人骨髓间质干细胞的分离培养和诱导分化为神经元样细胞的研究

目的:探索成人骨髓间充质干细胞(MSCs)的分离培养及诱导分化为神经元样细胞的体外条件。方法:采用Ficoll淋巴细胞分离液(1.077g·mL-1)密度梯度离心法从人的骨髓中分离出MSCs进行体外扩增。收集第2代细胞流式细胞仪检测MSCs表面标志。逐渐加入表皮生长因子(EGF)、β-巯基乙醇(BME)和全反式维甲酸(ATRA)诱导MSCs向神经元细胞分化,通过免疫细胞化学法鉴定诱导后细胞神经元烯醇化酶(NSE)、神经巢蛋白(nsetin)、胶质纤维酸性蛋白(GFAP)的表达情况。结果:流式细胞仪检测结果显示培养后的MSCs强表达CD29;较强表达CD44、CD166和CD105;不表达CD45、CD34和HLA-DR。诱导剂诱导后,MSCs分化为具有典型的神经元形态的细胞,免疫细胞化学显示78%的细胞NSE表达阳性;大约51%细胞nsetin表达阳性;所有细胞GFAP均阴性表达。结论:成人MSCs在体外可定向诱导分化为神经元样细胞,且具有较高的阳性分化率。     

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。     

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。     

碱性成纤维细胞生长因子等体外诱导成人骨髓间充质干细胞分化为神经元样细胞的研究

目的 成人骨髓间充质干细胞(hMSCs)体外定向诱导分化为神经元样细胞。方法 采用Percoll分离液离心分离hMSCs，体外扩增，分别采用含碱性成纤维细胞生长因子(bFGF)和2-巯基乙醇(2-ME)等无血清DMEM诱导hMSCs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)。结果 hMSCs在体外扩增传至5代后，流式细胞仪显示99．5％，97．8％，98．8％hMSCs表面抗原CD29、CD44、CD90表达阳性。接种到12孔板，3d后加入bFGF和2-ME联合或2-ME单种诱导剂诱导后，hMSCs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表达阳性，GFAP阴性。结论 成人骨髓间干细胞在体外可以分化为神经元样细胞。     

骨髓间充质干细胞分化为神经元样细胞后电生理特性的改变

背景：目前体外实验对骨髓间充质干细胞来源的神经元样细胞的研究多集中于形态学层面和神经标志物方面,对分化后的电生理功能研究较少。 目的：观察脑源性神经营养因子/碱性成纤维细胞生长因子/全反式维甲酸诱导Wistar大鼠骨髓间充质干细胞分化为神经元样细胞后电生理特性的变化。 设计、时间及地点：细胞学体外培养,对比观察,于2005-06/2007-10在天津市环湖医院细胞室和南开大学生命科学院完成。 材料：6周龄雄性Wistar大鼠3只,体质量160 g左右。 方法：贴壁培养法体外分离纯化间充质干细胞,用脑源性神经营养因子/碱性成纤维细胞生长因子/全反式维甲酸联合诱导间充质干细胞向神经元样细胞分化。诱导前和诱导3 d后分别用膜片钳技术检测细胞膜电流。 主要观察指标：流式细胞仪检测间充质干细胞表型;倒置显微镜观察诱导分化前后细胞形态变化;免疫细胞化学鉴定神经元特异性烯醇化酶的表达,以及全细胞电流测定结果。 结果：①流式细胞仪检测结果显示,CD90阳性率(99±3)%,CD31阳性率(3.4±0.8)%,CD34阳性率(0.3±0.1)%。说明这一细胞群大部分处于未分化的干细胞状态,其纯度可达95%。②光镜下可见未经诱导的间充质干细胞多为扁平形带突起的细胞,似纤维样细胞,诱导3 d后出现神经元样细胞。③免疫细胞化学结果显示,诱导前间充质干细胞的神经元特异性烯醇化酶呈弱阳性,诱导后呈强阳性。诱导72 h时分化率为(24.01±3.76)%。④诱导组神经元样细胞外向电流峰值及最大外向电流密度高于对照组(P < 0.05),但未发现内向钠电流。 结论：脑源性神经营养因子/碱性成纤维细胞生长因子/全反式维甲酸诱导方法可以诱导间充质干细胞向神经元方向分化,虽未发现具有成熟神经元电生理功能,但有向成熟神经元分化的趋势。     

