表皮生长因子对胎鼠纹状体神经干细胞的作用

目的：研究表皮生长因子（EGF）对胎鼠纹状体神经干细胞分裂增殖及分化的诱导作用。方法：应用EGF促使体外培养中的胎鼠神经干细胞分裂增殖并诱导其分化,随后使用透射电子显微镜及免疫组化方法研究干细胞的生物学特性。结果：胎鼠纹状体存在多潜能神经干细胞,EGF可以促使胎鼠纹状体神经干细胞分裂,并使其分化产生一定比例的神经元及胶质细胞。结论：EGF作为外源性信号因子作用于胎鼠纹状体神经干细胞的相应受体,对其分裂分化可产生重要作用。     

体外神经干细胞培养特性观察

目的 探讨神经干细胞(NSCs)体外分离培养和增殖的特性.方法 从新生24h内的SD大鼠脑组织分离NSCs,采用无血清悬浮培养法进行NSCs体外扩增培养.倒置相差显微镜观察细胞形态,通过绘制细胞生长曲线观察NSCs的自我更新和增殖能力,采用免疫细胞化学法检测NSCs标志物神经上皮干细胞蛋白(Nestin)的表达及分化后细胞神经元特异性烯醇化酶(NSE)、胶质纤维酸性蛋白(GFAP)和2,3-环核甘酸磷酸二脂酶(CNP)的表达.结果 从新生SD大鼠脑组织分离的细胞在无血清的培养基中形成悬浮的神经球.神经球具有自我更新和表达Nestin的能力,分化后的细胞能表达神经元、星型胶质细胞及少突胶质细胞的特异性抗原.结论 从新生大鼠的脑组织中成功分离出NSCs,其具有在体外自我更新和增殖、多向分化潜能及表达Nestin的能力.     

胚胎大鼠神经干细胞培养及分化鉴定的实验研究

目的建立胚胎大鼠神经干细胞体外培养及分化鉴定的方法。方法分离不问孕龄(13- 18d)SD胎鼠脑室下区、中脑及海马等部位组织,在含有表皮生长因子和碱性成纤维细胞生长因子及N-2 添加剂的DMEM培养基中悬浮培养,形成神经球体后撤除生长因子,经消化传代于含胎牛血清培养基中贴壁生长。应用免疫荧光方法检测原代细胞特异性标记巢蛋白、神经元标记微管蛋白-β、胶质细胞标记胶质纤维酸性蛋白,以及少突胶质细胞标记半乳糖脑苷的表达。结果胎鼠脑组织不同部位均可分离出神经干细胞,体外培养可以向神经元和神经胶质细胞分化,孕龄13d与18d胎鼠产生神经干细胞球体的能力不同,前者优于后者。结论 (1)胚胎大鼠脑内不同部位来源的组织生成神经干细胞球体数量和质量基本相同,但是不同胎龄的鼠脑组织产生神经干细胞球体的能力有所差异。(2)神经干细胞具有向神经元和神经胶质细胞分化的潜能。     

胎鼠纹状体神经干细胞分离培养及鉴定

背景：从胎鼠神经系统的多个部位如大脑半球、海马、皮质、脑室区等可分离出神经干细胞。 目的：探讨胚胎大鼠大脑纹状体神经干细胞的取材、分离、培养和鉴定方法,观察神经干细胞增殖、传代和分化的规律。 设计、时间及地点：观察性实验,于2008-03/08在烟台毓璜顶医院中心实验室完成。 材料：选用普通级健康成年Wistar大鼠12只,按雌雄1∶1比例于每晚6时合笼,次晨检查出阴道栓子(阴道内皮结晶)为妊娠0 d。 方法：在无菌条件下取孕13 d胚胎大鼠脑纹状体组织,用无血清培养技术在体外进行神经干细胞的培养、扩增和传代。分别用抗巢蛋白抗体、抗微管相关蛋白2抗体和抗胶质原纤维酸性蛋白抗体对培养的细胞进行干细胞特性和多分化潜能的免疫组织化学鉴定。 主要观察指标：①显微镜下观察细胞生长情况。②神经干细胞分化情况及免疫鉴定。 结果：①原代细胞种植后24 h可见大量分裂期的神经干细胞,呈对称或不对称分裂,细胞核较大,连续观察可见细胞分裂成2个完全独立的神经干细胞;48 h后出现许多由4~10个细胞疏松连接的细胞团;神经干细胞进入快速增殖期5 d后出现许多大小不等的细胞集落,即神经球,神经球呈规则的圆球状,细胞排列紧密,表面光滑没有突起。②培养4~6 h后悬浮的神经干细胞球逐渐贴壁,大部分细胞分化后胞体呈圆形或椭圆形,具有1个或2个长突起,少量细胞具有多个粗长突起。贴壁分化3 d左右,具有1个或2个突起的神经元样细胞逐渐减少,具有多个粗长突起的星形胶质细胞样细胞逐渐增多。悬浮的神经干细胞球经巢蛋白染色呈阳性反应,部分细胞呈微管相关蛋白2和胶质原纤维酸性蛋白染色阳性。 结论：从胎鼠纹状体中分离培养的神经干细胞具有自我增殖、自我更新能力,并能分化为神经元及神经胶质细胞。     

