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。     

液压性脑损伤后室下区神经干细胞的分离培养与鉴定

目的分离液压性脑损伤室下区巢蛋白（nestin）和胶质酸性纤维蛋白（GFAP）阳性（nestin^＋／GFAP^＋）共存细胞进行培养和诱导分化，观察其分裂、增殖和分化能力，以阐明损伤反应性星形胶质细胞增生过程中nestin^＋／GFAP^＋共存细胞是否具有神经干细胞特性。方法用液压冲击法建立动物模型，分离损伤成年SD大鼠室下区nestin^＋／GFAP^＋共存细胞，制成单细胞悬液，培养和诱导分化，以免疫荧光化学方法对原代和传代培养形成的神经球以及原代和传代培养诱导分化的细胞进行鉴定。结果结果显示培养及传代的细胞不断分裂增殖，可以形成悬浮生长nestin阳性的神经球；神经球诱导分化后可以分化为少突胶质细胞、神经元和星形胶质细胞。结论成年大鼠液压性脑损伤后分离的室下区细胞具有自我更新能力和多向分化潜能，是中枢神经系统神经干细胞。     

新生小鼠端脑组织神经干细胞诱导分化为胆碱能神经元的研究

目的探讨新生小鼠端脑组织神经干细胞是否能够分化成胆碱能神经元。方法取新生小鼠端脑组织．用无血清方法分离培养神经干细胞；用克隆培养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神经干细胞标志巢蛋白（nestin）及干细胞诱导分化后神经元标志微管相关蛋白2（MAP2）、星形胶质细胞标志胶质纤维酸性蛋白（GFAP）、胆碱能标志胆碱乙酰转移酶（CHAT）；比较不同的诱导分化条件（5％胎牛血清、5％胎牛血清＋碱性成纤维细胞生长因子）对胆碱能神经元分化的影响。结果从新生小鼠端脑组织分离培养出具有自我更新、扩增能力的神经球；各培养基中神经球均为nestin阳性。诱导分化后均能够产生MAP2阳性神经元、GFAP阳性星形胶质细胞以及ChAT阳性的胆碱能神经元。分化培养中加入碱性成纤维细胞生长因子能够提高胆碱能神经元分化的比例。结论新生小鼠端脑组织神经干细胞能够分化成胆碱能神经元。     

经枕大池神经干细胞移植治疗大鼠创伤性脑损伤的研究

目的 将神经干细胞经枕大池移植到创伤性脑损伤模型大鼠蛛网膜下腔中并观察其存活、迁移和分化,从而为神经干细胞的体内存活、迁移和分化机理研究和临床应用提供实验依据.方法 体外培养BrdU标记的胚胎神经干细胞并应用免疫荧光细胞化学染色对BrdU、神经干细胞标记物nestin的表达进行鉴定:采用Feeney自由落体撞击法制做大鼠脑损伤模型,伤后24 h将BrdU标记的胚胎神经十细胞经立体定向注射移植到蛛网膜下腔;制作大鼠脑绢织石蜡切片,应用免疫组织化学染色检测BrdU、微管相关蛋白2(MAP2)、胶质纤维酸性蛋白(GFAP)表达;伤前24h、伤后24 h及1、2周行动物运动神经功能评分.结果 免疫荧光检测显示神经球的表面细胞表达nestin及BrdU:免疫组织化学染色检测到脑内损伤灶存在BrdU阳性神经干细胞、MAP2阳性神经元和GFAP阳性胶质细胞;接受神经十细胞移植的大鼠神经运动功能评分的恢复较对照组有明显提高,差异有统计学意义(P<0.05).结论 经枕大池移植到脑损伤大鼠蛛网膜下腔中的神经干细胞能存活且具有远距离迁移能力,并明显有助于脑损伤大鼠神经运动功能的恢复.     

