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。     

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.     

胚胎大鼠神经干细胞的体外培养与鉴定

目的利用无血清培养和细胞克隆培养技术从孕鼠(14 d)胚胎中分离出神经干细胞,并进行传代培养和诱导分化。 方法 采用免疫细胞荧光化学染色方法观察不同代数细胞克隆球中nestin、neuN、GFAP阳性细胞比例,鉴定培养的原代和传代的细 胞类型结果随着传代次数增加,克隆球中nestin阳性细胞的比例无明显变化(P>0．05),不同代数的神经干细胞克隆球诱导分化 后均有一定比例的neuN与GFAP阳性细胞。结论 证实从胚胎大鼠大脑皮层分离培养的细胞是神经干细胞,提示体外培养体系中 培养的神经干细胞可以长期传代并保持于细胞的特性。     

神经干细胞原代培养及胰岛素对其增殖与分化的作用

目的原代培养获得神经干细胞并初步探讨胰岛素对神经干细胞增殖分化的影响.方法小鼠胎脑原代细胞培养,免疫组化检测鉴定.100 ng/ml胰岛素作用后,甲基噻基四唑(MTT)法及免疫组化观察胰岛素对神经干细胞增殖分化的影响.结果原代培养的神经球表达巢蛋白(nestin),神经球分化的细胞表达神经丝200(NF200)和胶质纤维酸性蛋白(GFAP).与对照组相比,胰岛素作用下的神经干细胞增殖能力强,且神经元分化率较高,可达(35.41±1.97)%.结论原代培养的细胞为神经干细胞,且在胰岛素作用下,其增殖能力和向神经元分化率均有增高.     

葡萄糖浓度对原代培养神经干细胞增殖、分化的影响

目的探讨不同浓度葡萄糖对神经干细胞增殖、分化的影响。方法胚胎小鼠脑细胞原代培养并鉴定。生理浓度和高浓度葡萄糖培养及诱导分化后．四唑盐比色（MTT）法及免疫荧光法观察两组不同葡萄糖浓度对神经干细胞增殖、分化的影响。结果原代培养的神经球表达巢蛋白（hestin），神经球分化的细胞表达神经丝200（NF200）和胶质纤维酸性蛋白（GFAP）。与生理浓度葡萄糖相比，高浓度葡萄糖降低了神经干细胞的增殖活力：分化3d后，高糖组的NF200阳性细胞和GFAP阳性细胞比例高于生理浓度组（P〈0．01），而nestin阳性细胞比例则反之（P〈0．01）；分化10d后，神经干细胞完全分化，免疫荧光检测发现生理浓度葡萄糖提高了NF200阳性细胞比例。结论高糖损害了神经干细胞的增殖活性。加速了神经干细胞的早期分化．降低了神经元的分化率。     

人小龄胚胎神经干细胞的分离培养、扩增及鉴定

目的 探索人胚胎神经干细胞的体外分离培养条件，从而在体外大量扩增神经干细胞；并观察神经干细胞增殖、分化的特点。方法 从人胚胎脑分离神经干细胞，部分细胞冻存，另一部分细胞进行体外培养。采用无血清培养液，加入表皮生长因子(EGF)和碱性成纤维细胞生长因子(bFGF)刺激细胞增殖，进行体外扩增、传代培养。采用免疫荧光法鉴定神经干细胞和分化的神经元及胶质细胞。结果 从人胚胎脑分离的细胞在含有EGF和bFGF的无血清培养液中能形成大量的神经干细胞球，这些神经干细胞球可在体外扩增及传代培养。免疫荧光法鉴定神经干细胞球中大部分为神经上皮干细胞蛋白(nestin)表达阳性细胞。贴壁后可以分化出神经元特异性烯醇化酶(NSE)、胶质纤维酸性蛋白(GFAP)表达阳性的细胞。经冻存后的胎脑细胞也能培养出具有同样特征的神经干细胞。结论 在含有EGF和bFGF的无血清培养液中，从人胚胎脑能分离培养出神经干细胞，并能在体外大量扩增。这为人类神经干细胞的进一步研究和应用提供了材料。     