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     

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。     

新生大鼠全大脑组织分离诱导分化神经干细胞的体外实验

背景：神经干细胞的供体一般以胎鼠和成年鼠为主，利用细胞培养技术分离步骤较繁琐。 目的：以新生大鼠为神经干细胞供体，拟建立一种较为简便、细胞获得率较高的分离培养方法。 设计、时间及地点：以细胞为对象观察性实验，于2006-10/2007-03在重庆医科大学完成。 材料：新生1~3 d 的Wistar大鼠全大脑。 方法：以胰蛋白酶消化、无血清、悬浮培养原代细胞，并加含体积分数为0.10胎牛血清的DMEM/F12培养液诱导其分化。 主要观察指标：应用相差显微镜观察神经干细胞的生长特点及分化后的细胞形态学变化。应用间接免疫细胞化学染色法鉴定神经干细胞及其分化后神经元和胶质细胞标志蛋白的表达。以BrdU标记神经干细胞，观察其增殖情况。 结果：新生大鼠脑组织分离的细胞具有连续传代和增殖的能力，能稳定表达神经干细胞特异性巢蛋白。诱导分化后的细胞能表达神经元细胞、星形胶质细胞、少突胶质细胞的特异性蛋白。 结论：从新生大鼠脑组织分离培养出的神经干细胞获得率高，保持了干细胞的未分化属性，具有自我更新和多项分化潜能。     

大鼠胎脑皮质神经干细胞体外培养及诱导分化的实验研究

目的 从孕龄15d SD胚胎鼠脑皮质中分离并培养神经干细胞（neural stem cells。NSCs），观察其生长、增殖及分化。方法 采用包含碱性成纤维细胞生长因子（bFGF）和表皮细胞生长因子（EGF）的无血清培养及单细胞克隆技术，对胚胎鼠脑皮质神经干细胞进行原代、传代培养及诱导其分化。用Nestin染色鉴定神经干细胞特性，用免疫组化方法（β-Ⅲ-tubulin、GFAP染色）检测神经干细胞分化为神经元及神经胶质细胞状况。结果 从孕龄15dSD胚胎鼠脑皮质中分离的组织，经原代及传代培养均可形成细胞克隆．切具有增殖能力。原代及传代培养细胞呈Nestin（神经上皮干细胞蛋白）表达阳性．诱导分化后的细胞表达神经元细胞、星形胶质细胞的特异性抗原。结论 本实验分离、培养的孕龄15dSD胚胎鼠脑皮质细胞Nestin表达阳性．分化后表达神经元和星形胶质细胞的标记物，是大鼠的神经干细胞，并具有多向分化潜能。     

