胚胎大鼠脑纹状体和中脑神经干细胞的培养

目的研究大鼠脑不同部位胚胎神经干细胞的增殖分化特性。方法采用无血清培养基分离和培养大鼠脑的纹状体和中脑的神经干细胞,通过巢蛋白(nestin)表达和5-溴脱氧尿嘧啶(5-bromo-2'-deoxyuridine BrdU)染色,鉴定细胞的增殖能力;通过新生神经元、星形胶质细胞和少突胶质细胞的特异性免疫细胞化学染色,鉴定培养细胞的多潜能性。通过酪氨酸羟化酶的染色(tyrosine hydroxylase,TH)鉴定多巴胺神经元。结果二者在体外培养都可增殖成球,并能分化成神经系统3个谱系的细胞。纹状体增殖传代3个月,中脑培养细胞增殖维持3周。中脑干细胞分化TH阳性细胞比例高于纹状体。结论培养的胎鼠脑细胞是神经干细胞。纹状体干细胞增殖能力高于中脑干细胞,中脑干细胞更倾向于分化成TH阳性细胞。     

无血清条件下体外分离培养鼠胚胎脑组织神经干细胞

背景：神经干细胞的体外培养成功为治疗中枢神经系统疾病提供了新的思路,但神经干细胞的分化和功能修复机制尚不甚清楚,移植后的细胞能否与体内细胞结合以及建立起正常的神经系统突触联系急需解决。 目的：在无血清条件下体外分离培养胚鼠神经干细胞,观察其生长及分化情况。 设计、时间及地点：细胞学体外观察,于2007-03/2008-01在天津市环湖医院神经干细胞室完成。 材料：孕14~16 d的SD大鼠,由北京维通利华实验动物中心提供。 方法：取孕鼠胚胎的脑海马组织,通过机械分离和胰蛋白酶消化结合法体外分离培养神经干细胞,在含有碱性成纤维细胞生长因子、表皮生长因子及B27的无血清DMEM/F12培养基中传代扩增。取原代培养7 d的神经干细胞,制备单细胞悬液,接种后加入含体积分数为10%胎牛血清的DMEM培养液诱导3 d。 主要观察指标：倒置显微镜下观察神经干细胞的增殖分化过程,并行免疫荧光染色鉴定。通过细胞免疫化学染色检测诱导分化后的细胞类型。 结果：分离培养的神经干细胞在无血清培养基中不断分裂增殖,8 d左右即可形成胞体透亮、折光性好的干细胞球,悬浮生长,免疫荧光染色巢蛋白呈阳性表达。神经干细胞球诱导5 d,免疫细胞化学染色后可见胶质纤维酸性蛋白及神经元特异性烯醇酶呈阳性表达的细胞。 结论：体外无血清条件下,分离培养的神经干细胞生长状态良好,且具有自我更新和增殖能力,诱导后能够向神经元及星形胶质细胞方向分化。     

大鼠中脑神经前体细胞的分离鉴定和培养

目的确认从E14.5SD大鼠中脑胚胎分离的细胞符合神经前体细胞特性,并在体外建立合适培养体系使神经前体细胞能够长期生长及传代。方法分离E14.5SD大鼠胚胎中脑腹侧组织细胞,在体外含有碱性纤维细胞生长因子(bFGF)的无血清培养液内种植并传代,行nestin免疫学检查,并在分化前后行神经元特异性烯醇化酶(NSE)和胶质原纤维酸性蛋白(GFAP)免疫学检查,同时做BrdU增殖实验。结果体外种植中脑神经细胞可以生长、分裂并长期传代,nestin染色及BrdU增殖实验为阳性,NSE和GFAP 在分化前阴性,而分化后为阳性。结论E14.5SD大鼠胚胎中脑分离的细胞符合神经前体细胞特性,并可在本实验所采用的培养液内长期生长、分裂和传代。     

