Collagen与胎脑神经细胞移植入脑缺血大鼠的形态学研究

目的：观察胶原蛋白（Collagen)与胎脑神经细胞（FBN）联合培养2天后，神经细胞与胶原蛋白的吸附情况；观察胶原蛋白与胎脑神经细胞（CFBN）移植入脑缺血大鼠后神经细胞的成活情况。方法：取孕14天的胎脑神经细胞，体外分离后与胶原蛋白进行联合培养；制备大脑中动脉闭塞（MCAO）模型。实验组在脑缺血2小时，再灌流48小时后植入CFBN，对照组同样条件未移植CFBN。实验组及对照组分别于2、6、10、30天后灌流取材，保证各时相点动物大于或等于3只。结果：CFBN植入2天后，机体发生轻微排斥反应，部分中性粒细胞和淋巴细胞浸润，6天后排斥反应消失。HE和尼氏染色可见实验组植入神经细胞生长良好。结论：CFBN在脑缺血大鼠中生长良好，CFBN可用作神经系统疾病的细胞移植治疗。     

人胚中脑神经干细胞增殖能力的研究

目的探讨神经球中细胞的分裂增殖能力。方法自孕13周人胚中脑腹侧分离出未成熟细胞,培养形成神经球,并对其进行扩增。应用单细胞显微操作法对神经球的细胞进行克隆分析;应用流式细胞仪测定神经球细胞的分裂增殖能力。结果大约在接种后7～10d,神经球开始形成;神经球细胞克隆形成率为1.26%±0.33%;流式细胞仪测定神经球中处于有丝分裂期的细胞占细胞总数的10.05%±1.43%。结论自人胚中脑腹侧组织中可以分离出形成神经球的细胞,这些神经球中的细胞具有一定的增殖能力。但并不是所有的细胞都具有增殖能力。     

高血压对大鼠脑动脉平滑肌细胞表型改变影响的研究

探讨高血压对脑动脉平滑肌细胞 (SMC)表型改变的影响。　　方法　将SD大鼠造成肾性高血压 ,并分为正常组和高血压 6、9及 1 2周组。各组动物脑标本分别做HE染色和α -Actin及增殖细胞核抗原 (PCNA)免疫组化染色。比较各组HE染色标本的动脉内径和管壁厚度的比值 ;对各组动物平滑肌细胞α -Actin和PCNA染色阳性和阴性的动脉进行比较。　　结果　随高血压时间延长 ,脑动脉硬化性改变逐渐明显 ;平滑肌PCNA染色阳性的脑动脉逐渐增多。　　结论　本实验提示高血压有可能促进颅内动脉SMC由收缩型向合成型转变。     

人胎脑神经干细胞在年幼大鼠脑内的成神经元分化★

目的：脑组织中不同部位的的神经元因功能不同具有特定的细胞形态，移植的神经干细胞能不能在相应部位分化成其相应的神经元还不明确。实验拟观察人胎脑神经干细胞植入年幼大鼠脑后的成神经元分化的作用，探讨神经干细胞替代治疗小儿脑病的可行性。 方法：实验于2007-04/07在海军总医院细胞实验室内完成。①实验材料：16周孕龄的人胎脑组织由海军医院妇产科提供，实验经孕妇及家属知情同意，并经医院伦理委员会批准。14只出生后10 d的同窝年幼SD大鼠，雌雄不分，由北京大学医学部实验动物中心提供，为二级清洁动物，实验过程中对动物处置符合动物伦理学标准。②实验方法：自孕16周的人胎脑组织分离培养神经干细胞球，在脑脊液中培养诱导分化实验以证明其分化潜能。将培养14 d的神经干细胞球移植于10 d龄大鼠侧脑室内，于移植后第4，7，14天行人神经丝特异性标志的免疫荧光分析，显示神经元的分布和细胞形态。 结果：①培养获得典型神经干细胞球，呈漂浮生长，在小儿脑脊液中能分化为神经元、星形胶质细胞和少突胶质细胞。②采用抗人神经丝混合单抗检测移植物的成神经元分化，移植后第4天，观察到阳性反应细胞在颗粒层表现为颗粒性细胞，锥体细胞层则出现长突起的锥体细胞，还有连接神经元样中间神经元，小脑内有单层排列的浦肯野细胞。对比各时间点的观察结果，阳性细胞分布位置未变，随着移植后天数的后延，阳性细胞数量呈减少趋势，但锥体细胞的突起明显加长。 结论：体外培养获得的人神经干细胞经脑室途径移植于年幼大鼠，在脑内发生迁移，并分化成形态上与其所在位置的宿主细胞一致的神经元。提示宿主脑组织微环境在引导移植物分化成神经元中发挥了重要作用，该结果对细胞替代治疗小儿脑病有重要启示意义。     

人Plecstrin同源域家族A成员1表达与胶质瘤恶性表型相关性研究

目的 研究人Plecstrin同源域家族A成员1(Pleckstrin homology like domain family A member 1,PHLDA1)与胶质瘤恶性表型关系.方法 收集马鞍山市中心医院神经外科和上海新华医院神经外科2015年1月至2020年4月65例原发脑胶质瘤的速冻标本.从接受尸检的非胶质...     

