大鼠大脑皮质神经前体细胞的分离培养及其增殖分化鉴定

目的:体外分离和培养大鼠大脑皮质神经前体细胞并进行增殖和分化鉴定。方法:分离2周龄大鼠皮质神经前体细胞,在含EGF、bFGF和GDNF的NSC培养基中进行体外培养,使用免疫细胞荧光染色技术对细胞的分化特性进行鉴定。结果:EGF、bFGF和GDNF可促进神经前体细胞的增殖及神经球的克隆形成,并获得了Nestin阳性的神经前体细胞,其可分化为分别表达β-Ⅲtubulin、GFAP和GalC的阳性细胞。结论:体外分离和培养的大脑皮质神经前体细胞,在含EGF、bFGF和GDNF的NSC培养基中培养,具有增殖分化的能力,有望应用于神经系统疾病的细胞移植治疗。     

慢病毒介导碱性成纤维细胞生长因子在大鼠羊膜上皮细胞中的表达和分泌

目的 探讨以慢病毒作为载体,将带有分泌肽的人碱性成纤维细胞生长因子(bFGF)基因片段转入大鼠羊膜上皮细胞(AECs)中,构建能稳定表达并分泌bFGF多肽的细胞载体. 方法 取妊娠晚期SD大鼠的羊膜组织进行AECs原代培养,用免疫荧光细胞化学和RT-PCR法鉴定;构建包含人神经生长因子(NGF)分泌肽-bFGF编码基因的慢病毒载体,并行慢病毒包装,用病毒上清对传代大鼠AECs进行感染并筛选,经免疫荧光细胞化学、RT-PCR及酶联免疫吸附法(ELISA)检测基因转染效果,体外培养观察基因转染后的细胞生长活性;用基因转染细胞的培养上清对PC12细胞进行培养,检测所分泌bFGF多肽的生物学活性. 结果 所培养的大鼠AECs经鉴定能表达上皮特异性标志物CK-19、神经类细胞标志物巢蛋白(nestin)、胶质纤维酸性蛋白(GFAP)以及细胞多能性标志分子SSEA-4、Oct-4、Nanog、Sox2、波形蛋白(vimentin)等;经感染并筛选后扩增培养的大鼠AECs在mRNA水平及多肽水平均能检测到目的 基因bFGF的表达,筛选后的转染细胞生长活性较未转染细胞明显提高,其培养上清能明显促进PC12细胞的生长和分化. 结论 用慢病毒作为载体能成功将bFGF基因转染入大鼠AECs中,所建立的基因修饰AECs能表达并分泌bFGF多肽,具有较强的生长活性及神经营养活性.     

骨髓基质细胞体外诱导分化成神经元和胶质细胞

目的　探索骨髓基质细胞 (bonemarrowstromalcells,BMSC)体外诱导分化为神经元和神经胶质细胞的可行性 ,为BMSC在神经科学领域内的应用奠定基础。方法　以成年犬BMSC为实验对象 ,利用碱性成纤维细胞生长因子 (bFGF)、表皮生长因子 (EGF)、维甲酸 (RA)、脑源性神经营养因子 (BDNF)、胶质细胞系来源神经营养因子 (GDNF)等作为增殖及分化诱导因子 ,进行增殖培养、分化诱导 ;免疫组化法进行细胞性质鉴定。结果　加入bFGF、EGF增殖培养 72h可见细胞分裂相 (成纤维细胞样细胞 )。加入RA、BDNF、GDNF诱导 3d ,部分细胞有NSE、GFAP成分表达 ;第 10d可见有神经元、神经胶质形态样细胞形成 ,经细胞成分 (NSE、GFAP)鉴定证实为神经元、神经胶质细胞。结论　BMSC在体外培养条件下 ,经过bFGF、EGF、RA、BDNF、GDNF等因子的“程序性”作用 ,可以分化成神经元和神经胶质细胞     