食蟹猴脑室下区成体神经前体细胞培养及鉴定

目的分离、培养成年食蟹猴脑室下区神经前体细胞并研究其生物学特性。方法取成年食蟹猴脑室下区组织,经木瓜蛋白酶和脱氧核糖核酸酶消化后,接种于DMEM-F12无血清培养基中。结果神经前体细胞可以增殖形成神经球,经免疫细胞化学方法检验这些细胞呈现巢蛋白nestin阳性,神经球分化后的细胞表达胶质细胞的标记物及神经元的标记物。脑室下区来源的神经球进行自主分化时可分化成神经元和胶质细胞,但分化成神经元的比例都很少。结论从食蟹猴脑室下区分离培养的细胞可以在体外增殖形成神经球,并可分化为神经元和胶质细胞,符合神经前体细胞的生物学性状。     

成年小鼠嗅球神经干细胞的培养和鉴定

目的 建立完善的成年小鼠嗅球神经千细胞分离培养和鉴定方法,探索新的成年神经干细胞种子来源. 方法 用无血清方法 分离培养成年小鼠嗅球来源的神经干细胞;用克隆培养、5-溴2-脱氧尿嘧啶核昔(BrdU)整合的方法 检验培养细胞的干细胞特性;用免疫荧光细胞化学的方法 检测BrdU、神经干细胞标记物巢蛋白(nestin)和SOX2、分化的细胞标记物Tuj1、胶质纤维酸性蛋白(GFAP)、04的表达. 结果 从成年小鼠嗅球能够分离、培养出具有自我更新、增殖能力的神经球.构成神经球的细胞呈nestin和SOX2阳性,它们分化后产生TuJ1阳性的神经元、GFAP阳性的星形胶质细胞、04阳性的少突胶质细胞. 结论 成年小鼠嗅球存在神经干细胞,其能够在体外进行培养、增殖、分化.是神经干细胞的新的种子来源.     

人胚神经干细胞的体外培养及鉴定

目的 探讨人胚神经干细胞的体外培养和诱导分化的条件。方法 从药物流产的12周到16周的人胚胎海马组织中分离神经干细胞，在EGF、bFGF和LIF联合作用下使其稳定增殖，并用10％的胎牛血清诱导其贴壁分化，应用免疫荧光染色方法行Nestin、NSE、MAP-2、GFAP和GalC免疫荧光染色，对神经干细胞及其分化的细胞进行鉴定。结果 体外培养的神经干细胞增殖成神经干细胞球并传代，鉴定为Nestin染色阳性细胞，并可诱导分化为神经细胞、星形胶质细胞和少突胶质细胞。结论 利用无血清培养技术和特定生长因子，可培养出在体外稳定增殖并有多向分化潜能的人胚神经干细胞。     

胚胎大鼠神经干细胞分离培养的实验研究

目的探讨胚胎大鼠神经干细胞的分离培养方法,并对其进行鉴定。方法从胚胎大鼠脑组织中分离神经干细胞,采用无血清培养技术,在生长因子的作用下使其在体外不断增殖克隆,同时利用免疫荧光染色的方法对培养的细胞进行鉴定。结果体外培养的细胞增殖成细胞克隆球并能稳定传代,经鉴定,均为nestin染色阳性细胞。结论利用无血清技术和特定生长因子,可以使来源于胚胎大鼠的神经干细胞在体外扩增并稳定传代。     

大鼠胚胎神经干细胞的体外培养及鉴定

目的探讨大鼠胚胎神经干细胞（NSC）的体外培养和诱导分化的条件和特点。方法从孕14～16d的大鼠胚胎脑皮质中分离NSC，在表皮生长因子、碱性成纤维生长因子和B27联合作用下使其稳定增殖，并用10％的胎牛血清诱导其贴壁分化，应用免疫荧光染色方法行巢蛋白（Nestin）、神经元特异性烯醇化酶（NSE）、胶质纤维酸性蛋白（GFAP）、Gale-C免疫荧光染色，对NSC及其分化的细胞进行鉴定。结果体外培养的NSC增殖成神经干细胞球并传代，鉴定为Nestin染色阳性细胞，并可诱导分化为神经元细胞（NSE染色阳性细胞）、星形胶质细胞（GFAP染色阳性细胞）和少突胶质细胞（Gale-C染色阳性细胞）。结论采用无血清培养基中加入特定生长因子的培养技术，可培养出在体外稳定增殖并有多向分化潜能的大鼠胚胎NSC。     

