分析显示NSE、NF阳性细胞分别为(79.5±2.5)%和(76.5±2.3)%.结论参芪液在体外可以诱导成人骨髓间质干细胞分化为神经元样细胞.     

多细胞因子诱导成年鼠骨髓基质细胞向神经细胞分化的实验研究

目的观察大鼠骨髓基质细胞(rBMSCs)的生长特点及诱导条件下分化成神经细胞的能力,并对其机制进行初步探讨。方法以密度梯度离心分离骨髓基质细胞,在神经干细胞培养液中培养,采用四唑盐(MTT)法观察在培养液中添加碱性成纤维细胞生长因子(bFGF)、表皮生长因子(EGF)对BMSCs增殖的影响;观察添加脑源性神经生长因子(BDNF)、神经生长因子(NGF)和维甲酸(RA)对rBMSCs的诱导分化情况;采用免疫组织化学法(ABC)检测诱导后的细胞表达神经元特异性烯醇化酶(NSE)、神经元核蛋白(NeuN)和胶质原性纤维酸性蛋白抗体(GFAP)等特异性标志物的情况;以流式细胞分选确定神经元的比例。结果bFGF和EGF能在体外促进rBMSCs增殖,BDNF、NGF和RA能诱导rBMSCs来源的神经干细胞(NSCs)表达NSE、GFAP等特异性标志物。结论EGF、bFGF、BDNF、NGF、RA及适宜的培养液可使rBMSCs定向转化为NSCs,获得足够的目的细胞,进而分化为神经元样和神经胶质样细胞。     

近交系胎鼠中脑多巴胺能神经元体外定向分化的初步研究

目的体外培养近交系大鼠胚胎腹侧中脑前体细胞(VMP)并诱导其分化为多巴胺能神经元(DN),为研究DN定向分化的分子机制提供细胞模型。方法取材胎龄11d的近交系大鼠胚胎VMP,体外用碱性成纤维细胞生长因子(bFGF)增殖培养7d后换用L-抗坏血酸-2-磷酸酯倍半镁盐(AA-2P)诱导分化为DN,随后进行免疫荧光染色鉴定。结果细胞总数扩增49.76倍,免疫荧光染色显示β-TubulinⅢ阳性的神经元中(71.33±20.42)%为TH阳性的DN,后者占细胞总数的(24.85±12.85)%。结论近交系大鼠VMP经体外原代培养能够得到较高比例的DN,可作为深入研究DN定向分化分子机制的细胞模型。     

骨髓基质干细胞向神经元样细胞分化前后BMP4基因差异表达的初步研究

目的初步探讨骨髓基质干细胞（BMSC）向神经元样细胞分化前后骨形态发生蛋白4（BMP4）基因的表达变化情况。方法体外培养大鼠BMSC,用碱性成纤维细胞生长因子（bFGF）和表皮生长因子（EGF）对其进行诱导,分别于诱导前和诱导后第7天行基因芯片检测BMP4基因表达水平,并采用实时定量PCR进行验证。结果在BMSC向神经元样细胞诱导分化后的芯片表达谱中,BMP4基因的表达明显上调,且实时定量PCR亦证实此结果。结论在BMSC向神经元样细胞的分化过程中,BMP4基因可能发挥着重要的作用。     