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。     

大鼠胎脑神经干细胞的培养分化及鉴定的实验研究

目的建立大鼠胎脑皮质神经干细胞的培养、扩增、诱导分化及鉴定的方法。方法选取孕14d胎鼠的大脑皮质作为细胞来源,在无血清培养基中添加B27、碱性成纤维细胞因子、表皮生长因子,建立胚胎神经干细胞的体外培养体系,用5%的胎牛血清诱导神经干细胞分化,用免疫荧光染色技术进行对神经干细胞及诱导分化细胞的鉴定。结果原代培养的神经干细胞折光性强,培养第2d开始形成细胞集落,第3d形成大的神经球。3～5代传代的细胞生长稳定。经胎牛血清诱导后第3d开始,神经球开始向周边发出突起,悬浮的细胞开始贴壁生长。原代及传代培养的神经球表达Nestin阳性,分化细胞分别表达NSE、GFAP和GALC阳性。结论应用无血清培养技术可在体外培养、扩增出神经干细胞。5%的胎牛血清可以诱导神经干细胞向神经元、星形胶质细胞和少突胶质细胞等成熟细胞分化。     

雪旺氏细胞促进共培养大鼠胚胎神经干细胞的分化

目的 探讨诱导神经干细胞分化的因素。方法 新生大鼠雪旺氏细胞与大鼠胚胎神经干细胞在无血清培养液中共培养，观察神经干细胞的形态变化及分别检测特异性标志蛋白tubulin-β、GalC和GAFP的表达。结果 相差显微镜下大部分神经干细胞伸出多个细长突起，且免疫莹光染色tubulin-β高度表达；少数细胞为有多个短小突起的小细胞，且GalC或GFAP阳性表达。结论 雪旺氏细胞与大鼠胚胎神经干细胞共培养可诱导神经干细胞分化。     

不同浓度胰岛素对小鼠神经干细胞分化的影响

目的 体外培养获得神经干细胞并初步探讨不同浓度胰岛素对神经干细胞分化能力的影响。方法 小鼠胎脑细胞体外培养获得神经干细胞,并行免疫荧光检测鉴定。25ng／ml、50ng／ml、100ng／ml、125ng／ml、150ng／ml胰岛素诱导分化后,免疫荧光染色观察不同浓度胰岛素对神经干细胞分化的影响。结果 体外培养的神经球表达巢蛋白（nestin）,神经球分化的细胞表达神经丝和胶质纤维酸性蛋白。不同浓度胰岛素作用下的神经元分化率有差别。其中100ng／ml组的分化率最高。结论 体外培养的细胞为神经干细胞,100ng／ml浓度的胰岛素可以起到较好的促进神经元分化的效果。     

Long-lasting coexpression of nestin and glial fibrillary acidic protein in primary cultures of astroglial cells with a major participation of nestin(+)/GFAP(-) cells in cell proliferation

Nestin, a currently used marker of neural stem cells, is transiently coexpressed with glial fibrillary acidic protein (GFAP) during development and is induced in reactive astrocytes following brain injury. Nestin expression has also been found in cultures of astroglial cells, but little is known about the fate and the mitotic activity of nestin-expressing cells in this in vitro model. The present study reveals a long-lasting expression of nestin in primary cultures of astroglial cells derived from the rat brain. Over 70% of the cells were nestin(+) at 12 weeks, with a large majority coexpressing the GFAP astrocytic marker. Time-course analyses supported a transition from a nestin(+)/GFAP(-) to a nestin(+)/GFAP(+) phenotype over time, which was further increased by cell cycle arrest. Interestingly, double staining with Ki67 revealed that over 90% of cycling cells were nestin(+) whereas only 28% were GFAP(+) in a population consisting of almost equivalent numbers of nestin(+) and GFAP(+) cells. These observations indicated that nestin(+)/GFAP(-) cells are actively engaged in mitotic activity, even after 2 weeks in vitro. Part of these cells might have retained properties of neural stem cells, insofar as 10% of cells in a primary culture of glial cells were able to generate neurospheres that gave rise to both neurons and astrocytes. Further studies will be necessary to characterize fully the proliferating cells in primary cultures of glial cells, but our present results reveal a major contribution of the nestin(+)/GFAP(-) cells to the increase in the number of astrocytes, even though nestin(+)/GFAP(+) cells proliferate also.     