一种分离培养少突胶质前体细胞的改进方法

目的:对少突胶质前体细胞的分离培养方法进行改良,为今后细胞移植治疗研究提供基础。方法:在大鼠少突胶质前体细胞分离纯化培养过程中添加不同浓度bFGF(5、10、20 ng/ml),显微镜观察少突胶质前体细胞在体外的生长情况,台盼蓝实验检测细胞存活率和获得率,免疫细胞化学技术进行细胞鉴定,流式细胞术检测细胞纯度。结果:在少突胶质前体细胞培养过程中添加10 ng/ml的bFGF,可明显提高少突胶质前体细胞的产出率和纯度。分离的少突胶质前体细胞呈A2B5、NG2阳性,能进一步分化为成熟少突胶质细胞且表达MBP和O4。结论:在培养中添加适当浓度的bFGF的方法能有效获得高纯度的少突胶质前体细胞。     

Sertoli细胞对大鼠大脑皮质神经前体细胞的诱导分化作用

目的:研究Sertoli细胞对体外培养大鼠皮质神经前体细胞生长发育的促进作用,及向多巴胺能神经元分化的诱导作用.方法:将大鼠Sertoli细胞与14 d大鼠皮质神经前体细胞体外共培养,免疫细胞化学检测神经干细胞标记物巢蛋白,神经元、星形胶质细胞和小胶质细胞标记物βⅢ-微管蛋白(βⅢ-tubulin)、胶质纤维酸性蛋白(GFAP)和半乳糖脑苷脂(GalC),及多巴胺能神经元标记物酪氨酸羟化酶(TH)的表达情况.结果:随着共培养时间的延长,神经前体细胞中GFAP阳性细胞数量逐渐减少,而β-Ⅲ tubulin 阳性细胞数增多,并出现TH阳性神经元.结论:Sertoli细胞抑制体外培养的大鼠皮质神经前体细胞的生长,但具有显著促神经元分化作用,并能够诱导与其共培养的大鼠皮质神经前体细胞分化为多巴胺能神经元.     

新生大鼠不同脑区内神经前体细胞生物学特性比较

为了观察海马、中脑和顶叶皮质内神经前体细胞的体外生长特性及其分化后所产生的神经元的形态特征,本研究使用碱性成纤维细胞生长因子(bFGF)体外扩增细胞技术和克隆形成实验分析了神经前体细胞的自我更新特性;采用神经前体细胞和骨髓基质细胞共培养方式,通过免疫细胞化学染色方法,研究了神经前体细胞的分化特点。结果发现:(1)新生大鼠海马、中脑和顶叶皮质内的神经前体细胞,在体外的分裂增殖能力无明显区别;(2)新生大鼠海马、中脑和顶叶皮质内的神经前体细胞在相同的条件下,其后代细胞中,神经元和胶质细胞所占的比例基本相同,但不同脑区内神经前体细胞所产生的神经元的形态却表现出明显不同。这些结果提示新生大鼠不同脑区内神经前体细胞的分化潜能已有了一定限制。     

Desert Hedgehog诱导大鼠胚胎中脑神经前体细胞定向分化为多巴胺能神经元

目的 研究Desert Hedgehog(DHH)对胚胎中脑神经前体细胞(NPCs)向多巴胺能神经元分化的诱导作用。方法 大鼠DHH病毒上清感染COS7、NIH/3T3和9L细胞, 用免疫荧光、实时定量PCR和Western blot方法检测DHH表达。分离培养大鼠胚胎中脑神经前体细胞，免疫荧光鉴定神经前体细胞的增殖分化特性。条件细胞表达DHH成功后分别与上述NPCs共培养10d，免疫荧光检测酪氨酸羟化酶(TH)的表达。结果 DHH在COS7、NIH/3T3和9L细胞中成功表达；同时，分离培养的大鼠胚胎中脑神经前体细胞具有良好的增殖分化特性，但两者共培养10d后偶见TH阳性的细胞。结论 DHH编码的蛋白不能或不能单独诱导与其共培养的NPCs定向分化为多巴胺能神经元。     

