Neuro-2a细胞替代神经元原代培养进行神经轴突测量实验研究

目的 建立使用小鼠神经母细胞瘤Neuro-2a细胞替代原代培养神经元进行神经轴突测量的方法,用于神经损伤研究.方法 使用浓度200 μmol/L、500 μmol/L、1 000μmol/L过氧化氢处理Neuro-2a细胞12h,戊二醛固定,生物染色,在光学显微镜下手动测量突起长度.结果 过氧化氢可剂量依赖性引起神经突起逐渐回缩变短,1 000 μmol/L过氧化氢处理组光镜下见不到明显突起,突起测量数据显示,过氧化氢处理后突起长度与未经处理细胞有显著差异.结论 Neuro-2a细胞在一定程度上可替代原代培养神经元进行轴突测量研究,该测量方法可用于神经轴突再生研究.     

Neuro-2α细胞替代神经元原代培养进行神经轴突测量实验研究

目的建立使用小鼠神经母细胞瘤Neuro-2α细胞替代原代培养神经元进行神经轴突测量的方法,用于神经损伤研究。方法使用浓度200μmol／L、500μmol／L、1000μmol／L过氧化氢处理Neuro-2α细胞12h,戊二醛固定,生物染色,在光学显微镜下手动测量突起长度。结果过氧化氢可剂量依赖性引起神经突起逐渐回缩变短,1000μmol／L过氧化氢处理组光镜下见不到明显突起,突起测量数据显示,过氧化氢处理后突起长度与未经处理细胞有显著差异。结论Neuro-2α细胞在-定程度上可替代原代培养神经元进行轴突测量研究,该测量方法可用于神经轴突再生研究。     

神经干细胞的端粒酶活性与其增殖分化的关系

目的探讨体外培养的神经干细胞端粒酶活性与细胞增殖、分化的关系,以及细胞分化后端粒酶逆转录酶的表达情况。方法采用无血清培养法从新生大鼠脑皮质分离培养神经干细胞；通过免疫荧光细胞染色鉴定神经干细胞；细胞计数法检测细胞的增殖情况；TRAP-ELISA法测定神经干细胞的端粒酶活性：RT-PCR法和Western-blot法测定细胞分化前后的端粒酶逆转录酶的表达。结果从新生大鼠脑皮质分离培养的神经干细胞具有端粒酶活性；体外培养12周内,神经干细胞的端粒酶活性未见变化,细胞的增殖速率亦未见明显不同；神经干细胞分化后端粒酶活性丧失,端粒酶逆转录酶的mRNA和蛋白质也均未见表达。结论在体外培养过程中,大鼠脑神经干细胞的端粒酶活性和细胞增殖速率未见变化；神经干细胞分化后端粒酶活性丧失,可能是由于端粒酶逆转录酶停止表达所致。     

神经干细胞原代培养及GABA能神经元的诱导分化

目的探讨神经干细胞的原代培养及γ-氨基丁酸(GABA)能神经元的诱导分化.方法取孕16 dWistar大鼠的胚鼠全脑,进行体外培养,并对培养的神经干细胞以及诱导分化的GABA能神经元进行鉴定.结果培养24h后,出现2～4个细胞的细胞球.分化2d后,神经球贴壁后伸出细长突起,并可和周边神经球伸出的突起连接.神经球周边可见大量散在贴壁的双极或多极细胞.免疫荧光染色可见神经球均有nestin、NF200、GFAP阳性细胞.取第3代神经球行GABA能神经元定向分化.分化24h后,实验组细胞球贴壁.3d后,实验组细胞球周边有大量散在分布细胞贴壁生长,胞体圆形较大,有1～2个细长突起.免疫荧光显示,实验组周边散在的贴壁细胞多为GAD65阳性细胞.GAD65阳性细胞分化率实验组(85.97±2.78)%、对照组(18.16±2.29)%,P<0.01.结论本实验利用寡核苷酸序列特异性阻断了bHLH基因家族的调控因子之一Hes1,解除了其对bHLH的抑制,促进了神经干细胞向神经元的分化.实验还发现,阻断Hes1后大大提高了神经干细胞向GABA神经元分化的比率.     

