Thrombospondin 1 promotes synaptic formation in bone marrow-derived neuron-like cells

In this study, a combination of growth factors was used to induce bone marrow mesenchymal stem cells differentiation into neuron-like cells, in a broader attempt to observe the role of thrombospondin 1 in synapse formation. Results showed that there was no significant difference in the differentiation rate of neuron-like cells between bone marrow mesenchymal stem cells with thrombospondin induction and those without. However, the cell shape was more complex and the neurites were dendritic, with unipolar, bipolar or multipolar morphologies, after induction with thrombospondin 1. The induced cells were similar in morphology to normal neurites. Immunohistochemical staining showed that the number of positive cells for postsynaptic density protein 95 and synaptophysin 1 protein was significantly increased after induction with thrombospondin 1. These findings indicate that thrombospondin 1 promotes synapse formation in neuron-like cells that are differentiated from bone marrow mesenchymal stem cells.     

人脑组织匀浆液诱导大鼠骨髓间质干细胞分化为神经细胞

目的 研究人脑组织匀浆液诱导大鼠骨髓间质干细胞向神经元细胞分化能力。方法从大鼠骨髓分离培养骨髓间质干细胞．经体外增殖，用人脑组织匀浆液诱导骨髓间质干细胞向神经元样细胞分化，应用免疫细胞化学方法对分化的细胞进行鉴定。结果大鼠骨髓间质干细胞可在体外增殖，经人脑组织匀浆液诱导，骨髓间质干细胞可向神经元样细胞分化，且分化率较高，24小时为45％，48小时为78．2％，72小时为88．3％。分化后的细胞表达神经元标志物-神经微丝(NF)和神经元特异性烯醇化酶(NSE)。结论人脑组织匀浆液可诱导大鼠骨髓间质干细胞向神经元细胞分化，从而为骨髓间质干细胞脑内移植与及其分化，以及神经功能的修复提供了基础。     

Differentiation of human adipose-derived stem cells into neuron-like cells by Radix Angelicae Sinensis

Human adipose tissues are an ideal source of stem cells. It is important to find inducers that can safely and effectively differentiate stem cells into functional neurons for clinical use. In this study, we investigate the use of Radix Angelicae Sinensis as an inducer of neuronal differentiation. Primary human adipose-derived stem cells were obtained from adult subcutaneous fatty tissue, then pre-induced with 10% Radix Angelicae Sinensis injection for 24 hours, and incubated in serum-free Dulbecco＇s modified Eagle＇s medium/Nutrient Mixture F-12 containing 40% Radix Angelicae Sinensis to induce its differentiation into neuron-like cells. Butylated hydroxyanisole, a common in- ducer for neuronal differentiation, was used as the control. After human adipose-derived stem cells differentiated into neuron-like cells under the induction of Radix Angelicae Sinensis for 24 hours, the positive expression of neuron-specific enolase was lower than that of the butylated hydroxyani- sole-induced group, and the expression of glial fibrillary acidic protein was negative. Alter they were induced for 48 hours, the positive expression of neuron specific enolase in human adipose-derived stem cells was significantly higher than that of the butylated hydroxyanisole-induced group. Our experimental findings indicate that Radix Angelicae Sinensis can induce human adipose-derived stem cell differentiation into neuron-like cells and produce less cytotoxicity.     