当归对宫内缺氧新生大鼠神经干细胞增殖、分化的影响

背景：实验小组前期研究发现孕鼠宫内缺氧可刺激胎鼠神经干细胞的增殖,缺氧6 h时增殖达高峰,在9 h也表现增殖,但能力开始下降。而缺氧达12 h时即表现为坏死或凋亡,但随缺氧天数的延长及时段的不同,对神经干细胞的影响又如何？ 目的：进一步探讨宫内缺氧对新生大鼠神经干细胞增殖、分化的影响及当归注射液的保护作用。 方法：孕 SD大鼠随机分为对照组、缺氧组和当归治疗组。孕14 d开始将当归组与缺氧组孕鼠置于三气培养箱中,制作缺氧性脑损伤新生鼠模型,此前1 h分别给于当归注射液和生理盐水尾静脉注射,对照组不缺氧,余同缺氧组。孕鼠分娩后立即取新生鼠大脑组织,经胶质纤维酸性蛋白、神经元特异性烯醇化酶免疫组织化学染色后行图像分析。 结果与结论：①缺氧组新生鼠海马胶质纤维酸性蛋白免疫组织化学阳性细胞的表达较相应对照组增加;而神经元特异性烯醇化酶免疫组织化学阳性细胞的表达较对照组减小。②当归治疗组新生鼠海马胶质纤维酸性蛋白免疫组织化学阳性细胞的表达较相应缺氧组减少;而神经元特异性烯醇化酶免疫组织化学阳性细胞的表达较对照组增大。结果表明,一定程度的缺氧可刺激神经干细胞增殖,并可刺激神经干细胞向神经胶质细胞分化,以及导致神经元的减少;当归注射液可减弱由于缺氧导致的神经干细胞的增殖和向胶质细胞分化的能力,并可缓解神经元的减少,提示当归可能对缺氧大鼠神经系统有一定的保护作用。     

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。     

Tumor suppressor gene BRCA-1 is expressed by embryonic and adult neural stem cells and involved in cell proliferation

BRCA-1 is a tumor suppressor gene that plays a role in DNA repair and cellular growth control. Here we show that BRCA-1 mRNA is expressed by embryonic rat brain and is localized to the neuroepithelium containing neuronal precursor cells. The expression of BRCA-1 decreases during rat brain development, but BRCA-1 is expressed postnatally by proliferating neuronal precursor cells in the developing cerebellum. Neural stem cells (NSC) prepared from embryonic rat brain and cultured in the presence of epidermal growth factor were positive for BRCA-1. Induction of NSC differentiation resulted in down-regulation of BRCA-1 expression as shown by RNA and protein analyses. In addition to embryonic cells, BRCA-1 is also present in NSC prepared from adult rat brain. In adult rats, BRCA1 was expressed by cells in the walls of brain ventricles and in choroid plexus. The results show that BRCA-1 is present in embryonic and adult rat NSC and that the expression is linked to NSC proliferation.     

EGF and FGF-2 responsiveness of rat and mouse neural precursors derived from the embryonic CNS

EGF and FGF-2 induce the proliferation of embryonic neural precursors (ENPs) in vitro from a number of different species. In this study, we demonstrate that embryonic age is a crucial determinant of the number and differentiation potential of rat embryonic neural precursor cells responding to either EGF and/or FGF-2, in that (i) there is a differential response to the two growth factors (both alone and in combination) according to the gestational age of isolation and (ii) when allowed to differentiate, there are temporal changes in the ability of these cells to produce neurons. Furthermore, for cultures of all gestational ages, there is a defined pattern of senescence, with cultures expanding longest when cells are isolated earlier in gestation. The suggestion is that rat ENPs in this study consist predominantly of neural progenitor cells with limited division potential rather than self-renewing multipotential neural stem cells. In contrast, mouse ENPs appeared to expand indefinitely and thus allow for longer studies to be carried out looking at the effects of growth factor concentrations. The effect of varying the concentration of EGF was assessed using mouse ENPs.     

Proliferation and differentiation of rat neuroepithelial precursor cells in vivo

Important features of adult neuronal number, location, and type are a consequence of early embryonic events that occur before neurons have differentiated. We have measured cell number during embryogenesis of the rat CNS. Markers that are expressed in the proliferating neuronal precursor are required to study the mechanisms controlling their proliferation and differentiation. By applying immunohistochemistry, fluorescence-activated cell sorting, and 3H-thymidine auto-radiography to dissociated rat CNS cells, we show that the monoclonal antibody Rat 401 recognizes a cell population with proliferative, temporal, and quantitative features expected of neuronal precursors.     

