兔胚胎神经干细胞的分离、培养和鉴别

目的：研究兔胎脑神经干细胞体外生长特性,为探讨神经干细胞的临床应用及神经系统的发育奠定基础。方法：采用含碱性成纤维细胞生长因子（bFGF）和表皮细胞生长因子（EGF）的N2无血清培养技术,取18天龄兔胚胎脑组织,分离神经干细胞,并观察分离的细胞体外培养、增殖、分化潜能,免疫组化鉴定。结果：从18天龄兔胎脑皮质和纹状体中成功分离出具有自我更新和多分化潜能的神经干细胞,在无血清培养时细胞呈半贴壁状态生长,形成神经球,可传代。细胞呈Nestin免疫反应阳性;在含血清培养基中培养时则分化,分化后的细胞表达神经元细胞、星形胶质细胞和少突胶质细胞的特异性抗原。结论：来自兔胎脑神经干细胞能在体外培养、增殖并保持传代能力。无血清N2EGF、bFGF培养基有利于兔胎脑神经干细胞的存活和增殖,含血清培养基能诱导兔胎脑神经干细胞分化。     

人胚与鼠胚神经干细胞体外培养的差异

为比较人胚与鼠胚神经干细胞体外培养的差异。实验采用具有丝裂原作用的细胞生长因子。结合无血清细胞培养技术从人胚和鼠胚皮层分离神经干细胞。在连续传代过程中观察其体外培养特性,免疫荧光染色检测Nestin抗原和分化后特异性成熟神经细胞抗原的表达,并用流式细胞仪检测神经干细胞分化情况。结果表明：（1）使用单一生长因子即可从鼠胚皮层分离神经干细胞,但在人胚却需同时使用多种生长因子,协同使用bFGF,EGF和LIF是人胚神经干细胞体外培养的较佳条件;（2）鼠胚皮层神经干细胞在连续传代过程中增殖速度快于人胚,其Nestin阳性率和BrdU标记的阳性率亦高于人胚,表明其增殖能力明显高于人胚,（3）人胚神经干细胞较鼠胚更易分化为神经元。     

体外神经干细胞克隆球的超微结构-透射电镜观察

为观察培养的神经干细胞克隆球内部的超微结构特征,采用无血清培养技术,在体外进行小鼠纹状体神经干细胞克隆球的培养传代,经过免疫细胞化学鉴定后,对单一的神经干细胞克隆球进行固定,常规透射电镜观察。结果表明,神经干细胞可以在bFGF等生长因子存在的情况下,在无血清培养液内增殖生成悬浮状态的神经干细胞克隆球,这种克隆可被诱导生成神经细胞和神经胶质细胞,电镜下,神经干细胞克隆球内部细胞相互间可形成特化的膜性结构,细胞内可有小泡出现,部分细胞有凋亡的形态。     

神经上皮干细胞的分离培养及其体外分化特性的观察

目的探讨大鼠胚胎神经管神经上皮干细胞的分离培养条件,并观察其在体外的分化特性.方法采用显微解剖、机械吹打、无血清悬浮培养方法分离培养神经上皮干细胞,采用巢蛋白(nestin)免疫细胞化学染色技术检测神经上皮干细胞,用NSE和GFAP免疫组化染色检测并计数神经细胞和神经胶质细胞.结果大鼠胚胎神经管神经上皮干细胞在无血清培养基中可形成大量呈nestin抗原阳性细胞构成的神经球,经传代有血清培养后分化为NSE阳性和GFAP阳性细胞,其中NSE阳性细胞占细胞总数的47.7%,GFAP阳性细胞占细胞总数的39.8%.结论胎鼠神经管神经上皮干细胞在无血清培养中可增殖和传代,在有血清培养中可分化为神经细胞和神经胶质细胞,两者之比为47.7∶39.8.     