依达拉奉体外定向诱导人间充质干细胞向神经元样细胞的分化

背景：依达拉奉作为新型氧自由基清除剂,一般用于抑制脂质过氧化反应,减轻脑水肿,保护神经细胞。 目的：观察依达拉奉体外定向诱导人骨髓间充质干细胞向神经元样细胞分化的可行性。 设计、时间及地点：细胞学体外观察,于2007-12/2008-09广东医学院附属医院中心实验室完成。 材料：骨髓来源于创伤所致闭合性股骨骨折的成年患者,由广东医学院附属医院骨科提供。依达拉奉由南京先声药业生产,批号P2007123144254453。 方法：无菌抽取的骨髓经肝素化后,采用密度梯度离心法及贴壁筛选法分离获得人骨髓间充质干细胞,传至第5代按1× 108 L-1接种于6孔板内,设立2组,依达拉奉组细胞达50%融合时用含碱性成纤维生长因子、胎牛血清的L-DMEM预诱导24 h,PBS洗涤后再用20 mg/L依达拉奉无血清L-DMEM诱导24 h;空白对照组始终用含体积分数为10%胎牛血清的L-DMEM培养,不加任何预诱导剂和诱导剂。 主要观察指标：诱导分化后细胞形态变化,SP法免疫细胞化学鉴定神经元烯醇化酶、巢蛋白、胶质纤维酸性蛋白及微管相关蛋白2的表达。 结果：体外诱导1 h后,依达拉奉组胞体收缩,2 h后形成较长突起,5 h后呈典型神经元样细胞;空白对照组细胞仍呈对称的梭形,无突起形成。免疫组化结果显示,诱导6 h后依达拉奉组神经元样细胞的胞体及部分突起呈棕黄色,强表达神经元烯醇化酶,弱表达胶质纤维酸性蛋白和巢蛋白,不表达微管相关蛋白2;空白对照组上述4种特异性抗原均呈阴性表达。 结论：人骨髓间充质干细胞经依达拉奉体外诱导后,所分化的细胞具有神经元表型,但还不够成熟,处于向成熟神经元分化的中间阶段。     

川芎嗪联合创伤性脑组织匀浆液诱导骨髓间充质干细胞向神经样细胞分化

背景：川芎嗪和创伤性脑组织匀浆液均可诱导骨髓间充质干细胞向神经样细胞分化。 目的：探讨川芎嗪与创伤性脑组织匀浆液诱导骨髓间充质干细胞向神经样细胞分化的联合效应。 方法：分离培养Wistar大鼠骨髓间充质干细胞后分为4组,加入不同的诱导培养基分别干预：空白对照组、川芎嗪诱导组、创伤性脑匀浆液诱导组和川芎嗪联合创伤性脑匀浆液诱导组。诱导后采用倒差显微镜观察细胞形态变化,并统计不同时段四组细胞分化率。分别取部分细胞进行神经元特异性烯醇化酶染色,胶质纤维酸性蛋白免疫细胞化学与免疫荧光细胞化学双标检测。 结果与结论：川芎嗪及受损大鼠脑匀浆液上清液可诱导大鼠骨髓间充质干细胞向神经元样细胞分化,随诱导时间的延长,诱导分化率越高,具有较重要的应用价值,而神经元特异性烯醇化酶与胶质纤维酸性蛋白在其分化信号中起重要作用。     

细胞密度与骨髓间充质干细胞向神经元样细胞的分化

景：细胞种植密度是影响干细胞分化的因素之一,对于细胞种植密度在骨髓间充质干细胞向神经元样细胞分化过程中的作用尚缺乏深入研究。 目的：观察细胞种植密度对骨髓间充质干细胞诱导向神经元样细胞分化的影响。 方法：采用贴壁培养法分离大鼠骨髓间充质干细胞,传至第4代后将其按2×102,2×103,4×103,8×103,2×104,4×104/cm2种植于六孔板,每组均加入碱性成纤维细胞生长因子+表皮生长因子+维甲酸诱导向神经元样细胞分化,并通过免疫组织化学染色鉴定,计算每组细胞出现神经元样细胞的比例,比较各组的分化率。 结果与结论：各组骨髓间充质干细胞加入诱导剂后均出现神经元样细胞,Nestin、NSE、GFAP细胞化学染色呈阳性。不同种植密度组出现神经元样细胞比例不同,以8×103/cm2组神经元样细胞比例最高,且神经元样存活时间最长,达7 d。结果说明骨髓间充质干细胞向神经元样细胞分化与细胞接种密度有关,过高或过低细胞密度均不利分化。     

碱性成纤维生长因子等诱导间质干细胞分化为神经元样细胞的研究

目的　体外定向诱导成人骨髓间质干细胞 (MSC)分化为神经元样细胞。方法　采用Ficoll Paque液 (10 77g/L)离心分离成人MSC ,体外扩增 ,分别采用含碱性成纤维细胞生长因子 (bFGF)和叔丁对甲氧酚 (BHA)或硫代甘油等试剂的无血清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)、巢蛋白 (nestin)的表达。结果　成人骨髓间质干细胞在体外扩增原代可获得 5× 10 5,10代可获得 2× 10 10 个细胞。加入bFGF和BHA等诱导剂或硫代甘油诱导后 ,MSC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、nestin表达阳性 ,GFAP阴性。结论　成人骨髓间质干细胞在体外可以分化为神经元样细胞。     