胚胎大鼠嗅神经干细胞的培养及分化特性

目的建立胚胎大鼠嗅神经干细胞(NSCs)体外培养方法,研究其增殖和分化特性.方法采用添加丝裂原的无血清培养基分离、培养胚胎14 d(E14)大鼠嗅球NSCs,应用免疫细胞化学方法鉴定培养的NSCs及自然分化为特异性神经细胞的类型,测定NSCs的生长曲线.结果从E14大鼠嗅球分离、培养出表达nestin,并能分化为神经元、星形胶质细胞和少突胶质细胞的NSCs.嗅NSCs的增殖依赖表皮生长因子(EGF)和碱性成纤维细胞生长因子(bFGF),其中EGF的促分裂增殖作用明显优于bFGF.结论从E14大鼠嗅球培养出具有自我增殖和多向分化潜能的NSCs.     

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。     

成年C57小鼠脊髓神经干细胞的培养与鉴定

目的　 从成年小鼠脊髓中培养神经干细胞 ,并对其进行鉴定和诱导分化。方法　 成年C5 7小鼠 ,悬浮培养神经干细胞技术。结果　培养 1～ 2周时 ,培养液中即可出现神经干细胞克隆球。该克隆球具有很强的自我增殖能力 ,可多次传代。免疫细胞化学技术证明该克隆球表达大量的神经干细胞特征性的中间丝———巢蛋白(Nestin) ;经 1%胎牛血清诱导后可表达神经元、星形胶质细胞和少突胶质细胞特征性标志物β tubulinIII、胶质纤维酸性蛋白 (Gliafibrillaryacidicprotein ,GFAP)和RIP ,提示它们可朝神经元、星形胶质细胞和少突胶质细胞方向分化。 结论　 本研究结果提示正常成年C5 7小鼠脊髓中含有神经干细胞 ,在体外条件下可以大量增殖 ,经诱导后可朝神经元和胶质细胞的方向分化     

多聚赖氨酸和明胶对神经干细胞增殖和分化的影响

摘要 背景：目前大鼠神经干细胞体外诱导分化的研究报道诸多,但其分化过程很难控制,很多实验的操作方法复杂,分化比率也很低。 目的：探索大鼠胚胎前脑神经干细胞体外原代及传代培养方法,并观察其分化规律。 方法：胎鼠在无菌条件下分离出前脑,制备单细胞悬液,以1×1011L-1接种于含N2的DMEM/F12培养基中培养,传代培养过程中加入BrdU,标记神经干细胞球。诱导分化实验分为多聚赖氨酸铺板组、明胶铺板组和无铺板组。采用体积分数20%胎牛血清刺激其分化。免疫细胞化学检测nestin、BrdU及在血清诱导条件下神经干细胞向神经细胞分化的能力。 结果与结论：细胞呈神经干细胞样生长,具有连续增殖能力,可以传代培养。传代神经球中的细胞均呈nestin阳性和BrdU阳性。多聚赖氨酸铺板组和明胶铺板组贴壁后分化为神经细胞能力强于无铺板组(P < 0.01)。多聚赖氨酸铺板组略强于明胶铺板组(P > 0.05)。神经谱系标记物神经胶质纤维酸性蛋白和微管相关蛋白2的免疫细胞化学结果均阳性。结果表明,大鼠胚胎前脑富含神经干细胞,其分化观察,多聚赖氨酸和明胶在诱导神经干细胞分化中作为细胞贴壁支持物提高分化细胞数量的作用,且多分化为星形胶质细胞。 关键词：多聚赖氨酸;神经干细胞;明胶;增殖分化;体外培养 doi:10.3969/j.issn.1673-8225.2010.47.004     

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。     

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。     

Olfactory bulb core is a rich source of neural progenitor and stem cells in adult rodent and human