骨髓间充质干细胞体外诱导分化为神经元样细胞的实验研究

目的　探索骨髓间充质干细胞 (MSCs)能否在体外诱导分化成神经样细胞 ,并初步探讨其分化机制。方法　培养大鼠MSCs,用二甲亚砜 (DMSO)和丁羟茴醚 (BHA)诱导分化 ,鉴定诱导分化前后的细胞是否表达神经细胞及神经干细胞的特异性标记蛋白 ,并研究其超微结构的变化。结果　诱导分化后 ,大部分MSCs变成双极、多极和锥形 ,并相互交织成网络结构 ,出现神经元特异性核蛋白 (NeuN)和巢蛋白 (Nestin)表达 ,无胶质纤维酸性蛋白(GFAP)和 2 3 环核苷酸磷酸二脂酶 (CNP)表达。部分MSCs转变为典型的神经元超微结构。结论　MSCs可以在体外诱导分化为神经元样细胞。     

大鼠骨髓间充质干细胞体外培养及分化为神经干细胞的实验研究

目的研究大鼠骨髓间充质干细胞(bone marrow mesenchymal stemcells,MSCs)体外分化为神经干细胞(neural stemcells,NSCs)的可能性。方法取4～6周SD大鼠双侧股骨和胫骨骨髓细胞进行体外培养,通过传代得到纯化的MSCs后换用分化液诱导,通过形态学观察诱导后细胞形态变化,并用免疫荧光检测不同天数细胞nestin的表达率;NSCs分化实验对所诱导的NSC的分化潜能进行检测。结果MSCs诱导后的细胞nestin表达率随天数增加逐渐升高,1周后形成高表达nestin的神经干细胞球形细胞团;在含血清培养基中可自然分化为神经元和神经胶质样细胞。结论MSCs能于体外分化出神经干细胞,且具有进一步的分化能力,骨髓来源的神经干细胞将可能作为种子细胞用于神经系统疾病的治疗。     

神经节苷脂对人骨髓间充质干细胞诱导分化为神经元样细胞生长状态的影响

目的探讨神经节苷脂对人骨髓间充质干细胞(hMSCs)诱导分化为神经元样细胞后生长状态的影响。方法MSCs由成年人骨髓经密度梯度离心加贴壁培养法获得,取第4～6代MSCs以10ng/ml bFGF预诱导24h后,用丁羟基茴醚(BHA)、二甲基亚砜(DMSO)和不同浓度神经节苷脂(25mmol/L、50mmol/L、75mmol/L)诱导分化,采用免疫细胞化学法鉴定神经细胞特异性抗原标记神经元特异性烯醇化酶(NSE)、微管相关蛋白-2(MAP2)和神经胶质相关蛋白(GFAP)的表达。唑盐比色实验(MTT法)检测不同浓度神经节苷脂对诱导后5h、24h、2d、3d细胞生长情况的影响。结果成功分离培养人MSCs,诱导分化后大部分MSCs变成神经元样细胞并表达NSE和MP2。中等浓度(50mmol/L)神经节苷脂组诱导后的细胞生长活力较低浓度和高浓度组好(P<0.05)。结论中等浓度神经节苷脂能使诱导后的细胞保持较好的生长状态。     

全反式维甲酸诱导大鼠骨髓间充质干细胞分化为神经元样细胞的研究

目的 体外探讨全反式维甲酸（ATRA）对大鼠骨髓间充质干细胞（MSCs）分化为神经元样细胞的作用。方法 从大鼠股髓分离出MSCs，采用贴壁法体外扩增、传代，取5代后细胞采用EGF共培养，二巯基乙醇（2-ME）预诱导，再以ATRA为诱导剂诱导，然后免疫细胞化学检测巢蛋白（nestin）、神经元特异性烯醇化酶（NSE）、胶质纤维酸性蛋白（GFAP）、乙酰胆碱酯酶（AchE）、酪氨酸羟化酶（TH）的表达情况。结果 MSCs诱导后具有神经元样细胞的形态，免疫细胞化学显示nestin、NSE、AchE有阳性表达，而GFAP、TH表达为阴性。结论 ATRA在体外能够有效的诱导大鼠骨髓间充质干细胞分化为神经元样细胞，且可表达AchE。     

人脐带间充质干细胞向神经细胞分化的研究

目的研究人脐带分离的间充质干细胞向神经细胞分化的可能性。方法将剔除动静脉的新鲜人脐带组织切成小块培养,得到贴壁细胞;经传代培养、细胞周期分析、流式细胞检测后,再以不同方案诱导向其神经细胞分化,并以免疫荧光和RT-PCR方法进行鉴定。结果培养5-7d后,有细胞从组织块中游出。细胞传代培养达23代后无明显的形态和增殖能力改变。细胞周期分析表明80%以上的细胞都处于G0~G1期。流式细胞检测表明这些细胞表达CD13、CD29、CD44、CD90、CD105和CD166等MSCs标志物。经神经分化诱导后,部分细胞呈现出与神经元或神经胶质细胞类似的形态;免疫荧光检测表明,第二神经分化诱导方案优于第一方案,其NSE和MBP阳性细胞分别达80.8%±3.9%、4.2%±1.3%,但未能见到GFAP阳性细胞。RT-PCR进一步证实了这些神经标志物的表达。结论人脐带间充质干细胞具有向神经细胞分化的潜能,可作为神经系统疾病细胞移植治疗的备选来源。     