Neural stem and progenitor cells in nestin-GFP transgenic mice

Neural stem cells generate a wide spectrum of cell types in developing and adult nervous systems. These cells are marked by expression of the intermediate filament nestin. We used the regulatory elements of the nestin gene to generate transgenic mice in which neural stem cells of the embryonic and adult brain are marked by the expression of green fluorescent protein (GFP). We used these animals as a reporter line for studying neural stem and progenitor cells in the developing and adult nervous systems. In these nestin-GFP animals, we found that GFP-positive cells reflect the distribution of nestin-positive cells and accurately mark the neurogenic areas of the adult brain. Nestin-GFP cells can be isolated with high purity by using fluorescent-activated cell sorting and can generate multipotential neurospheres. In the adult brain, nestin-GFP cells are approximately 1,400-fold more efficient in generating neurospheres than are GFP-negative cells and, despite their small number, give rise to 70 times more neurospheres than does the GFP-negative population. We characterized the expression of a panel of differentiation markers in GFP-positive cells in the nestin-GFP transgenics and found that these cells can be divided into two groups based on the strength of their GFP signal: GFP-bright cells express glial fibrillary acidic protein (GFAP) but not betaIII-tubulin, whereas GFP-dim cells express betaIII-tubulin but not GFAP. These two classes of cells represent distinct classes of neuronal precursors in the adult mammalian brain, and may reflect different stages of neuronal differentiation. We also found unusual features of nestin-GFP-positive cells in the subgranular cell layer of the dentate gyrus. Together, our results indicate that GFP-positive cells in our transgenic animals accurately represent neural stem and progenitor cells and suggest that these nestin-GFP-expressing cells encompass the majority of the neural stem cells in the adult brain.     

神经干细胞移植治疗大鼠脑缺血再灌注损伤实验研究

目的探讨大鼠胚胎神经干细胞移植治疗局灶性脑缺血再灌注损伤的可行性。方法孕龄8~10d的大鼠神经干细胞在体外扩增后,用免疫组织化学方法分别检测神经干细胞及其分化后代的特异性标志蛋白nestin、胶质纤维酸性蛋白(GFAP)和神经元特异性烯醇化酶(NSE)的表达。分别于缺血后不同时间窗将神经干细胞移植到局灶性脑缺血大鼠模型的缺血半暗带和梗塞中心,移植4w后比较不同移植部位神经干细胞存活、增殖和迁移的差异。结果从胎鼠中成功培养出悬浮生长的可表达nestin的神经球,其在含血清条件下可分化为表达GFAP的胶质细胞和表达NSE的神经元。神经干细胞移植4w后可见所有移植动物的细胞都存活,梗塞中心移植的细胞存活、增殖水平明显低于半暗带移植的细胞。结论大鼠胚胎神经干细胞移植到局灶性脑缺血再灌注损伤大鼠梗塞中心和半暗带均可长期存活,其增殖能力与移植部位密切相关。     

Neuronal differentiation of stem cells isolated from adult muscle

Lineage uncommitted pluripotent stem cells reside in the connective tissue of skeletal muscle. The present study was carried out with pluripotent stem cells (PPSCs) isolated from 6-month old rat muscle. Before differentiation, these cells were vimentin+, CD90+, CD45-, and varied in their expression of CD34. The PPSCs were expanded as non-adherent aggregates under similar conditions to those used to generate neurospheres from embryonic or neural stem cells. The PPSC-derived neurospheres were positive for nestin, an early marker present in neuronal precursors, and expressed the two alternative mRNA forms of the neuroectodermal marker Pax-6, as well as mRNA for Oct-4, a gene related to the pluripotentiality of stem cells. To confirm their neural potential, PPSC-derived neurospheres were plated on coated coverslips under varying conditions: Neurobasal medium with N2 or B27, and either NT3 or BDNF. After 4-6 days the cells expressed neuronal (Tuj1+, NF68), astrocytic (GFAP) and oligodendrocytic (MOSP+, MBP+) markers, both by immunocytochemistry and RT-PCR. In addition, PPSCs were cultured as monolayers under adherent conditions, exposed to growth factors and defined differentiating conditions for 5 hr, and subsequently kept for 2 days in a maturation medium. At this point they gave rise to a mixed population of early neural progenitors (Nestin+ or NG2+), immature and mature neurons (Tuj1+ and NF145+) and myelin producing oligodendrocytes (CNPase + and MOSP+). Our study shows that PPSCs present in adult muscle can overcome germ lineage restrictions and express the molecular characteristics of brain cells. Therefore, PPSCs isolated from adult muscle could provide a novel source for autologous cell replacement in neurodegenerative and demyelinating diseases.     