Primary neural precursor cell expansion, differentiation and cytosolic Ca(2+) response in three-dimensional collagen gel

To investigate the ability to culture neural precursor cells in a three-dimensional (3D) collagen gel, neuroepithelial cells were isolated from embryonic day 13 rat cortex, dispersed within type I collagen and maintained for up to 30 days in vitro. Cultured in Neuorobasal medium supplemented with B27 containing basic fibroblast growth factor, the collagen-entrapped precursor cells actively expanded and formed clone-like clusters. Many cells in the center of the cluster were proliferating as revealed by 5-bromo-2'-deoxyuridine uptake. Some cells began to migrate away from the center at 5 days and were labeled by either neuronal marker neuron-specific beta-tubulin (TuJ1) or astrocytic marker glial fibrillary acidic protein. The differentiated neurons (TuJ1(+)) exhibited characteristic cytosolic Ca(2+) oscillations in response to excitatory neurotransmitter glutamate. These findings suggest the suitability of the 3D culture system for the proliferation and differentiation of neural precursor cells.     

大鼠胚胎神经干细胞移植治疗脑出血的实验研究

目的 研究大鼠胚胎神经干细胞移植治疗脑出血的可行性。方法 从孕龄16天的大鼠胚胎脑组织中分离、培养神经干细胞并诱导其分化，通过免疫组化学技术研究其特性。制作大鼠脑出血模型，3天后将未分化的神经干细胞注入血肿同侧或对侧的尾状核内，记录损伤和移植后的大鼠运动功能。不同时间杀死大鼠，研究移植后的干细胞在体内分化和迁徙的情况。结果 实验中分离、培养的神经干细胞体外能够被诱导分化成神经元、光突胶质细胞和星形胶质细胞，血肿同侧移植干细胞的大鼠运动功能的改善显著好于血肿对侧移植干细胞组及未移植干细胞的对照组。免疫组化方法证实移植后的干细胞在体内可分化成神经元和胶质细胞，并向损伤区域迁徙。结论 大鼠胚胎神经干细胞体内、体外均具有多向分化潜能，其分化成各种类型神经细胞的比例与所处的外界环境有关，在脑内靠近损伤部位移植胚胎神经干细胞后能够有效改善脑出血动物的运动功能。     

In vitro analysis of a mammalian retinal progenitor that gives rise to neurons and glia

In vivo lineage studies have shown that retinal cells arise from multipotential progenitors whose fates are regulated by cell–cell interactions. To understand the mechanism underlying their maintenance and differentiation, we have analyzed the differentiation potential of progenitors derived from embryonic rat retina in vitro. These progenitors proliferate and remain undifferentiated in vitro in the presence of epidermal growth factor (EGF) and display properties similar to stem cells. In addition to expressing nestin, the neuroectodermal stem cell marker, retinal progenitors are multipotential. Upon withdrawal of EGF and addition of serum, the progenitors downregulate the expression of nestin and express cell-type specific markers corresponding to neurons and glia. In addition to expressing cell-type specific markers, retinal progenitors and their progeny could be distinguished on the basis of their distinct voltage gated current profile. A proportion of progenitors is lineage restricted and the fate of these cells can be influenced by the microenvironment, suggesting that stage-specific interactions mediated by the local environment influence the progression of progenitors towards acquisition of differentiated phenotypes.     

Grafted lineage-restricted precursors differentiate exclusively into neurons in the adult spinal cord

Multipotent neural stem cells (NSCs) have the potential to differentiate into neuronal and glial cells and are therefore candidates for cell replacement after CNS injury. Their phenotypic fate in vivo is dependent on the engraftment site, suggesting that the environment exerts differential effects on neuronal and glial lineages. In particular, when grafted into the adult spinal cord, NSCs are restricted to the glial lineage, indicating that the host spinal cord environment is not permissive for neuronal differentiation. To identify the stage at which neuronal differentiation is inhibited we examined the survival, differentiation, and integration of neuronal restricted precursor (NRP) cells, derived from the embryonic spinal cord of transgenic alkaline phosphatase rats, after transplantation into the adult spinal cord. We found that grafted NRP cells differentiate into mature neurons, survive for at least 1 month, appear to integrate within the host spinal cord, and extend processes in both the gray and white matter. Conversely, grafted glial restricted precursor cells did not differentiate into neurons. We did not observe glial differentiation from the grafted NRP cells, indicating that they retained their neuronal restricted properties in vivo. We conclude that the adult nonneurogenic CNS environment does not support the transition of multipotential NSCs to the neuronal commitment stage, but does allow the survival, maturation, and integration of NRP cells.     