体外定向诱导人骨髓基质细胞分化为神经元样细胞三种方法的比较

目的观察比较不同的体外定向诱导培养方法对人骨髓基质细胞(骨髓间充质干细胞)分化为神经元样细胞的影响。方法分离、扩增培养人骨髓基质细胞,分别采用人重组纤维母细胞生长因子(FGF-2)和人重组脑源性神经营养因子(BDNF)、2-巯基乙醇(2-ME)以及硫代甘油(TG)等3种不同的诱导剂,诱导骨髓基质细胞分化为神经元样细胞。用神经元特异性烯醇化酶(NSE)和胶质纤维酸性蛋白(GFAP)免疫组织化学方法对已分化的神经元样细胞进行鉴定和分化率分析。结果经3种诱导剂诱导的人骨髓基质细胞均出现神经元样细胞的分化,胞体呈神经元状,伸出较长轴突样和树突样突起且有分支。免疫组化鉴定显示,诱导分化后的神经元样细胞NSE染色呈阳性,GFAP免疫组化染色均呈阴性,其中FGF-2+BDNF诱导组神经元样细胞分化率为(68.220±5.743)%;2-ME诱导组为(50.700±3.374)%;TG诱导组为(32.240±3.800)%,3组之间差异具有显著性意义(P<0.05)。结论人骨髓基质细胞能够在诱导剂的诱导下发挥横向分化能力,3种体外诱导培养方法均可诱导人骨髓基质细胞分化为神经元样细胞。     

成人骨髓基质细胞体外诱导成神经细胞实验研究

目的探讨骨髓基质细胞的体外培养、扩增及诱导其向神经元样细胞定向分化的方法。方法采用改进的全骨髓培养法,培养来源于正常成人献髓者的骨髓基质细胞。传至第5代,用全反式维甲酸(RA)等细胞因子诱导分化,于诱导后第7d和第14d行神经元细胞特异性抗原神经元特异性烯醇化酶(NSE)、微管相关蛋白-2(MAP-2)、神经丝蛋白(β-tublin)和胶质纤维酸性蛋白(GFAP)鉴定,计数诱导前后的细胞。结果成人骨髓基质细胞经RA 碱性成纤维细胞生长因子(bFGF) 脑源性神经营养因子(BDNF)诱导后可见神经元样细胞出现,经NSE、MAP-2及β-tublin免疫组化和荧光鉴定阳性,NSE表达率达到48.5%±0.2%。结论骨髓基质细胞在体外扩增迅速,纯化需时短,并可在全反式维甲酸等细胞因子诱导下向神经元样细胞分化。     

骨髓基质细胞分化为神经元样细胞后与皮质神经元间突触的建立

目的 观察与大脑皮质神经元共培养的骨髓基质细胞(BMSCs)经诱导分化成神经元样细胞后,与脑皮质神经元之间形成功能性突触的情况.方法 无菌条件下取绿色荧光蛋白(GFP)转基因小鼠骨髓,用贴壁筛选法体外培养获得GFP转基因小鼠BMSCs(GFP-GM-BMSCs),在体外培养、扩增、纯化.取第3代GFP-GM-BMSCs,种植到源于小鼠大脑的原代皮质神经元和胶质细胞中,培养介质为加有20 ng/mL表皮生长因子(EGF)、20 ng/mL碱性成纤维细胞生长因子(bFGF)的无血清培养基(Neurobasal-A+2%B27),体外模拟建立细胞移植的共培养体系.共培养第10天,利用FM1-43荧光染料染色活动突触小泡的特性,通过荧光显微镜观察共培养的两种细胞之间形成的突触.结果 与神经元共培养的GFP-GM-BMSCs在含有EGF、bFGF的无血清培养基中7 d后分化为神经元样细胞.共培养10 d后,FM1-43染色阳性的突触囊泡明显增加,主要位于神经元样细胞胞体、突起及其末端结构上.结论 在体外模拟细胞移植共培养体系中,分化自GFP-GM-BMSCs的神经元样细胞能与神经元之间形成突触样连接.     

胚鼠室管膜及成鼠骨髓神经干细胞分离培养及分化鉴定实验研究

目的 :探寻胚鼠室管膜和成鼠骨髓分离培养神经干细胞 (NSC)的可行性。方法 :将分离的胚脑室管膜组织或成年骨髓细胞分别特殊培养。以细胞克隆及免疫细胞化学方法判断NSC增殖并鉴定NSC、神经元和神经胶质细胞。结果 :两种组织来源细胞在相应培养条件下NSC均有快速增殖。可得到具有长突起并建立有网状联系的神经元及胶质细胞。结论 :由胚鼠室管膜和成鼠骨髓诱导分化NSC是可行的     