The olfactory bulb (OB) core is an extension of the rostral migratory stream and thus is a potential source of neural progenitor and neural stem cells. We characterized in vivo and in vitro neuronal progenitor and neural stem cells in the adult OB core. In mouse and rat, bromodeoxyuridine (BrdU) labeling showed that the OB core accumulates newly replicated cells. Nestin, a neuroepithelial stem cell marker, was enriched in the OB core. BrdU-positive cells were immunolabeled for nestin and TUC4, a marker for early postmitotic neurons. The distributions of cells labeled for BrdU, TUC4, and nestin were similarly concentrated in the OB core. Nestin- and TUC4-positive cells were also found in the OB of young and aged humans. Isolated and cultured OB core cells from adult rat and mouse had the capacity to generate numerous neurospheres. Adult OB core neurospheres were cryopreserved and subsequently cultured. Single cell clonal analysis of neurospheres revealed the capacity for self-renewal and multipotency. Cultured adult OB core cells differentiated into neurons, astrocytes, and oligodendrocytes. Some neurons expressed choline acetlytransferase, substance P, and glutamic acid decarboxylase. Basic fibroblast growth factor potentiated the self-renewal of cells and beta-nerve growth factor stimulated differentiation. OB-derived neural stem cells in coculture with skeletal muscle cells were induced to become neurons expressing choline acetyltransferase and substance P and formed neuromuscular synaptic junctions on myocytes displaying acetylcholinesterase-positive motor end plates. Cocultured OB-derived neural stem cells with myoblast cells also generated nonneural cell progeny. We conclude that the adult mammalian OB core is a reservoir of neural progenitor cells and pluripotent neural stem cells.     

Prospective characterization of neural stem cells by flow cytometry analysis using a combination of surface markers

Neural stem cells (NSCs) with self-renewal and multilineage differentiation properties can potentially repair degenerating or damaged neural tissue. Here, we have enriched NSCs from neurospheres, which make up a heterogeneous population, by fluorescence-activated cell sorting (FACS) with antibodies against syndecan-1, Notch-1, and integrin-beta1, which were chosen as candidates for hematopoietic cell-or somatic stem cell-markers. Antigen-positive cells readily initiated neurosphere formation, but cells lacking these markers did so less readily. Doubly positive cells expressing both syndecan-1 and Notch-1 underwent neurosphere formation more efficiently than did singly positive cells. The progeny of sorted cells could differentiate into neurons and glial cells both in vitro and in vivo. These antibodies were also useful for isolating cells from the murine embryonic day 14.5 brain that efficiently formed neurospheres. In contrast, there was no distinct difference in neurosphere formation efficiency between Hoechst 33342-stained side population cells and main population cells, although the former are known to have a stem cell phenotype in various tissues. These results indicate the usefulness of syndecan-1, Notch-1, and integrin-beta1 as NSC markers.     

Nestin-EGFP transgenic mice: visualization of the self-renewal and multipotency of CNS stem cells

We generated transgenic mice carrying enhanced green fluorescent protein (EGFP) under the control of the nestin second-intronic enhancer (E/nestin:EGFP). Flow cytometry followed by in vitro assays revealed that in situ EGFP expression in the embryonic brain correlated with the mitotic index, the cogeneration of both neurons and glia, and the frequency of neurosphere formation in vitro. High-level EGFP expressors derived from embryos included a distinct subpopulation of cells that were self-renewable and multipotent, criteria that define neural stem cells (NSCs). Such cells were largely absent among lower-level or non-EGFP expressors, thereby permitting us to enrich for NSCs using EGFP expression level. In adults, although E/nestin:EGFP-positive cells included the NSC population, the frequency of neurosphere formation did not correlate directly with the level of EGFP expression. However, moderately EGFP-expressing cells in adults gained EGFP intensity when they formed neurospheres, suggesting embryonic and adult NSCs exist in different microenvironments in vivo.     