JAK-STAT3信号通路调节大鼠骨髓间充质干细胞神经定向分化

探索体外诱导大鼠骨髓间充质干细胞（rat mesenchymal stem cells,MSCs）神经诱导分化的内在分子机制。方法：体外贴壁法培养MSCs,纯化培养传至3代后,诱导组采用含有表皮生长因子（EGF）,碱性成纤维细胞生长因子（bFGF）,脑源性神经神经生长因子（BDNF）的L-DMEM作为神经诱导培养基,诱导MSCs向神经细胞分化;含AG490组采用含有AG490（JAK-STAT3通路抑制剂）5uM的诱导组神经诱导培养基,同等条件下诱导MSCs神经分化。倒置显微镜下观察细胞形态变化、免疫细胞化学检测神经元特异性烯醇化酶(NSE),微管相关蛋白2（MAP2）,胶质原纤维酸性蛋白（GFAP）阳性率。Western blot检测JAK-STAT3通路中总STAT3蛋白和酪氨酸(Tyr705) STAT3磷酸化蛋白表达水平。结果：神经诱导培养基可以诱导MSCs向神经元和神经胶质细胞转分化,诱导5天后细胞轴突逐渐变长,呈典型神经细胞样形态,并且细胞轴突间相互接触。Western blot结果表明在神经诱导过程中酪氨酸(Tyr705)STAT3磷酸化逐渐增强,JAK-STAT3信号通路被激活。而JAK-STAT3通路特异性抑制剂AG490处理后,再经神经诱导培养基诱导后,MSCs向神经胶质细胞分化比例明显下降,而MSCs向神经元分化没有受到影响, 从而提高了 MSCs向神经元分化的比例。 结论 JAK2/STAT3信号通路参与了MSCs神经定向分化的调节,抑制此信号通路可以减少向星形胶质细胞分化的比例,提高了MSCs向神经元分化的比例。     

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。     

脑脊液中细胞源性囊泡对间充质干细胞向神经前体细胞分化的影响

目的探讨脑脊液微囊泡在间充质干细胞(MSCs)向神经干细胞分化中的作用。方法从人脑脊液中分离微囊泡,从小鼠骨髓中分离原代MSCs。将MSCs传代后在神经分化诱导培养基和脑脊液微囊泡作用下神经分化诱导。细胞形态学观察细胞突起数目和长度,western blot检测细胞神经特异性蛋白表达水平变化。结果单纯神经诱导液组单位面积内细胞突起长度为(2.2±0.4)mm,突起数量为(94±12)个;神经诱导液加脑脊液微囊泡诱导的细胞中细胞突起更长(5.7±1.2)mm,数目也更多(178.2±32)个,差异具有统计学意义(P0.01)。微囊泡组细胞中NSE、Nestin和MAP-2表达水平高于单纯神经诱导液组细胞,差异具有统计学意义(P0.01)。结论脑脊液微囊泡可有效促进MSCs向神经干细胞分化,有望为干细胞移植治疗神经功能缺损提供了一个新的思路。     

维甲酸诱导胚胎大鼠脑海马神经干细胞向神经元的分化

背景：目前认为直接进行神经干细胞移植后细胞虽能存活,但是只分化成神经胶质细胞,并不能分化成有功能的神经元。 目的：探讨维甲酸诱导对胚胎大鼠脑海马神经干细胞向神经元分化的作用。 设计、时间及地点：细胞学体外实验,于2008-09/2009-02在辽宁医学院科技实验楼完成。 材料：胚龄13.5 d的SD大鼠由辽宁医学院实验动物中心提供。 方法：分离胎鼠脑海马组织,胰蛋白酶消化法体外培养获得神经干细胞。将原代和传代细胞以1×107 L-1接种到培养孔中,分别进行常规贴壁分化培养和维甲酸诱导分化培养。 主要观察指标：神经干细胞的鉴定,光镜及免疫组化检测神经干细胞诱导分化结果。 结果：免疫组织化学检测结果显示,原代和传代后得到的神经干细胞团均呈巢蛋白阳性。常规贴壁分化培养7 d后,神经元多呈椭圆型和近似三角形,胞体大,细胞边缘清楚,胞体上有多个突起;而维甲酸诱导分化培养后,神经元数量增加,形态清楚,但细胞胞体上突起较少。与常规贴壁分化培养比较,维甲酸诱导分化培养后神经干细胞向神经元分化率明显升高(P < 0.01),神经干细胞向神经胶质细胞分化率明显降低(P < 0.01)。 结论：从大鼠胚胎脑海马组织中分离得到可自我复制和多向分化的神经干细胞,维甲酸体外诱导后可以增加其向神经元方向分化的比例。     