A quantitative morphometric analysis of rat spinal cord remyelination following transplantation of allogenic Schwann cells

Quantitative morphometric techniques were used to assess the extent and pattern of remyelination produced by transplanting allogenic Schwann cells into demyelinated lesions in adult rat spinal cords. The effects of donor age, prior culturing of donor cells, prior lesioning of donor nerves, and host immunosuppression were evaluated by transplanting suspensions of 30,000 acutely dissociated or cultured Schwann cells from neonatal, young adult, or aged adult rat sciatic nerves into X-irradiation and ethidium bromide-induced demyelinated dorsal column lesions, with or without co-transplantation of neonatal optic nerve astrocytes. Three weeks after transplantation, spinal cords were processed for histological analysis. Under all Schwann cell transplant protocols, large areas containing many Schwann cell-like myelinated axon profiles could be readily observed throughout most of the lesion length. Within these "myelin-rich" regions, the vast majority of detectable axons showed a peripheral-like pattern of myelination. However, interaxonal spacing also increased, resulting in densities of myelinated axons that were more similar to peripheral nerve than intact dorsal columns. Freshly isolated Schwann cells remyelinated more axonal length than cultured Schwann cells, and cells from younger donors remyelinated slightly more axon length than cells from older donors, but all Schwann cell transplant protocols remyelinated tens of thousands of millimeters of axon length and remyelinated axons at similar densities. These results indicate that Schwann cells prepared under a variety of conditions are capable of eliciting remyelination, but that the density of remyelinated axons is much lower than the myelinated axon density in intact spinal cords.     

Extraordinary synapses of the unipolar brush cell: An electron microscopic study in the rat cerebellum

A neglected type of neuron, termed the unipolar brush cell, was recently characterized in the granular layer of the mammalian cerebellar cortex with several procedures, including light and electron microscopic immunocytochemistry utilizing antibodies to calretinin and neurofilament proteins. Although certain features of the unipolar brush cells were highlighted in these studies, the internal fine structure was partially obfuscated by immunoreaction product. In this study, rat cerebella were prepared for electron microscopy after perfusion fixation and Araldite embedding, and folia of the vestibulo-cerebellum, where unipolar brush cells are known to be enriched, were studied by light microscopy in semithin (0.5–1 μ) sections and by electron microscopy in ultrathin sections. Unipolar brush cells were easily identified in semithin sections immunostained with antibodies to GABA and/or glycine, and cbunterstained with toluidine blue. The unipolar brush cells have a pale cytoplasm and are GABA and glycine negative, while Golgi cells are darker and appear positive for GABA and, for the most part, also for glycine. Sets of identification criteria to differentiate unipolar brush cells from granule and Golgi cells in standard electron micrographs are presented. The unipolar brush cells possess many distinctive features that make them easily distinguishable from other cerebellar neurons and form unusually conspicuous and elaborate synapses with mossy rosettes. The unipolar brush cell has a deeply indented nucleus containing little condensed chromatin. The Golgi apparatus is large and the cytoplasm is rich in neurofilaments, microtubules, mitochondria, and large dense core vesicles, but contains few cisterns of granular endoplasmic reticulum. In addition, unipolar brush cells contain an unusual inclusion, which invariably lacks a limiting membrane and is made up of peculiar ringlet subunits. The cell body usually emits a thin axon and is provided with a single, large dendritic trunk that terminates with a paintbrush-like bunch of branchlets. Numerous nonsynaptic appendages emanate from the cell body, the dendritic stem, and the branchlets. The appendages contain rare organelles and lack neurofilaments. The branchlets contain numerous mitochondria, neurofilaments, large dense core vesicles, and clusters of clear, small, and round synaptic vesicles. They form extensive asymmetric synaptic junctions with of a or two mossy fibers, which indicates minimal convergence of excitatory inputs. Under the postsynaptic densities, the branchlet cytoplasm displays a microfilamentous web. Besides their contact with mossy rosettes, the branchlets form symmetric and asymmetric synaptic junctions with presumed Golgi cell boutons that contain pleomorphic synaptic vesicles, indicating that the unipolar brush cells receive an inhibitory modulation. Some of these junctions are unusually extensive. The branchlets also form asymmetric synapses with granule cell dendrites, in which they represent the presynaptic elements, a feature never described before in the normal cerebellum. A minority of the unipolar brush cells receive mossy fiber contacts directly on the cell body, or on short dendritic branchlets emanating directly from the cell body. Such “enmarron” synapses were previously attributed to Golgi cells. Thus, the unipolar brush cells have complex synaptic features: Besides being specialized to form a powerful link with mossy rosettes, they may also have a paracrine function, and they participate with presynaptic dendrites in the cerebellar microcircuit. © 1994 Wiley-Liss, Inc.     