人胚海马神经干细胞体外培养及分化研究

目的 研究人胚胎海马神经干细胞体外长期培养的条件和其在自主分化条件下的分化能力和分化特点。方法 从人胚胎海马分离神经干细胞。采用无血清培养法，进行体外培养、扩增，形成神经球。使神经球贴壁分化，分化培养基不含有任何细胞有丝分裂促进剂。使用5-溴脱氧尿嘧啶核苷(BrdU)标记分裂增生的细胞，观察细胞的分裂增殖情况。使用免疫细胞化学法鉴定神经干细胞及其在不加诱导剂下的自主分化能力。结果 从人胚胎海马分离的神经干细胞具有增殖能力，细胞倍增时间为3．2d。BrdU检测有正在分裂、增殖的细胞。细胞贴壁分化后可以出现Nestin、GFAP、Tuj-1表达阳性的细胞。神经干细胞共培养6个月，传代14代。结论 分离培养的海马神经干细胞具有自我更新和增殖能力，可以长期培养。在不加任何诱导剂的自主分化条件下可以向神经元、胶质细胞分化。少突胶质细胞的培养需要不同的培养条件。分离培养的干细胞具有神经干细胞的特征。可用于基础和临床的相关研究。     

胎鼠海马神经干细胞生物学特性的研究

目的研究胎鼠脑组织中海马神经干细胞的自我繁殖特性及多向分化潜能.方法在碱性成纤维生长因子(FGF-2)作用下从胎鼠脑组织中分离、培养神经干细胞并诱导其分化,然后采用间接免疫荧光和免疫组化技术研究神经干细胞的特性.结果由胎鼠海马中分离出的神经干细胞在体外分裂增殖形成干细胞团,分化产生神经元和胶质细胞,它们分别对神经上皮干细胞蛋白、神经元特异性烯醇化酶、胶质纤维酸性蛋白等抗原标记呈现阳性.结论胎鼠脑海马中存在神经干细胞,其具有自我增殖特性和多向分化潜能,在FGF-2的作用下分裂增殖,并分化成神经元、胶质细胞.     

人胚额叶皮层和海马干细胞的自主分化研究

目的 研究人胚胎额叶皮层和海马组织神经干细胞的自主分化特性。观察额叶皮层神经干细胞和海马神经干细胞特性的异同。方法 从人胚胎额叶皮层和海马组织分别分离提出神经干细胞，经无血清体外培养、扩增，形成神经球。神经球贴壁进行不加诱导剂的自主分化。采用细胞生长曲线检测神经干细胞的增殖能力。使用5-溴脱氧尿嘧啶核苷（BrdU）标记分裂增生的细胞，观察细胞的分裂增殖情况。免疫细胞化学法鉴定神经干细胞的自主分化能力，比较额叶皮层和海马神经干细胞的分化特点。结果 从人胚胎额叶皮层和海马分离的神经干细胞具有增殖能力，额叶皮层神经干细胞的细胞倍增时间为3．9d，海马神经干细胞的细胞倍增时间为3．2d。细胞贴壁分化后出现Nestin、GFAP、Tuj-1表达阳性的细胞。皮层和海马神经干细胞分化产生的Tuj-1阳性细胞分别是40．7％和19．3％；皮层和海马神经干细胞分化产生的GFAP阳性细胞分别是59．3％和80．7％。结论 分离培养的额叶皮层和海马神经干细胞具有自我更新和增殖能力，可以向神经元、胶质细胞分化。额叶皮层神经干细胞与海马神经干细胞的倍增时问、自主分化特点和分化为神经细胞和胶质细胞的比率各有不同。     

次声对成年大鼠齿状回神经前体细胞增殖的影响

目的研究次声对成年大鼠海马齿状回神经前体细胞增殖的影响。方法大鼠随机等分为正常对照组、假次声组和次声组（每组16只）。次声组暴露于8Hz、130dB次声环境7d（2h／d），暴露结束后第1、3、7、14d处死，采用抗5-溴脱氧尿嘧啶尿苷（BrdU）免疫组化方法，观察齿状回BrdU阳性细胞数的变化。结果次声作用结束后第1d，齿状回BrdU阳性细胞数与假次声组和正常对照组相比均无统计学差异；第3d及第7d，BrdU阳性细胞数减少（P〈0．05），第14d恢复正常水平。结论8Hz、130dB次声可抑制正常成年大鼠海马神经前体细胞增殖，可能与次声引起大鼠脑内微环境改变有关。     