不同ES细胞系体定量神经分化的比较

利用视黄酸（RA）诱导小鼠胚胎干细胞通过悬浮类胚培养体外定量分化为神经样细胞,结果显示能够进行种系嵌合的MESPU22细胞系与不能进行种系统嵌合的MESPU13细胞系的体外神经分化比例在统计学上没有明显差异,此结果对于人的胚胎干细胞全能性的鉴定和检测有重要启示。     

猕猴神经干细胞的诱导分化、干性维持及同种脑内移植

为探索猕猴神经干细胞分化及特性维持,推进神经干细胞临床应用研究,该实验以绿色荧光蛋白(green fluorescence protein,GFP)为标记探讨猕猴胚胎干细胞向玫瑰花环(rosettes)结构神经干细胞的分化及其碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)和表皮生长因子(epidermal growth factor,EGF)的扩增培养。结果表明:1)建立了稳定高效的猕猴神经干细胞分化体系,在该分化体系下,GFP标记猕猴胚胎干细胞在分化的第12天时,95%以上的细胞分化为神经干细胞;2)分化得到的Rosettes结构神经干细胞经bFGF/EGF扩增后,能够较好地维持其Rosettes结构;3)经bFGF/EGF扩增后的rosettes结构神经干细胞移植到猕猴脑内后能够较好的存活并向神经元分化,即bFGF/EGF扩增培养能较好地维持Rosettes结构的神经干细胞,且移植到猕猴脑内的该细胞亦能够较好地存活并向神经元分化,该结果为神经干细胞应用于临床提供了基础理论依据。     

诱导小鼠胚胎干细胞在体外分化为骨骼肌细胞的实验研究

小鼠胚胎干细胞是从胚泡未分化的内部细胞团中得到的干细胞,它在体外培养的环境中具有无限增殖、自我更新以及多向分化的特性。将小鼠胚胎干细胞在体外诱导分化为肌肉细胞,并且利用这些分化得来的肌肉细胞治疗肌肉退行性疾病,是干细胞研究领域的热点。该实验的目的在于筛选小鼠胚胎干细胞向骨骼肌细胞定向分化的实验条件,有效地将体外单层贴壁培养的小鼠胚胎干细胞诱导分化成骨骼肌细胞。最终发现,10-8mol/L维甲酸(retinoid acid,RA)+0.5%二甲基亚砜(dimethyl sulfoxide,DMSO)组诱导小鼠胚胎干细胞在体外分化成骨骼肌前体细胞的效率最高,分化得到的骨骼肌前体细胞经进一步纯化,能分化为多核的肌管。该实验为治疗肌肉退行性疾病提供了细胞来源,也为研究小鼠胚胎干细胞分化为骨骼肌细胞的机制提供了有利的条件。     

人胚不同脑区神经前体细胞的分离培养及分化特性的比较

目的研究人胚不同脑区神经前体细胞(neural progenitor cells,NPCs)培养及增殖分化特性。方法取14-17周人胚脑区组织,分为新皮质、纹状体、间脑、中脑、后脑和延髓组,悬浮培养。鉴定细胞球巢蛋白抗原的表达,分化及自我更新能力。观察各脑区培养细胞的生长、增殖状况。新皮质、纹状体及间脑来源的神经球分化后,运用免疫荧光细胞化学法比较神经元及星形胶质细胞的比例。结果各脑区培养出的悬浮细胞球巢蛋白抗原阳性,可分化为MAP2或GFAP阳性细胞,且BrdU掺入实验阳性。体外培养第3d,纹状体及间脑组均可见大量神经球,且纹状体组明显多于间脑组;新皮质组传代后可见较多神经球;其它组仅见个别神经球。新皮质、纹状体、间脑来源的NPCs诱导分化后,MAP2或GFAP阳性细胞率各组间比较差异无显著性。结论人胚不同脑区均可培养出NPCs,从易到难依次为纹状体、间脑、新皮质及其它脑区。新皮质、纹状体、间脑来源的NPCs体外分化比例一致。     

PKH26示踪神经干细胞移植急性脑损伤的初步观察

严重的颅脑损伤导致神经细胞和神经组织坏死,无法再生恢复,致残率高,至今缺乏有效疗法。本实验将体外培养的神经干细胞（NSC）移植入急性脑损伤大鼠模型脑内,观察移植后NSC在宿主鼠脑内的自然存活、迁移及分化情况。实验用NSC来源于孕15d Wister胎鼠脑组织,于DMEM／F12（1：1）培养基（含有bFGF,EGF和N2添加剂）中获取生长旺盛的神经干细胞球,     