SD大鼠胚胎神经干细胞分离、培养及鉴定

目的 体外培养并鉴定神经干细胞，为相关实验研究奠定基础。方法 分离SD大鼠胎鼠的间脑，加入神经生长因子EGF和bFGF在神经干细胞条件培养基中克隆培养。用免疫细胞化学方法鉴定分离的神经干细胞。结果 分离培养的细胞具有不断增殖的能力，表达神经巢蛋白（nestin），并能经过诱导分化为神经元和神经胶质细胞。结论 成功建立了神经干细胞的分离培养方法，可用于进一步的实验研究。     

Properties of the ON bistratified ganglion cell in the rabbit retina

The identity of the types of different neurons in mammalian retinae is now close to being completely known for a few mammalian species; comparison reveals strong homologies for many neurons across the order. Still, there remain some cell types rarely encountered and inadequately described, despite not being rare in relative frequency. Here we describe in detail an additional ganglion cell type in rabbit that is bistratified with dendrites in both sublaminae, yet spikes only at light onset and has no response bias to the direction of moving bars. This ON bistratified ganglion cell type is most easily distinguished by the unusual behavior of its dendritic arbors. While dendrites that arborize in sublamina b terminate at that level, those that ascend to arborize in sublamina a do not normally terminate there. Instead, when they reach the approximate radius of the dendrites in sublamina b, they dive sharply back down to ramify in sublamina b. Here they continue to course even further away from the soma at the same level as the branches wholly contained in sublamina b, thereby forming an annulus of secondary ON dendrites in sublamina b. This pattern of branching creates a bistratified dendritic field of approximately equal area in the two sublaminae initially, to which is then added an external annulus of dendrites only in sublamina b whose origin is entirely from processes descending from sublamina a. It is coupled to a population of wide‐field amacrine cells upon which the dendrites of the ganglion cell often terminate. J. Comp. Neurol. 521:1497–1509, 2013. © 2012 Wiley Periodicals, Inc.     

心肌条件培养液与5-氮胞苷诱导骨髓基质干细胞向心肌样细胞的分化

背景：骨髓基质干细胞可以明显改善心肌缺血时的心脏功能，但其直接分化为心肌细胞并参与心功能恢复的机制尚不明确。 目的：探讨心肌条件培养液与5-氮胞苷诱导骨髓基质干细胞分化为心肌样细胞的作用。 方法：全骨髓贴壁法分离培养骨髓基质干细胞，将第6代骨髓基质干细胞随机分为4组：5-氮胞苷+心肌条件培养液联合诱导组；5-氮胞苷组；心肌条件培养液组；基础培养基组(空白组)。用心肌条件培养液和5-氮胞苷诱导3周后用免疫组化法测定各组心肌肌钙蛋白表达，并采用单因素方差分析检验进行统计学分析。 结果与结论：在体外心肌条件培养液可以诱导骨髓基质干细胞向心肌细胞分化，提示其中细胞因子可能起关键作用，但心肌条件培养液的这种作用要弱于化学诱导剂5-氮胞苷的诱导作用，且联合诱导作用更强。     