Effects of Abeta25-35 on neurogenesis in the adult mouse subventricular zone and dentate gyrus

It has been demonstrated that neuorgenesis driven by neural precursor cells persists well into the adult period. This study was to observe the effects of Amyloid-beta (25-35) peptide (Abeta(25-35)) on neurogenesis in the subventricular zone and dentate gyrus of adult mouse brain. Aggregated Abeta(25-35)(1 mg/ml, 3 microl) was injected into the lateral ventricle of adult mouse. Animals were transcardially perfused with 4% paraformaldehyde in PBS, respectively at 5, 10, 20, 30 days after the Abeta(25-35) injection. All the animals were injected with BrdU (50 mg/kg, i. p) to label the neural precursor cells 24 h before the each perfusion. NeuN immunofluorescence and BrdU immunohistology were performed. It was found that Abeta(25-35) could injure the mature neurons and decrease the number of NeuN positive neurons. It also showed that Abeta(25-35) inhibited neurogenesis and significantly decreased the number of BrdU positive cells in the dentate gyrus of hippocampus, but it had no obvious effects on neurogenesis in the subventricular zone. The present results indicated that Abeta(25-35) could impair neurogenesis in the hippocampus of adult mouse brain.     

Isolation of multipotent neural precursors residing in the cortex of the adult human brain.

Multipotent precursors able to generate neurons, astrocytes, and oligodendrocytes have previously been isolated from human brain embryos and recently from neurogenic regions of the adult human brains. The isolation of multipotent neural precursors from adult human should open new perspectives to study adult neurogenesis and for brain repair. The present study describes the in vitro isolation from adult human brains of a progenitor responsive to both epidermal and basic fibroblast growth factors that forms spheres as it proliferates. Single spheres derived from various regions of the brain generate in vitro neurons, astrocytes, and oligodendrocytes. The clonal origin of the spheres was revealed by genomic viral insertion using lentiviral vector. Interestingly, this vector appears to be a potent tool for gene transfer into human neural progeny. Ninety-six percent of the spheres investigated were multipotent. Multipotent precursors were isolated from all brain regions studied, including the temporal and the frontal cortex, the amygdala, the hippocampus, and the ventricular zone. This study is the first evidence that primitive precursors such as multipotent precursors exist in the adult human cortex and can reside far from the ventricles. Neurogenesis derived from adult human progenitors differ to murine neurogenesis by the requirement of laminin for oligodendrocyte generation and by the action of basic-fibroblast growth factor and platelet derived growth factor that prevented the formation of oligodendrocytes and neurons. Moreover, the differentiation of human adult precursors seems to differ from fetal ones: adult precursors do not necessitate the removal of mitogen for differentiation. These results indicate that the study of adult multipotent precursors is a new platform to study adult human neurogenesis, potentially generate neural cells for transplantation, and design protocols for in vivo stimulation.     

成年大鼠脑创伤后神经前体细胞的增殖及迁移

目的 研究液压冲击性脑损伤后成年大鼠神经前体细胞的增殖及迁移。方法 制作液压冲击性脑损伤模型，免疫组织化学方法动态检测巢蛋白（Nestin）和5溴脱氧尿苷（BrdU）的表达。BrdU标记方法确定增列殖的前体细胞；Nestin的表达用于确定神经前体细胞。结果 同正常对照组相比较，伤侧皮层、海马及室下区的Nestin阳性细胞数于伤后1d明显增多，7d达高峰,30d消失；BrdU阳性细胞数于作后3d达高峰，而7d以后逐渐减小，室下区BrdU阳性细胞及Nestin阳性细胞经胼胝体向对侧迁移。结论 液压冲击性脑损伤可激发成年大鼠神经前体细胞增殖及迁移。     

Prolonged voluntary wheel‐running stimulates neural precursors in the hippocampus and forebrain of adult CD1 mice