朗飞结处神经突起生长抑制因子

朗飞结以及结侧区是有鞘轴突上的一些极化区域,越来越多的证据表明胶质细胞分泌的某些抑制中枢神经系统损伤后轴突再生过程中神经突起生长的分子如粘蛋白（tenascins）、硫酸软骨素蛋白聚糖（chondroitin sulphate proteoglycans）、髓鞘相关糖蛋白（myelin-associated glycoprotein,MAG）、轴突生长抑制因子（Nogo）以及少突胶质细胞髓鞘糖蛋白（OMGP）等非常特异性的富集于朗飞结区域。这些分子在体外组织培养过程中显示出强烈的神经突起生长抑制作用。在一些基因无义突变的动物模型,能够观察到朗飞结处轴突的生长,表明这些抑制分子能够生理性地保持轴突的完整性并且阻止轴突间随机和错误的联结,然而,大部分的基因无义突变动物模型显示不出明显的中枢神经系统再生改善。这些被称为抑制因子的分子是否是神经再生失败的真正元凶这些抑制因子体内体外实验结果的不一致以及它们特异的定位分布让我们有理由对它们在其他生理作用和功能方面进行重新评价。考虑到轴突-胶质细胞相互作用的双向特性,本综述认为这些抑制因子不仅通过神经元上的受体信号通路调节轴突的极化、离子通道的功能以及轴突的分枝,另一方面轴突产生的化学分子也能反馈性的通过朗飞结区域寡突胶质细胞上的胶质细胞受体信号通路影响寡突胶质细胞的发育。     

苯丙氨酸及其代谢物对P19细胞神经分化的影响

探讨苯丙氨酸及其代谢物对P19细胞分化的影响及机制.实验各组从P19细胞神经分化诱导期开始分别加入苯丙氨酸、苯乙酸和苯乳酸,观察神经分化期细胞形态,MTT法检测神经分化期细胞存活率和LDH漏出量的变化,并通过流式细胞仪检测诱导后细胞周期有无变化.结果显示:苯丙氨酸及其代谢物可使P19神经元肿胀、崩解,神经突起纤细且突起数少,可降低P19细胞存活率,但对LDH漏出量并无影响,对诱导后的P19细胞周期也无影响.以上结果提示,苯丙氨酸及其代谢物可干扰P19细胞的神经诱导过程,母源性苯丙酮尿症中枢神经系统损伤可能与其在脑发育早期阶段神经诱导及分化过程中受到高苯丙氨酸及其代谢物影响有关.     

Axonogenesis in neuro-2a cells correlates with GM1 upregulation in the nuclear and plasma membranes.

GM1 ganglioside was previously shown to occur in the nuclear membrane, as well as the plasma membrane, of central nervous system (CNS) and peripheral nervous system (PNS) neurons undergoing morphological differentiation in culture. NG108-15 neuroblastoma cells showed the same phenomenon when induced to extend axon-like but not dendrite-like processes, although in both cases terminal differentiation was evidenced by failure of extended neurites to retract following washout of neuritogenic agent. The present study of Neuro-2a neuroblastoma cells subjected to similar treatments has revealed both similarities and differences compared to NG108-15 cells. Similar to the latter, Neuro-2a cells responded to neuraminidase and ionomycin with axon-like outgrowth together with upregulation of nuclear GM1, and to three other agents (retinoic acid, dibutyryl cyclic AMP, exogenous GM1) with dendrite-like outgrowth that was unaccompanied by nuclear GM1 increase. Although both cell types responded to low serum by extending neurites of mixed axonal-dendritic properties, Neuro-2a, in keeping with its greater tendency to extend some neurites of axonal character in low serum, showed elevated nuclear GM1 in a significant number of such differentiated cells. All three axonogenic agents induced parallel upregulation of GM1 in plasma-, nuclear-, and Golgi membranes, and these increases were stable to washout. Neurites generated in Neuro-2a cells by the three dendritogenic agents lacked stability, unlike those produced by the same agents in NG108-15 cells. This study also amplified the differences in response triggered by exogenous GM1 compared to that resulting from enzyme-mediated elevation of endogenous GM1.     