胚胎大鼠中枢神经系统神经干细胞分离培养方法研究

为了建立经济实用的神经干细胞培养方法 ,以Wistar大鼠胚龄 1 4～ 1 5天的胚胎为实验材料 ,选取中枢神经系统的大脑和小脑神经组织 ,经 0 1 2 5 %胰酶消化处理 ,在生长因子 (aFGF和bFGF)作用下 ,采用无血清培养法进行培养。通过倒置相差显微镜进行观察 ,可观察到明显的干细胞的不对称分裂图像。神经干细胞在培养一段时间后均出现细胞团 ,细胞团贴壁后可进行分化 ,形态上表现为神经元样和神经胶质样细胞。利用激光扫描共聚焦显微镜对其进行鉴定 ,可检测到神经干细胞标志性蛋白-Nestin蛋白。采用此法可以分离得到具有干细胞特征和多分化潜能的神经干细胞 ,建立了一套神经干细胞的分离、培养和鉴定的方法 ,这将对更好地理解神经系统的发育机制 ,及临床治疗神经系统疾病具有重要意义。     

Derivation and characterization of ovine embryonic stem-like cell lines in semi-defined medium without feeder cells

Domestic animal embryonic stem (ES) cells would provide an invaluable research tool for genetic breeding and the production of transgenic animals. Unfortunately, authentic domestic animals ES cells have not been established despite progress made over more than two decades. Here, we show that ovine ES-like cells can be efficiently derived and propagated in a semi-defined medium that contains N2, B27, GSK3 inhibitor (CHIR99021), and basic fibroblast growth factor (bFGF). These ovine ES-like cells had a characteristic three-dimensional appearance, showed a bFGF dose-dependence, expressed specific markers such as alkaline phosphatase (AP), Oct-4, Sox2, Nanog and can be maintained for 30 passages. Moreover, these cells differentiated in vitro into neuronal cells, and formed teratomas containing a variety of different tissues including cartilage and neural tissue when injected into kidney capsules of severe combined immunodeficiency (SCID) mice. But the cell lines fail to contribute to embryonic development upon blastocyst transplantation. To our knowledge, this is the first experiment to use semi-defined medium without feeder-cells to derive ES-like cells from ovine blastocysts, and opens the door to deriving authentic ES cells from domesticated ungulates.     

Alternative splicing generates a CaM kinase IIβ isoform in myocardium that targets the sarcoplasmic reticulum through a putative αKAP and regulates GAPDH

In the postgenomic era the elucidation of the physiological function of genes has become the rate-limiting step in the quest to understand the development and function of living organisms. Double-stranded RNA (dsRNA) interferes with gene expression in various species, a phenomenon known as RNA interference (RNAi). We show here that RNAi is also effective in modifying gene expression in neural stem cell differentiation. The progenitor cells were obtained from E14 mouse embryonic forebrain and maintained using N-2 medium containing basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and B27.A gene (NM017084.1) was previously discovered and validated to express obviously differently between differentiated and undifferentiated neural stem cells in our laboratory. Here we report a long double-stranded RNA to knock out or knock down this gene. The results demonstrated that following RNAi inhibition of expression of the NM017084.1 gene, the differentiation of neural stem cells is accelerated. Thus the NM017084.1 gene may play a pivotal role in the process of differentiation of neural stem cells.     

Angiogenic potential of CD44+ CD90+ multipotent CNS stem cells in vitro

Although it is now clear that several subpopulations of neural stem cells (NSCs) exist during early development and adulthood, the angiogenic potential of NSCs remains a subject of debate. Here, we report that CD44(+) CD90(+) cells isolated from primary neurospheres can form vascular-tube structures in vitro. NSCs isolated from the mouse embryonic cortex formed neurospheres when cultured in serum-free medium containing 20ng/ml basic fibroblast growth factor (bFGF). CD44(+) CD90(+) cells were enriched from the neurospheres using an EPICS ALTRA flow cytometer, and antibodies against CD44 and CD90. The purified CD44(+) CD90(+) cells generated neurospheres, and differentiated into neurons and astrocytes. When the cells were inoculated into collagen gels and cultured with 20% fetal bovine serum plus bFGF for 7 days, vascular tube-like structures were formed. These results indicate that CD44(+) CD90(+) cells have the ability to generate neurospheres and to form vascular tubes.     