Improvement of neurological deficits by intracerebral transplantation of human adipose tissue-derived stromal cells after cerebral ischemia in rats

Mesenchymal stem cells can be expanded rapidly in vitro and differentiated into multiple mesodermal cell types. In addition, their differentiation into neuron-like cells expressing markers typical for mature neurons has been reported. We isolated human adipose tissue stromal cells (hATSCs) from human liposuction tissues and induced neural differentiation with azacytidine. Following neural induction, hATSCs changed toward neural morphology and displayed expression of MAP2 and GFAP. hATSCs, which were labeled with LacZ adenovirus, were injected into the lateral ventricle of the rat brain. Transplanted cells migrated to various parts of the brain, and ischemic brain injury by middle cerebral artery occlusion (MCAo) increased their migration to the injured cortex. Some of the transplanted cells expressed MAP2 and GFAP. Transplantation of hATSCs improved functional deficits in ischemic brain injury induced by MCAo. Intracerebral grafting of BDNF-transduced hATSCs significantly improved motor recovery of functional deficits in MCAo rats. These data indicate that transplanted hATSCs survive, migrate, and improve functional recovery after stroke and that genetically engineered hATSCs can express biologically active gene products and, therefore, can function as effective vehicles for therapeutic gene transfer to the brain.     

音速波状蛋白促进骨髓间充质干细胞向多巴胺能神经元分化

目的探讨音速波状蛋白（Shh）促进人骨髓间充质干细胞（MSCs）体外定向分化为多巴胺能神经元样细胞的作用。方法体外分离、扩增和鉴定人骨髓MSCs。采用不同诱导方案诱导MSCs向神经元和多巴胺能神经元样细胞定向转化后,进行抗神经巢蛋白（Nestin）、神经元特异烯醇化酶（NSE）、神经胶质纤维酸性蛋白（GFAP）、酪氨酸羟化酶（TH）和多巴胺转运体（DAT）等免疫细胞化学染色,并计算阳性细胞百分率。结果实验组诱导后MSCs能分化为具有典型神经元形态的细胞,可见NSE、Nestin、GFAP、TH和DAT等神经细胞标志表达;对照组MSCs细胞形态无明显变化,上述特异性标志物表达均为阴性。实验2组（诱导方案含Shh）与1组（诱导方案不含Shh）的NSE、Nestin、GFAP阳性细胞百分率的差异无统计学意义,但实验2组TH和DAT阳性细胞百分率明显高于实验1组,差异具有统计学意义（P〈0.05）。结论Shh可促进MSCs分化为多巴胺能神经元样细胞。     

Protein hairy enhancer of split-1 expression during differentiation of muscle-derived stem cells into neuron-like cells

Muscle-derived stem cells were isolated from the skeletal muscle of Sprague-Dawley neonatal rats aged 3 days old. Cells at passage 5 were incubated in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) fetal bovine serum, 20 μg/L nerve growth factor, 20 μg/L basic fibroblast growth factor and 1% (v/v) penicillin for 6 days. Cells presented with long processes, similar to nerve cells. Connections were formed between cell processes. Immunocytochemical staining with neuron specific enolase verified that cells differentiated into neuron-like cells. Immunofluorescence cytochemistry and western blot results revealed that the expression of protein hairy enhancer of split-1 was significantly reduced. These results indicate that low expression of protein hairy enhancer of split-1 participates in the differentiation of muscle-derived stem cells into neuron-like cells.     

Effects of olfactory ensheathing cells on the proliferation and differentiation of neural stem cells

BACKGROUND:Olfactory ensheathing cells can promote oriented differentiation and proliferation of neural stem cells by cell-secreted neural factors. OBJECTIVE:To observe the effect of olfactory ensheathing cells on the differentiation and proliferation of neural stem cells. DESIGN,TIME AND SETTING:Cytology was performed at the Department of Neurology,Tongji Medical College,Huazhong University of Science and Technology,China,from September 2007 to October 2008. MATERIALS:Mouse anti-nestin polyclonal antibo...     