神经干细胞移植治疗大鼠脑缺血再灌注损伤的实验研究

目的探讨胎鼠皮质培养的神经干细胞移植治疗大鼠脑缺血再灌注损伤的可行性及疗效。方法取孕龄15d Sprague-Dawley(SD)胎鼠皮质细胞培养为神经干细胞;用线栓法制备大鼠脑缺血再灌注损伤模型;将24只健康SD大鼠分为假手术组、缺血对照组、缺血移植组,在移植后2周、4周依据Garcia的18分评分法对各组大鼠的神经功能进行评分;行脑灌注固定取材,免疫组化染色观察移植后神经干细胞的分化、迁移和整合情况。结果用胎鼠皮质培养的细胞nestin表达阳性;缺血移植组大鼠的神经功能评分明显高于缺血对照组(P<0.05);缺血移植组免疫组织化学染色能够检测到存活的BrdU阳性细胞,移植后4周时可见移植细胞向周围迁移、分化、参与血管形成,并可见梗死区边缘微血管明显增生;缺血移植组大鼠脑GFAP阳性细胞数较缺血对照组明显增多(P<0.05)。结论分离、培养胎鼠皮质细胞Nestin表达阳性,即是大鼠的神经干细胞;移植体外培养的神经干细胞能在脑缺血大鼠脑内存活、迁移、分化,并且对脑梗死大鼠的神经功能修复起到了积极作用。     

Tumour necrosis factor-α impairs neuronal differentiation but not proliferation of hippocampal neural precursor cells: Role of Hes1

Tumour necrosis factor-α (TNFα) is a pro-inflammatory cytokine, which influences neuronal survival and function yet there is limited information available on its effects on hippocampal neural precursor cells (NPCs). We show that TNFα treatment during proliferation had no effect on the percentage of proliferating cells prepared from embryonic rat hippocampal neurosphere cultures, nor did it affect cell fate towards either an astrocytic or neuronal lineage when cells were then allowed to differentiate. However, when cells were differentiated in the presence of TNFα, significantly reduced percentages of newly born and post-mitotic neurons, significantly increased percentages of astrocytes and increased expression of TNFα receptors, TNF-R1 and TNF-R2, as well as expression of the anti-neurogenic Hes1 gene, were observed. These data indicate that exposure of hippocampal NPCs to TNFα when they are undergoing differentiation but not proliferation has a detrimental effect on their neuronal lineage fate, which may be mediated through increased expression of Hes1.     

Toluene inhibits muscarinic receptor-mediated cytosolic Ca2+ responses in neural precursor cells

Toluene is widely used as a component in industrial solvents and many toluene-containing products are abused via inhalation. While many studies have demonstrated its inhibitory effects on neuronal activity, the effects of toluene on receptor signaling in proliferating and differentiating neural precursor cells are presently unclear. Here, using digital video microscopy and Ca2+ imaging, we investigated the effects of acute exposure to toluene on the function of muscarinic acetylcholine receptors (mAChRs) expressed in neural precursor cells. The neural precursor cells were isolatedfrom embryonic day 13 (E13) rat cortex and expanded in serum-free medium containing basic fibroblast growth factor (bFGF). We found that the acetylcholine (ACh) analog carbachol (CCh) induced a dose-dependent increase in cytosolic Ca2+, which was blocked by the muscarinic receptor antagonist atropine in a reversible manner. Toluene was added to the perfusion medium and concentrations of toluene in the medium were determined by gas chromatographic analysis. Following imaging, the cells were fixed and processed for 5-bromo-2'-deoxyuridine (BrdU, cell proliferation marker) and beta-tubulin (TuJ1, neuronal marker) immunostaining. In the 5 day culture, most cells continued to divide (BrdU+), while afew cells differentiated into young neurons (TuJ1-). The CCh-induced Ca2+ elevations in proliferating (BrdU+TuJ1-) neural precursor cells were significantly reduced by acute exposure to 0.15 mM toluene and completely blocked by 10 mM toluene. Toluene's inhibition of muscarinic receptor-mediated Ca2+ signaling was rapid, reversible and dose-dependent with an IC50 value 0.5 mM. Since muscarinic receptors mediate cell proliferation and differentiation during neural precursor cell development, these results suggest that depression of muscarinic signaling may play a role in toluene's teratogenic effect on the developing nervous system.