Neural precursors derived from human embryonic stem cells

Before the successful isolation of human embryonic stem (hES) cells, many investigations had shown that mouse embryonic stem (mES) cells can be induced to differentiate into neural precursors which could be purified and differentiated to mature dopamine, motor, serotonin, GABA neurons, and oligodendrocytes and astrocytes in vitro[1―3]. mES cell-derived dopamine neurons have been shown capable of integrating into host brains after transplanting to the rodents of Park-inson’s disease model …     

低氧促进神经干细胞向多巴胺能神经元分化

神经干细胞（neural stem cells,NSCs）作为具有多向分化潜能的神经前体细胞,被广泛应用于细胞移植等研究,而低氧不但调节干细胞的体外增殖,在干细胞分化中也具有重要的作用。本文着重探讨了低氧对NSCs分化的调节作用。采用Wistar孕大鼠（E13．5d）,分离胚胎中脑NSCs,加入无血清DMEM／F12培养液（含20ng／mL EGF、20ng／mL bFGF、1％ N2和B27）,3～5d后传代,细胞培养至第三代进行诱导分化,分别在低氧（3％O2）和常氧（20％O2）条件下诱导分化3d,然后在常氧条件下分化成熟5～7d（DMEM／F12含1％FBS、N2和B27）后进行检测。Nestin、NeuN以及TH免疫组织化学鉴定NSCs;流式细胞术分析测定NSCs向TH阳性神经元方向的分化;高效液相色谱测定细胞培养上清液中多巴胺（dopamine,DA）含量。结果显示,分离培养的NSCs均为nestin阳性细胞;低氧可明显促进NSCs向神经元方向的分化;TH阳性神经元比例在常氧和低氧组分别为（10．25±1．03）％和（19．88±1．44）％。NSCs诱导分化7d后,低氧组细胞培养上清液中DA浓度明显增加,约为常氧组的2倍（P〈0．05,n=8）。上述结果表明,3％低氧可促进NSCs向神经元方向,特别是向DA能神经元方向分化。这为NSCs应用于临床治疗帕金森病提供了基础。     

Neural precursors derived from human embryonic stem cells

Human embryonic stem (hES) cells provide a promising supply of specific cell types for transplantation therapy. We presented here the method to induce differentiation of purified neural precursors from hES cells. hES cells (Line PKU-1 and Line PKU-2) were cultured in suspension in bacteriological Petri dishes, which differentiated into cystic embryoid bodies (EBs). The EBs were then cultured in N₂ medium containing bFGF in poly-L-lysine-coated tissue culture dishes for two weeks. The central, small cells with 2–3 short processes of the spreading outgrowth were isolated mechanically and replated. The resulting neurospheres were cultured in suspension for 10 days, then dissociated into single cell suspension with a Pasteur pipette and plated. Cells grew vigorously in an attached way and were passed every 4–5 days. Almost all the cells were proved nestin positive by immunostaining. Following withdrawal of bFGF, they differentiated into neurons expressing β-tubulin isotype_III, GABA, serotonin and synaptophysin. Through induction of PDGF-AA, they differentiated into astrocytes expressing GFAP and oligodendrocytes expressing O₄. The results showed that hES cells can differentiate into typical neural precursors expressing the specific marker nestin and capable of generating all three cell types of the central nervous system (CNS)in vitro.     

脊髓源神经干细胞诱导分化为胆碱能神经元过程中Aldoc和Stmn1表达的研究

目的探讨脊髓源神经干细胞在诱导分化为胆碱能神经元过程中Aldoc和Stmn1的表达变化情况。方法从孕17天Wistar胚胎大鼠取出脊髓组织,制成细胞悬液,采用含EGF和bFGF的无血清限定性培养基培养,然后进行诱导分化,观察脊髓源神经干细胞向胆碱能神经元分化的情况,应用荧光定量PCR方法分析Aldoc和Stmn1基因在脊髓源神经干细胞诱导分化为胆碱能神经元的过程中的表达变化情况。结果神经干细胞在诱导分化为胆碱能神经元后,经免疫荧光检测有ChAT阳性细胞表达;Aldoc基因的表达量在胆碱能神经元较神经干细胞低(P<0.05);Stmn1基因的表达量则在诱导分化后较神经干细胞升高(P<0.05)。结论 Aldoc对神经干细胞的干性维持有重要作用,Stmn1在胆碱能神经元的成熟过程中起作用。