大鼠视网膜干细胞的增殖和多向分化**

背景：国内对视网膜干细胞的体外分离培养及鉴定仍处于初步探索阶段。 目的：体外分离、培养及鉴定新生大鼠视网膜干细胞,探讨其多向分化潜能。 方法：用神经干细胞无血清培养方法分离和培养新生24 h的SD大鼠睫状体细胞,第6代视网膜干细胞经胎牛血清诱导分化,应用免疫细胞化学方法检测视网膜干细胞的分化特性。 结果与结论：体外培养的细胞球具有连续克隆能力,Nestin抗原阳性,BrdU 标记结果显示悬浮细胞团主要由分裂增殖的细胞组成,并表达胚胎发育早期视网膜内原始细胞的特异性抗原Chx-10;诱导分化后的细胞表达星形胶质细胞特异性标志物GFAP、神经元特异性标志物NSE、感光细胞标志物Opsin、双极细胞特异性抗原PKC和节细胞特异性抗原β-tubulin,实验初步证实培养的视网膜干细胞具有神经干细胞特性,能自我更新和增殖分化成为感光细胞类型的细胞。 关键词：视网膜干细胞;细胞培养;无血清;细胞分化;大鼠 doi:10.3969/j.issn.1673-8225.2012.06.015     

成年大鼠纹状体区神经干细胞的分离培养

目的 从成年大鼠纹状体区分离并鉴定神经干细胞。方法 利用无血清增减和单细胞克隆技术在成年大鼠纹状体区分离具有单细胞克隆能力的细胞群,选取单个克隆进行连续伟代培养获得大量亚克隆,采用兔疫荧光细胞化学技术检测神经上皮干细胞蛋白（Ｎｅｓｔｉｎ）和分化后牧民性成熟神经细胞抗原的表达。结果 从纹状体区分离的细胞群具有连续克隆能力,表达神经上皮干细胞蛋白,分化后的细胞表达神经元、星形胶质细胞和少突胶质细胞的牧     

碱性成纤维细胞生长因子诱导大鼠受损视神经胶质细胞去分化

目的研究碱性成纤维细胞生长因子(bFGF)促进大鼠受损视神经修复及诱导胶质细胞去分化的机制。方法成年SD大鼠55只随机分为正常对照组、损伤组、bFGF组。术后7d取眶内段视神经行Agilent基因芯片、实时荧光定量PCR检测;术后7d、14d取眶内段视神经行HE、免疫组织化学等检测。结果术后7d,bFGF组与损伤组相比有645条基因上调,458条下调,其中包括与神经干细胞或前体细胞及神经发育、增殖凋亡、染色质构型、转录调节、信号转导、神经生长相关基因等。与损伤组相比,bFGF组受损视神经远侧段细胞核增大,细胞数量增加,巢蛋白、细胞外信号调节激酶1/2(ERK1/2)、胶质纤维酸性蛋白(GFAP)、髓鞘碱性蛋白(MBP)阳性物质增加,近侧段神经丝(NF)阳性物质增加。结论外加bFGF能促进受损视神经细胞增殖,增强神经胶质细胞去分化变化,同时改善神经再生的抑制性微环境,促进视神经再生。     

The influence of piezoelectric scaffolds on neural differentiation of human neural stem/progenitor cells

Human neural stem/progenitor cells (hNSCs/NPCs) are a promising cell source for neural tissue engineering because of their ability to differentiate into various neural lineages. In this study, hNSC/NPC differentiation was evaluated on piezoelectric, fibrous scaffolds. These smart materials have an intrinsic material property where transient electric potential can be generated in the material upon minute mechanical deformation. hNSCs/NPCs cultured on the scaffolds and films differentiated into β-III tubulin-positive cells, a neuronal cell marker, with or without the presence of inductive factors. In contrast, hNSCs/NPCs cultured on laminin-coated plates were predominantly nestin positive, a NSC marker, in the control medium. Gene expression results suggest that the scaffolds may have promoted the formation of mature neural cells exhibiting neuron-like characteristics. hNSCs/NPCs differentiated mostly into β-III tubulin-positive cells and had the greatest average neurite length on micron-sized, annealed (more piezoelectric), aligned scaffolds, demonstrating their potential for neural tissue-engineering applications.     