Expression and distribution of microtubule-associated protein 2 (MAP2) in neuroblastoma and primary neuronal cells

We examined the expression and distribution of microtubule-associated protein 2 (MAP2) during the differentiation in culture of both mouse NB2a neuroblastoma and primary embryonic rat neurons. The differentiation of NB2a cells was induced with retinoic acid (RA) which stimulated the extension of a highly branched neuritic network and dibutyryl cAMP which stimulated the outgrowth of long bipolar or monopolar processes. We found that although monoclonal antibodies to MAP2 stained the cell bodies of control and differentiated cells, only the RA-induced neurites were positive for this antigen. These data support our ultrastructural studies indicating that the RA-induced neurites were dendrite-like and that the dibutyryl cAMP-induced processes were axon-like. Studies on the biosynthesis of MAP2 indicated that RA induced a 2-3-fold increase in MAP2 synthesis in 24 h; however, this effect was transient, with the synthesis of MAP2 in RA-treated cells returning to control level by 72 h. Although biosynthetic studies suggested the synthesis of species at 250-300 kdalton, the major molecular weight form in the neuroblastoma cells was 230 kdalton. Immunocytochemical analysis of primary neurons showed staining of neuronal cell bodies and of short processes, but virtually no staining of the long axon-like processes. The staining of neuronal cell bodies and processes was evident at all stages of cell differentiation. This finding was corroborated by immunoblots which showed significant amounts of MAP2 throughout cell development. The molecular weight of the immunoreactive material was ca. 300 kdalton in both primary neurons and rat brain. Immunoblots also revealed that embryonic neurons expressed only MAP2B as they differentiated in culture for 14 days. Biosynthesis studies suggested that early in culture there was a modest increase in MAP2 synthesis, but no detectable change was observed thereafter. We concluded therefore that both neuroblastoma cells and primary neurons can differentiate neuritic processes, which show dendritic properties in terms of morphology and preferential distribution of MAP2.     

Telomerase and neuronal marker status of differentiated NT2 and SK-N-SH human neuronal cells and primary human neurons

Upon treatment with retinoic acid, NTera-2 (NT2) human teratocarcinoma and SK-N-SH neuroblastoma cells can be induced to terminally differentiate into postmitotic neuronal cells. The neuronal cell yield obtained from the NT-2 cells is partially dependent on the time of differentiation (24-55 days). SK-N-SH cells differentiate into a mixed population of neuronal and epithelium-like cells. Here we report modified protocols that increase the number of differentiated NT-2 and SK-N-SH cells and that establish an enriched neuronal SK-N-SH-derived cell population essentially devoid of nonneuronal cells. Differentiated cells express the cytoskeleton-associated protein tau and other typical neuronal markers, such as Map2, Ngn1, NeuroD, Mash1, and GluR which are also expressed in primary human fetal neurons. Telomerase activity is down-regulated in differentiated cells, which is consistent with the telomerase status of primary fetal human neurons. Thus, differentiated NT2 and SK-N-SH cells may represent an excellent source for studies investigating the role of telomerase or other survival-promoting activities in protecting human neuronal cells from cell death-mediating stresses associated with neurodegenerative diseases.     