Evaluation of progenitor cell cultures from human embryos for neurotransplantation

Human neural stem cells (HNSCs) are used in studies of neural development and differentiation, and are regarded as an alternative source of tissue for neural transplantation in degenerative diseases. Selection and standardization of HNSC samples is an important task in research and clinical approaches. We evaluated embryonal brain matter obtained from human 8-12-week-old fetuses by means of flow cytometry on a panel including: nestin; vimentin; NeuN; GFAP; beta-tubulin III; CD56; N-Cad; OB-Cad; HLA-ABC; HLA-DR; CD34, and annexin. Samples from embryos of even the same gestation differ dramatically regarding neural cell development, their phenotype and viability. The samples containing the highest proportion of stem cells and multipotent progenitors of neural types, and the least of definitive cells and antigens of histocompatibility, were selected for further expansion in serum-free medium. Secondary phenotyping 14 days later revealed again a marked heterogeneity of the cultures. For the final culturing for 24 h in a serum-containing medium we selected only samples having following phenotype: nestin+, and vimentin+ no less than 25%; HLA-DR+ and CD34+ no more than 5%; GFAP+ no more than 10%; beta-tubulin+ no more than 20%; CD56+, N-Cad+, OB-Cad+, HLA-A,B,C+, and annexin+ no more than 15%; cell viability no less than 60%. Immunocytochemical study of selected samples proved that numerous neural stem cells, and neuro- and glioblasts necessary for transplantation were present. Our results demonstrate that the flow cytometry phenotyping allows the screening and standardization of HNSC samples for further expansion and transplantation.     

Isolation of neural stem cells from the spinal cords of low temperature preserved abortuses

In the present study, we show that neural stem cells can be obtained from the spinal cords of low temperature preserved abortuses. Fourteen weeks old abortuses were stored in a refrigerator at 4 degrees C for 2 h, 6 h and 12 h before use. Neural stem cells were isolated from cervical cord, thoracic cord and lumbar/sacral cord separately and induced to differentiate with fetal bovine serum. Clonal culture was carried out to demonstrate that the isolated cells met the standard of stem cells. Fluorescent immunocytochemistry was used to examine the expression of neural stem cell marker (nestin), neuronal marker (MAP2), astrocyte marker (GFAP) and cholinergic marker (ChAT). The stem cells in different cultures were compared. As a result, neural stem cells were obtained from all the spinal cord segments with different postmortem intervals. The lumbar/sacral cord cultures gave rise to the most abundant primary neurospheres. When the preservation was prolonged to 12 h, the number of primary neurospheres decreased sharply. Neurospheres in all cultures showed nestin positive immunoreactivity and could yield astrocytes and neurons including cholinergic neurons in differential cultures. The clonal formation and phenotype capacity were similar in all cultures. In conclusion, spinal neural stem cells can be isolated from low temperature preserved abortuses and represent an alternative source for both experimentation and potential therapeutic uses.     