Adult Bone Marrow Stromal Cells Differentiate into Neural Cells in Vitro

Bone marrow stromal cells (BMSC) normally give rise to bone, cartilage, and mesenchymal cells. Recently, bone marrow cells have been shown to have the capacity to differentiate into myocytes, hepatocytes, and glial cells. We now demonstrate that human and mouse BMSC can be induced to differentiate into neural cells under experimental cell culture conditions. BMSC cultured in the presence of EGF or BDNF expressed the protein and mRNA for nestin, a marker of neural precursors. These cultures also expressed glial fibrillary acidic protein (GFAP) and neuron-specific nuclear protein (NeuN). When labeled human or mouse BMSC were cultured with rat fetal mesencephalic or striatal cells, a small proportion of BMSC-derived cells differentiated into neuron-like cells expressing NeuN and glial cells expressing GFAP.     

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。     

兔间充质干细胞诱导纹状体神经干细胞分化为神经细胞的研究

目的研究兔纹状体间充质干细胞诱导神经干细胞分化为神经细胞的可行性和机制。方法培养骨髓间充质干细胞(BMSCs)融合达90%时,更换培养基无血清培养24h,收集细胞培养液即为其条件培养基。取成年兔纹状体细胞进行神经干细胞培养,加BMSC条件培养基进行神经干细胞的诱导分化。结果2～4h后神经干细胞开始贴壁,随后有突起从干细胞长出,7d后出现大量不同形态的分裂增殖新生神经细胞,较对照组神经元数量增加,有显著性差异,细胞折光性强,突起长,生存时间延长。结论间充质干细胞能提高神经干细胞诱导分化为神经元的数量,并能延长神经元的生存时间。     

内皮祖细胞与卒中

内皮祖细胞（endothelial progenitor cells,EPC）作为成年干细胞的一种,近些年对其研究较热,但多数集中在心血管病方面,在卒中方面的研究有限。对动物和人类的研究表明EPC可作为血管病的预测因子,并且EPC以及对EPC的调节有可能用于脑血管病的治疗。本文对EPC在卒中方面的研究进展做一综述。     

Immunohistochemical, Morphological and Ultrastructural Resemblance between Dendritic Cells and Folliculo-Stellate Cells in Normal Human and Rat Anterior Pituitaries

Immunolabeling of cryo-sections of human anterior pituitaries obtained at autopsy, and of cryo-sections of freshly prepared rat anterior pituitaries, with a panel of monoclonal antibodies against markers of the monocyte/dendritic cell/macrophage lineage, reveals in both species a characteristic pattern of immunopositive cells, among which many cells with dendritic phenotype are found. Cells characterized by marker expression of MHC-class II determinants and a dendritic morphology are present in both human and rat anterior pituitary. Markers characteristic of dendritic cells such as the L25 antigen and the OX6 antigen were present in anterior pituitaries from human and rat respectively. The population of MHC-class II expressing dendritic cells of the rat anterior pituitary is compared at the ultrastructural level with the folliculo-stellate cell population, which cell type has been previously characterized by its distinctive ultrastructure and immunopositivity for the S100 protein. Using immuno-electron microscopy of rat anterior pituitaries fixed with periodate-lysine-paraformaldehyde, we were able to distinguish non-granulated cells expressing MHC-class II determinants, whereas no MHC-class II expression was found in the granulated endocrine cells. Using double immunolabeling of cryo-sections of these rat AP with 25 nm and 15 nm gold labels, we demonstrated an overlap between the populations of MHC-class II-expressing and S100 protein-expressing cells. Furthermore, MHC-class II-expressing and S100-positive cells showed ultrastructural characteristics that have been previously ascribed to folliculo-stellate cells. At the light microscopical level in the rat AP, a proportion of 10 to 20% of the S100-positive cells was found immunopositive for the MHC-class II marker OX6. In the human AP, S100-positive folliculo-stellate cells and cells expressing the leukocyte common antigen CD45 were found to occupy predominantly different tissue compartments in the human anterior pituitary, namely the epithelial parenchyme cords and perivascular compartments respectively. A proportion of CD45⁺ cells was found in the parenchyme compartment and, vice versa, indicating an overlap of the tissue compartments in which both cell types occur. However, at the light microscopical level we could not find cells expressing both the S100 and CD45 marker. The present finding of a proportion of S100-positive pituitary cells with ultrastructural and immunohistochemical characteristics of both dendritic cells and folliculo-stellate cells, confirms the suggested heterogeneity of the latter cell group with respect to their ultrastructural phenotype and putative function. The possibility of a myeloid origin of part of the folliculo-stellate cell group in the AP, is discussed and might elucidate some of the discrepancies in the literature concerning the embryological origin of this cell group.     