端粒、端粒酶与干细胞

摘要：端粒、端粒酶与干细胞密切相关,在维持干细胞自我更新和增殖能力中起重要作用。端粒是真核细胞染色体末端的DNA重复序列和特异结合蛋白的复合体,富含鸟嘌呤,具有保护染色体的作用,端粒长度反映细胞的复制史及复制潜能。影响端粒长度的因素包括：端粒结合蛋白、端粒帽蛋白、端粒酶及 DNA复制酶等,其中端粒酶是最主要的因素。端粒酶位于端粒末端,作用是合成端粒DNA序列,以抵消或延缓端粒随细胞分裂的不断缩短。端粒酶活性的丧失及其增殖相关基因表达的改变是造成干细胞体外复制和扩增受限的主要原因。随着组织细胞工程学的兴起,体外定向诱导干细胞分化为各种所需组织细胞已经成为研究的焦点,因此诱导和增加端粒酶的活性,维持干细胞分化、自我更新和增殖能力,延长干细胞的寿命具有重要意义。     

Dynamics of telomerase activity in response to acute psychological stress

Telomerase activity plays an essential role in cell survival, by lengthening telomeres and promoting cell growth and longevity. It is now possible to quantify the low levels of telomerase activity in human leukocytes. Low basal telomerase activity has been related to chronic stress in people and to chronic glucocorticoid exposure in vitro. Here we test whether leukocyte telomerase activity changes under acute psychological stress. We exposed 44 elderly women, including 22 high stress dementia caregivers and 22 matched low stress controls, to a brief laboratory psychological stressor, while examining changes in telomerase activity of peripheral blood mononuclear cells (PBMCs). At baseline, caregivers had lower telomerase activity levels than controls, but during stress telomerase activity increased similarly in both groups. Across the entire sample, subsequent telomerase activity increased by 18% one hour after the end of the stressor (p < 0.01). The increase in telomerase activity was independent of changes in numbers or percentages of monocytes, lymphocytes, and specific T cell types, although we cannot fully rule out some potential contribution from immune cell redistribution in the change in telomerase activity. Telomerase activity increases were associated with greater cortisol increases in response to the stressor. Lastly, psychological response to the tasks (greater threat perception) was also related to greater telomerase activity increases in controls. These findings uncover novel relationships of dynamic telomerase activity with exposure to an acute stressor, and with two classic aspects of the stress response – perceived psychological stress and neuroendocrine (cortisol) responses to the stressor.     

骨髓间充质干细胞向神经元样细胞诱导分化过程中端粒酶的变

背景：已知端粒酶活性变化与组织工程应用的安全性密切相关，而目前端粒酶在骨髓间充质干细胞诱导分化过程中的变化及机制研究尚少。 目的： 观察体外人骨髓间充质干细胞向神经元样细胞诱导分化过程中端粒酶的变化。 设计、时间及地点：细胞学开放性实验，于2006-12/2007-11在吉林大学第三临床医院实验中心及基础医学院病理生物学教育部重点实验室完成。 材料：无菌条件下采集非血液系统疾病的志愿者髂骨骨髓，体外分离培养人骨髓间充质干细胞。 方法：取第3代培养的人骨髓间充质干细胞，应用二甲基亚砜/丁羟茴香醚（DMSO/BHA）联合诱导法诱导其向神经元样细胞分化。 主要观察指标：绘制体外培养的人骨髓间充质干细胞的生长曲线。免疫荧光及细胞化学染色法分别检测诱导后细胞的巢蛋白和尼氏体的表达，TRAP-ELISA方法检测人骨髓间充质干细胞诱导前后端粒酶活性的变化。 结果： 体外培养的人骨髓间充质干细胞在第3~8代生长较快,第10代以后细胞的生长能力明显减弱。经DMSO/BHA诱导分化后的细胞能够表达具有神经元特征的巢蛋白及尼氏体结构。此外，TRAP-ELISA结果显示诱导2 h细胞端粒酶活性变化不明显，当诱导6 h以后细胞端粒酶活性明显降低，诱导至24 h时，细胞端粒酶活性已近阴性（P < 0.05）。 结论：人骨髓间充质干细胞在体外成功诱导分化为神经元样细胞的过程中端粒酶活性逐渐降低直至消失。