Generation of developmental patterns in the neuroepithelium of the developing mammalian eye: the pigment epithelium of the eye

Culture experiments with eye anlages of mouse embryos were performed to study developmental traits of the neuroepithelial cells of the prospective pigment epithelium in the eye anlage of pigmented mice. Between the neural plate stage on embryonic day 8 (ED 8, developmental stage 12) and the neural tube stage on embryonic day 9 1/2 (stage 15), the cultured neuroepithelium of the eye generated neurons and glia, identified by morphological and immunocytochemical evidence, but no pigmented cells. In contrast, eye anlages did produce pigment epithelium when cultured in their natural position in a head tissue fragment. A minority of developing neurons displayed tyrosine hydroxylase immunoreactivity, whereas GABAergic, serotoninergic and substance P-ergic neurons, which are common in the mature neuroretina, were not observed. When neuroepithelial cells from embryonic eyes older than stage 15 (ED 9 1/2) were cultured, they differentiated into pigment cells but not into nerve cells or glia. This developmental sequence indicates that the pigment cells derive from the neural lineage. Pigment cell fate dominates over the neural fate beginning at about stage 15 (ED 9 1/2-10). That is at least 2 days before the pigment cell phenotype becomes apparent in vivo (ED 11 1/2-12).     

小鼠胚胎干细胞来源的神经干细胞的稳定传代体系探索

目的探讨体外诱导、获取均一的神经干细胞群(NSCs)的有效方法,并建立神经干细胞体外稳定传代扩增体系。方法首先采用无血清的诱导培养基贴壁诱导mESCs形成神经上皮祖细胞(NPCs)。然后将经添加表皮生长因子(EGF)和成纤维细胞生长因子-2(FGF2)的无血清培养基短暂悬浮培养后的NPCs再贴壁培养,诱导形成NSCs。通过细胞系46C监测NPCs的形成,同时对分化细胞进行定量PCR和免疫荧光染色,在不同水平检测细胞分化效果。结果 mESCs神经诱导5 d出现大量Sox1+的NPCs;NPCs悬浮培养后,进一步诱导可得到形态均一的NSCs。第2代和第6代NSCs的神经干细胞标志定量PCR检测结果为:Pax6、Nestin、Mash1、BLBP高表达。第8代NSCs免疫荧光染色显示90%以上的细胞均为Nestin、RC2和Pax6阳性。结论成功诱导mESC生成神经干细胞群,并且可以在体外连续稳定的传代。     

腺病毒载体介导的LacZ基因在神经前体细胞的转染和表达

为了观察含报告基因 L ac Z的重组 5型腺病毒载体 ( Ad5CMVLac Z)在神经前体细胞转染和表达的量效关系并探讨用该载体构建基因修饰细胞的可能性 ,本研究用不同滴度 ( 1× 10 3～ 1× 10 1 0 PFU/ml)的 Ad5CMVLac Z转染体外培养的 SD胎鼠(胎龄 12 d)海马神经前体细胞 ,用β-半乳糖苷酶 (β-gal)免疫组化反应检测转染效率。结果显示 :当病毒滴度为 1× 10 7时 ,转染率约为 5 0 % ,当滴度增加到 1× 10 1 0时 ,转染率达 10 0 %。Ad5CMVL ac Z在神经前体细胞的转染率具有滴度依赖性的量效关系。表明高滴度的 Ad5CMVLac Z成功地转染了大部分神经前体细胞 ,Lac Z基因也得到充分表达。提示神经前体细胞是该载体转染的适宜靶细胞 ,并有可能通过该载体转导目的基因 ,构建出基因修饰细胞     

Olfactory ensheathing cells promote proliferation and inhibit neuronal differentiation of neural progenitor cells through activation of Notch signaling