脊髓来源神经干细胞分离培养及向雪旺氏细胞的诱导分化

目的 探讨新生大鼠脊髓来源神经干细胞(NSCs)的分离培养及在体外一定条件下向周围神经雪旺氏细胞分化的可行性. 方法 分离新生大鼠的脊髓组织,在含有B27(终浓度1%)、碱性成纤维细胞生长因子(bFGF)和表皮生长因子(EGF)(终浓度均为20 μg/L)培养基中分离培养出NSCs.用复合诱导因子(10%FBS+5 μmol/L血小板凝集抑制剂+10 ng/mL bFGF+5 ng/mE血小板源性生长因子)在体外诱导NSCs分化为雪旺氏细胞.免疫荧光细胞化学方法[一抗为p75、S-100、神经胶质纤维酸性蛋白(GFAP)]鉴定体外诱导分化结果.结果 培养的新生大鼠脊髓组织细胞nestin染色表达阳性;分离培养的大鼠脊髓来源NSCs经诱导分化后形态类似雪旺氏细胞,免疫荧光细胞化学方法显示诱导后细胞表达雪旺氏细胞的表面标志,GFAP、S-100和P75表达阳性.结论 新生大鼠脊髓来源NSCs可以在体外诱导分化为雪旺氏细胞.     

大鼠脂肪基质细胞的分离培养及二步法诱导分化神经细胞

目的 研究脂肪基质细胞体外培养扩增及向神经细胞诱导分化的新方法.方法 取SD大鼠的腹膜后脂肪组织,以Ⅰ型胶原酶消化离心,含10%胎牛血清的DMEM/F12培养基重悬细胞,增殖传代,取第5代细胞进行二步法诱导分化.首先以无血清Neurobasal培养基联合应用碱性成纤维细胞生长因子(bFGF) 20 ng/mL、表皮生长因子(EGF)20 ng/mL和N2(1:100)添加剂将细胞先诱导成神经球;再取第二代神经球加入1%胎牛血清、5%马血清、0.5μmol/L的维甲酸(RA)和50ng/mL脑源性神经营养因子(BDNF),促进其向神经元和胶质细胞方向分化;免疫荧光染色鉴定细胞分化结果.结果 初接种的细胞大多呈圆球状,悬浮在培养基中,24 h后贴壁.随着细胞密度加大,形态变成单层成纤维样细胞,呈漩涡状排列.无血清Neurobasal培养基诱导5～7 d可见悬浮的神经球出现;神经球迅速增殖,14d时直径可达100μm.分化后的大部分细胞呈现典型的神经元样改变,细胞体为圆形,内可见明显的细胞核,细胞伸出细长突起或网状突起,可见较多的细胞形成网络连接.免疫荧光染色鉴定结果证实,除了神经球可以高表达巢蛋白(nestin)(阳性率75.62%±1.34%)之外,分化细胞还可有效表达β-mbulinⅢ(未成熟神经元标记物,阳性率达57.62%±4.92%)或微管相关蛋白2(MAP2ab)(成熟神经元标记物,阳性率11.25%±3.87%);有少量细胞表达胶质纤维酸性蛋白(GFAF)(阳性率18.34%±3.87%)和GalC(阳性率14.35%+3.98%).结论 从成年大鼠脂肪中分离得到的脂肪基质细胞可在体外培养得到大量分化潜能稳定的子代细胞.应用本实验二步诱导法,可得到较高比例的nestin及β-mbulinⅢ阳性细胞,并有一定比例MAP2ab阳性细胞出现.     

Adult human spinal cord harbors neural precursor cells that generate neurons and glial cells in vitro

Adult human and rodent brains contain neural stem and progenitor cells, and the presence of neural stem cells in the adult rodent spinal cord has also been described. Here, using electron microscopy, expression of neural precursor cell markers, and cell culture, we investigated whether neural precursor cells are also present in adult human spinal cord. In well-preserved nonpathological post-mortem human adult spinal cord, nestin, Sox2, GFAP, CD15, Nkx6.1, and PSA-NCAM were found to be expressed heterogeneously by cells located around the central canal. Ultrastructural analysis revealed the existence of immature cells close to the ependymal cells, which display characteristics of type B and C cells found in the adult rodent brain subventricular region, which are considered to be stem and progenitor cells, respectively. Completely dissociated spinal cord cells reproducibly formed Sox2(+) nestin(+) neurospheres containing proliferative precursor cells. On differentiation, these generate glial cells and gamma-aminobutyric acid (GABA)-ergic neurons. These results provide the first evidence for the existence in the adult human spinal cord of neural precursors with the potential to differentiate into neurons and glia. They represent a major interest for endogenous regeneration of spinal cord after trauma and in degenerative diseases.