嗅鞘细胞对脂肪干细胞诱导分化的影响

目的 比较2种不同方法共培养脂肪干细胞后神经元特异性核蛋白(neuron specific nuclear protein,NeuN)的表达.方法 以transwell小室为共培养载体,实验组中将脂肪干细胞接种于上室,嗅鞘细胞接种于下室,对照组下室无嗅鞘细胞,只加入嗅鞘细胞条件培养液,共培养12 d后观察脂肪干细胞NeuN的表达.结果 共培养12 d后2组都有部分脂肪干细胞表达NeuN,实验组NeuN阳性率为303/345(87.8%),而对照组为120/320(37.5%),差异有统计学意义(χ~2=181.6,P＜0.05).结论 脂肪干细胞在与嗅鞘细胞共培养时更容易向神经元样细胞分化.     

Human placental decidua basalis-derived mesenchymal stem cells differentiate into dopamine neuron-like cells with no response to long-term culture in vitro

Human placental decidua basalis-derived mesenchymal stem cells (DBMSCs) have been identified as valuable sources for cell transplantation. In this study, we found that DBMSCs could be induced to form neural stem cells in the form of neurospheres. These neurospheres were further differentiated into dopamine neuron-like cells with a cocktail of cytokines. The differentiated DBMSCs were verified through the presence of a neuron-like morphology, the expression of specific dopamine neuron makers, and the production of dopamine. In addition, this differentiation capacity of DBMSCs was not affected by long-term culture, and the cells maintained a normal karyotype in vitro. The dopamine neuronal differentiation and the relative safety transplantation potential of DBMSCs may facilitate stem cell therapeutic approaches to Parkinson's disease.     

胰腺导管上皮细胞转分化为胰岛样细胞*☆

目的:培养小鼠胰腺导管上皮细胞并使之转分化为胰腺干细胞及胰岛样细胞,为胰岛移植治疗糖尿病提供细胞来源。方法 分离培养昆明小鼠胰腺导管上皮细胞,以添加有KGF、HGF和烟酰胺的DMEM/F12培养基培养,不同时间取样本于光镜和电镜下观察,检测第1 d和16 d时CK-19、PDX-1免疫化学染色变化并以半定量RT-PCR检测第1d和16d时胰岛素和胰高血糖素基因表达情况,21d时行双硫腙染色试验及葡萄糖刺激的胰岛素释放试验以检验胰岛样细胞的生理功能。 结果 分离第1d,大部分细胞CK-19染色阳性,偶可见PDX-1阳性细胞,16d后,CK-19阳性细胞快速增殖形成细胞团,大部分细胞PDX-1染色阳性; RT-PCR显示培养细胞胰岛素和胰高血糖素表达明显增强,分别增加了5.4倍和6.1倍（P<0.01）;21d时胰岛样细胞团更加成熟,双硫腙着色阳性,且对高糖（15mmol/l）刺激的胰岛素释放较低糖（5.6mmol/l）时增加了1.6倍（P<0.05）。 结论 小鼠胰腺导管上皮细胞在体外培养条件下可增殖,并具有干细胞潜能,可转分化胰岛素分泌细胞。     

体外培养大鼠骨髓间充质干细胞向神经元样细胞分化：Wnt3a信号分子的诱导作用**★

背景：Wnt信号通路是细胞增殖分化的关键调控环节。已有证据显示此通路参与了对神经前体细胞增殖、分化以及决定细胞命运的调控。目前有关Wnt信号通路对间充质干细胞向神经元样细胞分化的作用还少见报道。 目的：寻找促进间充质干细胞向神经元样细胞分化的Wnt信号分子。 方法：采用密度梯度离心法在体外分离培养SD大鼠股骨间充质干细胞并培养。传代后通过形态学和流式细胞学检测细胞表面标志物CD29、CD44、CD34、CD45,筛选并鉴定培养细胞。采用神经营养因子碱性成纤维细胞生长因子分别联合Wnt3a和Wnt5a诱导方案，通过免疫组化和RT-PCR的方法比较Wnt3a、Wnt5a在间充质干细胞向神经元样细胞分化过程中的作用，以碱性成纤维细胞生长因子单独培养为对照。 结果与结论：间充质干细胞经培养、传代后，细胞贴壁生长，形态均一，呈长梭形，流式细胞学检测细胞表面标志物CD29、CD44高表达, CD34、CD45低表达。Wnt3a诱导后细胞巢蛋白、神经元特异性烯醇化酶呈阳性，胶质纤维酸性蛋白无明显表达，诱导后细胞的活力良好；Wnt5a诱导组及对照组巢蛋白呈弱阳性表达，神经元特异性烯醇化酶及胶质纤维酸性蛋白阴性。RT-PCR结果显示，Wnt3a诱导组巢蛋白在诱导前后均有表达，神经元特异性烯醇化酶在诱导后5 d可见明显的扩增条带，10 d后更加明显；胶质纤维酸性蛋白在诱导10 d后有比较弱的扩增条带出现。结果说明Wnt3a分子能够促进体外培养的间充质干细胞向神经元样细胞分化。     