A population of neural progenitor cells (NPCs) has been known to exist in adult spinal cord and migrate toward the lesion regions during spinal cord injury (SCI). Although there are some positive effects of the transplanted olfactory ensheathing cells (OECs) on axonal regeneration in SCI, little is known about the effects and the underlying mechanism of these grafted OECs on NPCs. In this study, we have investigated how soluble factors derived from rat OECs regulate the proliferation and differentiation of rat NPCs. The conditioned medium from cultured OECs showed its ability to promote proliferation and inhibit neuronal differentiation of NPCs. Notch signaling was apparently involved in this process. With the addition of DAPT, which inhibited Notch signaling, the effects of OEC-conditioned medium on NPCs were blocked. We thus conclude that diffusible factors released from OECs activate the Notch signaling pathway to stimulate the proliferation and suppress neuronal differentiation of NPCs. These findings reveal the likely limitation of using OECs transplantation for SCI repair.     

不同时期视网膜前体细胞的培养

目的 研究不同时期视网膜前体细胞的培养.方法 分离E14和E18 SD大鼠的视网膜前体细胞,用改进DMEM/F12无血清培养基悬浮培养并利用体外诱导分化、免疫细胞化学、扫描电镜和透射电镜等方法对细胞进行鉴定.结果 视网膜前体细胞在DMEM/F12无血清培养基中悬浮成球,贴壁后,细胞逐渐从细胞球向周围迁移分化.扫描电镜可见细胞球和分化细胞的形态,透射电镜可见细胞球和分化细胞的超微结构.免疫细胞化学显示,悬浮培养的细胞球中大部分细胞表达神经外胚层源性干细胞标志物nestin和细胞分裂增殖标志物BrdU.贴壁后,视网膜前体细胞能分化为多种视网膜细胞类型,包括Thy1.1阳性的视网膜神经节细胞.E14和E18视网膜神经节细胞的百分比分别为16.91%±4.05%和4.65%±1.88%,两组比较,差异有统计学意义(t=15.04,P＜0.001).结论 改进DMEM/F12无血清培养基可成功培养视网膜前体细胞,培养的细胞具有无限增殖和多向分化潜能.早期视网膜前体细胞向视网膜神经节细胞分化的百分比较高.     

Expression of Multipotent and Retinal Markers in Pigment Epithelium of Adult Human in Vitro

Immunoperoxidase and molecular genetic analysis showed that retinal pigment epithelial cells from adult human eye undergo morphogenetic changes in vitro. They lose expression of tissue-specific protein RPE65 and start to express stem cell markers: Oct4 (POU5F1), Nanog, Prox1, Musashi 1, and Pax6, which indicates their differentiation. Expression of Musashi 1 and Pax6 attest to neural differentiation, which is also confirmed by the expression of βIII-tubulin, a neuroblast marker, and markers of differentiated neuronal cells, tyrosine hydroxylase and neurofilament proteins. These findings attest to the capacity of retinal pigment epithelium from adult human eye to transdifferentiation into neural lineage cells, which makes them an interesting object for cell therapy in neurodegeneration.     

Expression of chemokine receptors CXCR4, CCR2, CCR5 and CX3CR1 in neural progenitor cells isolated from the subventricular zone of the adult rat brain

We have studied the expression of chemokine receptors CXCR4, CCR2, CCR5, and CX3CR1 at the mRNA and protein levels in adult neural progenitor cells (NPCs) in neurosphere cultures using RT-PCR and immunocytochemistry methods. NPCs were isolated from the subventricular zone of adult rat brain and propagated in vitro as neurospheres. The neurospheres showed immunoactivity of nestin, an intermediate filament marker for NPCs. NPCs in the neurosphere cultures differentiated into NeuN-, GFAP-, or GalC-positive cells in vitro. Using cultured cortical microglial cells as positive control, we demonstrated the mRNA expression of CXCR4, CCR2, CCR5, and CX3CR1 in neurospheres by RT-PCR. Double immunofluorescent staining further confirmed the co-localization of nestin with either CXCR4, CCR2, CCR5, or CX3CR1 on neurospheres. These results suggest that adult NPCs in the neurosphere cultures express chemokine receptors CXCR4, CCR2, CCR5, and CX3CR1.