大鼠骨髓基质细胞生物学特性研究

目的 观察大鼠骨髓基质细胞的生物学特性。方法 取SD大鼠骨髓，分离培养骨髓基质细胞，相差显微镜下观察其形态，传代后MTT法绘制其生长曲线。结果 原代培养20天后可分离得到骨髓基质细胞，典型的骨髓基质细胞可分为两个类型，传代后2小时贴壁率达70％以上。结论 骨髓基质细胞具有多态性和贴壁生长特性,通过贴壁培养方法能够较容易地对其进行分离扩增，可作为多种疾病细胞治疗和基因治疗的载体。     

Synaptic Integration in Cerebellar Granule Cells

To understand the function of cerebellar granule cells, we need detailed knowledge about the information carried by their afferent mossy fibers and how this information is integrated by the granule cells. Recently, we made whole cell recordings from granule cells in the non-anesthetized, decerebrate cats. All recordings were made in the forelimb area of the C3 zone for which the afferent and efferent connections and functional organization have been investigated in detail. Major findings of the study were that the mossy fiber input to single granule cells was modality- and receptive field-specific and that simultaneous activity in two and usually more of the afferent mossy fibers were required to activate the granule cell spike. The high threshold for action potentials and the convergence of afferents with virtually identical information suggest that an important function of granule cells is to increase the signal-to-noise ratio of the mossy fiber–parallel fiber information. Thus a high-sensitivity, noisy mossy fiber input is transformed by the granule cell to a high-sensitivity, low-noise signal.     

脑源性神经营养因子诱导大鼠骨髓基质细胞成为神经干细胞及其分化作用的实验研究

目的探讨脑源性神经营养因子（BDNF）诱导大鼠骨髓基质细胞（BMSCs）成为神经干细胞及其分化作用。方法取大鼠BMSCs。分别以BDNF和BDNF＋RA（维甲酸）作为诱导物诱导，于诱导3d、7d后行巢蛋白（Nestin）、神经元特异烯醇化酶（NSE）、胶质纤维酸性蛋白免疫细胞化学染色。结果后BDNF和BDNF＋RA诱导组均有大量Nestin染色阳性细胞，BDNF＋RA组阳性率高于BDNF组（P〈0．01）。NSE、GFAP免疫阳性细胞在诱导3d后也有少量表达。诱导7d后BDNF和BDNF＋RA诱导组Nestin阳性细胞明显减少．与诱导3d后比较差异有显著性（P〈0．01），而NSE、GFAP阳性细胞敷增多，与诱导3b比较差异有显著性（P〈0．01），且BDNF＋RA组阳性率高于BDNF组（P〈0．01）。结论联合应用BDNF与RA可提高BMscs神经转化．并促进其向神经元及星形胶质细胞细胞分化。     

Enrichment of Neurons and Neural Precursors from Human Embryonic Stem Cells

Human embryonic stem (hES) cells proliferate and maintain their pluripotency for over a year in vitro (M. Amit, M. K. Carpenter, M. S. Inokuma, C. P. Chiu, C. P., Harris, M. A. Waknitz, J. Itskovitz-Eldor, and J. A. Thomson. 2000. Dev. Biol. 227: 271-278) and may therefore provide a cell source for cell therapies. hES cells were maintained for over 6 months in vitro (over 100 population doublings) before their ability to differentiate into the neural lineage was evaluated. Differentiation was induced by the formation of embryoid bodies that were subsequently plated onto appropriate substrates in defined medium containing mitogens. These populations contained cells that showed positive immunoreactivity to nestin, polysialylated neural cell adhesion molecule (PS-NCAM) and A2B5. After further maturation, these cells expressed additional neuron-specific antigens (such as MAP-2, synaptophysin, and various neurotransmitters). Calcium imaging demonstrated that these cells responded to neurotransmitter application. Electrophysiological analyses showed that cell membranes contained voltage-dependent channels and that action potentials were triggered by current injection. PS-NCAM and A2B5 immunoselection or culture conditions could be used to produce enriched populations (60-90%) which could be further differentiated into mature neurons. The properties of the hES-derived progenitors and neurons were found to be similar to those of cells derived from primary tissue. These data indicate that hES cells could provide a cell source for the neural progenitor cells and mature neurons for therapeutic and toxicological uses.