活体取材山羊皮层组织神经干细胞的培养与鉴定

目的通过手术从活体山羊中获取小块皮层脑组织,进行体外神经干细胞（NSC）的培养与鉴定。方法选用健康成年山羊,全麻后通过手术开颅取大小约2cm×2cm×1cm皮层脑组织块,在体外用无血清培养基自然筛选法进行分离、培养得到NSC,并采用免疫荧光细胞染色法进行NSC特异性抗原巢蛋白（nestin）及多向分化潜能的鉴定。结果皮层脑组织块在体外通过无血清培养基自然筛选法培养2w后可以成功地培养得到大量细胞球,经nestin鉴定为NSC球;所得到的NSC球在体外分化后经微管相关蛋白-2（MAP2）、胶质纤维酸性蛋白（GFAP）及少突胶质细胞蛋白标志物1（O1）免疫荧光组化染色后确定可分化为神经元、星形胶质细胞及少突胶质细胞。结论对活体山羊通过手术获取其少量皮层脑组织,在体外培养后可得到相当数量的NSC,有望用于自体NSC的移植治疗。     

神经干细胞原代培养方法的优化

目的 探讨低浓度胰酶不同消化分离时间对体外培养新生大鼠海马NSCs增殖与凋亡的影响.方法 取出生24 h内SD大鼠海马组织,以1.25 g/L胰酶37℃分别消化5、10、15、20和25min(依次为A～E组),获得单细胞悬液后进行培养.通过台盼蓝染色计数、细胞形态观察和神经球数目比较不同消化时间对NSCs活力和生长的影响;用5-溴-2脱氧尿嘧啶核苷(BrdU)标记法检测NSCs的增殖能力;用免疫荧光细胞化学法检测BrdU、nestin的表达:用Annexin V-FITC/PI染色和流式细胞仪检测细胞凋亡率.结果 原代和传代培养的NSCs都能快速增殖并形成神经球;免疫荧光染色结果显示神经球细胞均表达NSCs特异性标志物nestin;所获得的细胞能将BrdU结合到细胞核中;各组培养3、5、7 d后,C组(消化15 min)NSCs成球数最多,BrdU标记克隆率最高,细胞凋亡率最低,与其他组比较差异有统计学意义(P<0.05).结论 体外分离培养的新生大鼠海马NSCs具有增殖能力,1.25 g/L胰酶在不同消化时间对NSCs增殖能力和凋亡率的影响有所不同,消化时间过长或过短都会抑制NSCs增殖,诱导NSCs凋亡,且消化时间越长NSCs的凋亡率越高.

Abstract：

Objective To study the influence of digestion times of low concentration trypsin on the proliferation and apoptosis of neural stem cells (NSCs) in the hippocampus of neonate rats.Methods Hippocampus of neonatal rats (within 24 h) were taken out, and treated with trypsin at 1.25g/L concentration and 37 ℃ for 5, 10, 15, 20 and 25 minutes; unicellular suspension was then successfully got and primary culture and subculture were performed. Effects of trypsinization on cell viability and growth of NSCs were compared by observing the cell morphology and Trypan blue staining.The 5-bromodeoxyuridine labeling was performed to assess the self-renewing and proliferative activities of NSCs. Fluorescence immunocytochemistry was carried out to examine the expressions of BrdU and nestin. Apoptosis was measured by Annexin V-FITC/PI assay and flow cytometry. Results Primary and passage culture of NSCs enjoyed rapid proliferation and formation of neurospheres. The neurosphere cells expressed NSCs specific marker nestin by immunofluorescence; all the neurosphere cells could incorporate BrdU into the nucleus; of the neurospheres obtained from the 3rd, 5th and 7th d, those digested for 15 rain enjoyed the highest level of NSCs neurospheres, the highest BrdU labeled clone and the lowest cell apoptosis as compared with those digested for 5, 10, 20 and 25 min (P<0.05). Conclusion The NSCs isolated from the hippocampus of neonatal rats have the ability of proliferation in vitro. And 1.25 g/L concentration of trypsin with digestion times could positively change the proliferative and apoptosis capacity of NSCs: too short or long digestion times can inhibit the proliferation of NSCs and induce the apoptosis of NSCs; the longer the digestion time, the higher the apoptosis of NSCs.