Voluntary wheel‐running induces a rapid increase in proliferation and neurogenesis by neural precursors present in the adult rodent hippocampus. In contrast, the responses of hippocampal and other central nervous system neural precursors following longer periods of voluntary physical activity are unclear and are an issue of potential relevance to physical rehabilitation programs. We investigated the effects of a prolonged, 6‐week voluntary wheel‐running paradigm on neural precursors of the CD1 mouse hippocampus and forebrain. Examination of the hippocampus following 6 weeks of running revealed two to three times as many newly born neurons and 60% more proliferating cells when compared with standard‐housed control mice. Among running mice, the number of newly born neurons correlated with the total running distance. To establish the effects of wheel‐running on hippocampal precursors dividing during later stages of the prolonged running regime, BrdU was administered after 3 weeks of running and the BrdU‐retaining cells were analyzed 18 days later. Quantifications revealed that the effects of wheel‐running were maintained in late‐stage proliferating cells, as running mice had two to three times as many BrdU‐retaining cells within the hippocampal dentate gyrus, and these yielded greater proportions of both mature neurons and proliferative cells. The effects of prolonged wheel‐running were also detected beyond the hippocampus. Unlike short‐term wheel‐running, prolonged wheel‐running was associated with higher numbers of proliferating cells within the ventral forebrain subventricular region, a site of age‐associated decreases in neural precursor proliferation and neurogenesis. Collectively, these findings indicate that (i) prolonged voluntary wheel‐running maintains an increased level of hippocampal neurogenesis whose magnitude is linked to total running performance, and (ii) that it influences multiple neural precursor populations of the adult mouse brain. © 2009 Wiley‐Liss, Inc.     

不同程度的性发作对成年大鼠空间学习记忆影响的研究

目的研究不同程度的性发作对成年大鼠空间学习记忆的影响。方法采用氯化锂和匹罗卡品联合诱导大鼠不同程度的癫模型(轻型和重型)。于造模后第6d给所有大鼠腹腔注射5-溴脱氧尿苷嘧啶(BrdU+)标记海马齿状回增殖的内源性神经前体细胞;用免疫组化方法观察各组大鼠注射BrdU+后第1d和第28d齿状回BrdU+阳性细胞数以及第28d的BrdU+/神经元核性蛋白(NeuN+)阳性细胞数及分布情况;利用Morris水迷宫评价大鼠的学习记忆功能。结果与正常组及轻型组比较,在各个时间点重型组海马齿状回BrdU+细胞数均增加(P<0.05),28d时BrdU+/NeuN+细胞数相应增多,但其占BrdU+细胞数的比例明显下降(P<0.05)。28d时重型组大鼠的学习记忆功能较正常组及轻型组明显下降(P<0.05)。结论严重的癫发作造成大鼠对空间学习记忆功能的损害,可能与其刺激大鼠海马齿状回内源性神经前体细胞增殖水平,抑制其分化为新生的成熟神经元有关。     

Postnatal neurogenesis in hippocampal slice cultures: early in vitro labeling of neural precursor cells leads to efficient neuronal production

Neurogenesis continues throughout life in the hippocampus. To study postnatal neurogenesis in vitro, hippocampal slices from rats on postnatal day 5 (P5) were cultured on a porous membrane for 14 or 21 days. In the initial experiments, precursor cells were labeled with bromodeoxyuridine (BrdU) after 7 days in culture because hippocampal slices are generally used in experiments after 1-2 weeks in culture. Fourteen days after labeling, however, only about 10% of BrdU-labeled cells expressed neuronal markers, although in living rats, about 80% of cells labeled with BrdU on P5 had become neurons by P19. Next, rats were injected with BrdU 30 min before culture, after which hippocampal slices were cultured for 14 days to examine the capacity of in vivo-labeled neural precursors to differentiate into neurons in vitro. In this case, more than two-thirds of BrdU-labeled cells expressed neuronal markers, such as Hu, NeuN, and PSA-NCAM. Furthermore, precursor cells underwent early in vitro labeling by incubation with BrdU or a modified retrovirus vector carrying EGFP for 30 min from the beginning of the culture. This procedure resulted in a similar high rate of neuronal differentiation and normal development into granule cells. In addition, time-lapse imaging with retrovirus-EGFP revealed migration of neural precursors from the hilus to the granule cell layer. These results indicate that in vivo- and early in vitro-labeled cultures are readily available ex vivo models for studying postnatal neurogenesis and suggest that the capacity of neural precursors to differentiate into neurons is reduced during the culture period.