稳定表达人CNTF诱导PC12细胞分化作用的研究

目的:探讨CNTF对PC12细胞的诱导分化作用。方法:应用转基因技术将pcDNA-S-hCNTF质粒转入COS7细胞进行表达,并对CNTF表达进行检测,然后将hCNTF修饰过的COS7细胞与PC12细胞进行联合培养,4天后对CNTF作用后的PC12进行形态学观察,β-tubulin免疫细胞化学染色及不同培养时间4天,5天后阳性细胞的指数进行分析,同时通过MTF实验对诱导培养后的PC12细胞内增殖进行检测。结果:PC12细胞与hCHTF修饰过的COS7细胞经过4天的联合培养,细胞大部分形态呈现出类似于神经元的形态,胞体立体感较强,并且细胞长出较长的突起。另外细胞呈现β-tubulin反应阳性,并且随培养时间延长,β-tubulin阳性细胞的数目有增加的趋势(从76.6％增加到88.3％),最后,反映细胞增殖程度的OD值为0.328±0.019,明显低于对照组0.586±0.028,0.597±0.032。结论:hCNTF对PC12细胞向神经元分化有明显的促进作用。     

Overlap in the distribution of cholinergic and catecholaminergic neurons in the upper brainstem of the ferret

The distribution of catecholaminergic and cholinergic neurons in the upper brainstem of the ferret were mapped by staining immunohistochemically two adjacent series of sections of brainstem for tyrosine hydroxylase and choline acetyltransferase, respectively. As in other species, large numbers of tyrosine-hydroxylase-positive neurons are localized in the ventral tegmental area (A10), the substantia nigra (A9), and in A8. Tyrosine-hydroxylase-positive neurons in the dorsolateral pontine tegmentum (A4, A6, and A7--the locus coeruleus complex) of the ferret are rather diffusely distributed, as has been observed in other carnivore species such as the cat and the dog, but unlike the cat, these cells in the ferret display a relative uniformity in size and morphology. Choline-acetyltransferase-positive neurons which extend in the ferret's pedunculopontine tegmental nucleus and ventral parabrachial area (Ch5) are relatively large cells that stain intensely for choline acetyltransferase, and their dendrites form prominent bundles in regions where unstained fibre tracts are prevalent. Choline-acetyltransferase-positive neurons distributed in the laterodorsal tegmental nucleus (Ch6) are smaller than the cholinergic cells of Ch5, and they stain less intensely for choline acetyltransferase. Rostrally, there is little overlap between the catecholaminergic cell groups A8, A9, and A10 and the cholinergic cell groups of Ch5 and Ch6. Caudally, the Ch5 neurons extend some considerable extent into the locus coeruleus complex. In the region of overlap, no cells with staining for both tyrosine hydroxylase and choline acetyltransferase were observed, as was ascertained with a double staining method employing a combination of tyrosine hydroxylase immunofluorescence and choline acetyltransferase peroxidase-antiperoxidase immunohistochemistry. In conclusion, the ferret has a typically carnivore pattern for the distribution of catecholaminergic cells in the upper brainstem, and there is a significant overlap between the catecholaminergic and cholinergic cell groups in the dorsolateral pontine tegmentum.     

Thymic innervation in the rat: A light and electron microscopical study

The innervation of the rat thymus was studied by light and electron microscopy in juvenile and aged rats. By light microscopy numerous fine nerves were found in the connective tissue septa penetrating between the thymic lobules. These septa were clearly delineated in the juvenile animals, but indistinct in the aged rats, thus creating the spurious impression that thymic parenchyma contains nerves. In the aged animals the nerves are thicker, tortuous, and more branched than in juvenile animals. Electron microscopy confirms the light microscopic observations: no nerves were found within the thymic parenchyma. The thymic capsule and larger connective tissue septa contain bundles of myelinated and unmyelinated axons, surrounded by a perineural sheath. Within the extraparenchymal compartment, which is greatly enlarged in aged animals, efferent and sensory nerves, devoid of perineurium, were found to contact mainly reticular cells, and in rare instances plasma cells and lymphocytes. The majority of axonal varicosities are not closely related to cellular elements, and, in general, vesicles are relatively infrequent. The possible functional significance of these observations is discussed.