能谱-X线微束分析检测新生大鼠额叶皮质神经干细胞定向分化为神经元样细胞各时段α辅肌动蛋白的表达

BACKGROUND： Alpha-actinin （ a -actinin） plays a key role in neuronal growth cone migration during directional differentiation from neural stem cells （NSCs） to neurons. OBJECTIVE： To detect in situ microdistribution and quantitative expression of a -actinin during directional differentiation of NSCs to neurons in the temporal lobe cerebral cortex of neonatal rats. DESIGN, TIME AND SETTING： Between January 2006 and December 2008, culture and directional differentiation of NSCs were performed at Department of Histology and Embryology, Preclinical Medical College, China Medical University. Immune electron microscopy was performed at Department of Histology and Embryology and Department of Electron Micrology, Preclinical Medical College, China Medical University. Spectrum analysis was performed at Laboratory of Electron Microscopy, Mental Research Institute, Chinese Academy of Sciences. MATERIALS： Basic fibroblast growth factor, epidermal growth factor, brain-derived nerve growth factor, type-1 insulin like growth factor, and a -actinin antibody were provided by Gibco BRL, USA; rabbit-anti-rat nestin monoclonal antibody, rabbit-anti-rat neuron specific enolase polyclonal antibody, and EDAX-9100 energy dispersive X-ray analysis were provided by PHILIPS Company, Netherlands. METHODS： NSCs, following primary and passage culture, were differentiated with serum culture medium （DMEM/F12 ＋ 10% fetal bovine serum ＋ 2 ng/mL brain-derived nerve growth factor ＋ 2 ng/mL type-1 insulin like growth factor）. MAIN OUTCOME MEASURES： Expression of a -actinin in neuron-like cells was quantitatively and qualitatively detected with immunocytochemistry using energy dispersive X-ray analysis. RESULTS： Immunocytochemistry, combined with electron microscopy, indicated that positive α -actinin expression was like a spheroid particle with high electron density. In addition, the expression was gradually concentrated from the nuclear edge to the cytoplasm and expanded into developing neurites, during differentiation of neural stem cells to neurons. Conversely, energy dispersive X-ray analysis indicated that the more mature the neural differentiation was, and the greater the expression of α -actinin. CONCLUSION： The gradual increase of α -actinin expression is related to growth, development, and maturity of differentiated neuron-like cells, in neonatal rat frontal lobe cortex, at different differentiating time points of NSCs to neurons.     

人脐带间充质干细胞体外扩增和向神经元样细胞定向诱导分化的研究

目的探讨人脐带间充质干细胞(MSCs)的体外分离、纯化、扩增和向神经元样细胞的定向诱导分化,以期为脐带MSCs的神经移植提供理论依据。方法无菌条件下收集剖宫产新生儿脐带,酶消化法获取MSCs,进行培养。用流式细胞仪检测MSCs的表面标志。取扩增3,5,10代的MSCs分别向神经元样细胞诱导,用免疫组化和RT-PCR法检测神经元样细胞特异性标志。结果脐带富含MSCs,且脐带MSCs(UCMSCs)强表达CD13、29、CD44、CD105,弱表达CD106,不表达CD34、CD11a、CD14、CD33、CD45。神经条件培养基诱导后的细胞平均有70%左右呈现典型的神经元样表型。免疫组化法检测发现不同代数的MSCs经诱导后均表达nestin,NSE,NeuN,NF-M,弱表达GFAP。RT-PCR显示诱导后NSEmRNA表达增加。结论MSCs存在于人脐带中,并且在体外有较强的增殖能力,特定条件下能够分化为神经元样细胞。     

猫骨髓源性神经干细胞诱导分化的实验研究

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。