大鼠胎脑神经细胞与几丁质多孔体联合培养及移植治疗脑损伤的实验研究

目的：评价几丁质多孔体的细胞相容性；研究几丁质与神经细胞复合体脑内移植对脑损伤的修复作用，为脑组织工程的研究做探索工作。方法：将大鼠胎脑神经细胞与几丁质多孔体联合体外培养，进行光镜、电镜观察。并将体外联合培养4天的几丁质－神经细胞复合体移植到大鼠皮质损伤模型的大鼠脑损伤部位，于术后3、6、10及15天观察脑组织对移植物的反应、移植细胞生长状态及载体在体内的降解情况等。结果：胎脑神经细胞能伸出突起附着在几丁质多孔体内，几丁质对神经细胞生长无不良影响；脑组织对移植物无明显的免疫排斥反应；载体与周围脑组织整合好，载体内有大量存活神经细胞，神经细胞之间有突起相连，形态接近正常脑组织细胞。结论：几丁质具有良好的细胞相容性和组织相容性，是神经组织工程的一种较为理想的生物载体材料，但其降解周期较长，还需进一步观察长期疗效；将胎脑神经细胞或神经干细胞与几丁质多孔体联合培养，移植来治疗中枢神经系统损伤是一个很有前景的新方法。     

骨形态发生蛋白2对14 d胎鼠端脑神经干细胞分化为胆碱能神经元的影响*☆

背景：骨形态发生蛋白2可能是参与胆碱能神经元前体细胞分化的细胞外调控因子。 目的：观察骨形态发生蛋白2在孕14 d胎鼠端脑神经干细胞诱导成胆碱能神经元过程中的作用。 方法：取孕14 d胎鼠端脑,用含EDTA的胰酶和Ⅰ型胶原酶消化,无血清培养基培养细胞,种植于涂有多聚赖氨酸的培养板,细胞原代培养24 h后半量换液,加入10 μg/L骨形态发生蛋白2继续培养。 结果与结论：胶原酶消化得到的神经干细胞呈单层贴壁生长;Nestin免疫荧光鉴定细胞为阳性,获取的神经干细胞纯度大于99%;ChAT免疫荧光鉴定骨形态发生蛋白2可以将孕14 d胎鼠端脑神经干细胞诱导成胆碱能神经元,细胞纯度大于97%。     

人胚脑神经细胞感染柯萨奇B族病毒的形态学研究

采用人胚脑皮质神经细胞原代培养,接种100TCID50柯萨奇B3病毒（CVB3）后用光镜、组织染色法及酶学测定观察病变全过程。人胚脑神经细胞受到病毒感染后48h逐渐出现细胞固缩、折光性增强、脱落、碎裂样改变。肌酸激酶增高。ＣＢＶ３感染所致的人胚脑皮质细胞病变,反映了人脑在自然状态下感染ＣＶＢ３后的神经细胞病变过程。     

人胎脑神经生长因子4基因与质粒载体重组体的体外构建

BACKGROUND： Neurotrophin-4 （NT-4） can promote neuronal growth, development, differentiation, maturation, and survival. NT-4 can also improve recovery and regeneration of injured neurons, but cannot pass through the blood-brain barrier, which limits its activity in the central nervous system. Delivering NT-4 into the central nervous system v/a cells or vectors may have therapeutic benefit. OBJECTIVE： To construct a recombinant vector with a human embryonic brain-derived NT-4 gene and pEGFP-NI. DESIGN, TIME AND SETTING： Neural genetic engineering experiment. The study was performed at the Neuroscience Institute of Kunming Medical College between October 2007 and March 2008. MATERIALS： The pEGFP-N1 plasmid vector was provided by Kunming Institute of Zoology, Chinese Academy of Sciences; embryonic brain tissues were provided by the First Affiliated Hospital of Kunming Medical College. TRIzol RNA extraction Kit was purchased from Sigma （USA）, One Step RNA PCR Kit （AMV） etc. were from Takara （Dalian, China）. METHODS： Total RNA was extracted from human embryonic brain tissues using Trizol. The agarose gel electrophoresis showed two bands： 18 S and 28 S, which were essential subunits of total RNA. The human NT-4 DNA was obtained via RT-PCR and inserted into the pEGFP-N1 vector using ligation and transformation reaction. MAIN OUTCOME MEASURES： The sequencing results of the DNA in the recombinant of NT-4- pEGFP-NI. RESULTS： The NT-4-pEGFP-N1 vector was sequence-verified and showed the expected molecular weight. CONCLUSION： The recombinant of NT-4-pEGFP-N1 was constructed successfully in vitro.     

纤维蛋白原、纤维蛋白及其降解产物对共培养血管平滑肌细胞表型转换的影响

目的：研究不同浓度纤维蛋白原（Fg）、纤维蛋白（Fb）及其降解产物（FDPs）对共培养血管平滑肌细胞（SMCs）表型转换的影响。方法：建立兔主动脉内皮细胞（ECs）-SMCs共培养体系，分别观察在正常增殖和不同浓度Fg、Fb和FDPs干预后共培养SMCs表型转换标志物——α-SM-actin mRNA的表达。结果：在共培养条件下从第3天开始SMCs呈增殖表型的形态特征，第6天后，呈收缩表型的形态特征；较高浓度（3.0-6.0mg·mL^-1）的Fg、Fb和FDPs均可显著下调α-SM-actin mRNA的表达，抑制SMCs向收缩表型转换。结论：增殖过程中SMCs表型发生有规律的转换；Fg、Fb和FDPs通过影响SMCs的异常表型转换，参与了动脉粥样硬化进程。     

Neurosphere and neurosphere-forming cells: morphological and ultrastructural characterization

Despite recent advances in our understanding of neural stem cell (NSC) biology, the free-floating structures generated by these cells in vitro, the "neurospheres", have not been fully characterized. To fill this gap, we examined neurospheres and neurosphere-derived NSCs by confocal microscopy, electron microscopy (EM) and cytofluorimetry. Here, we show that neurospheres and neurosphere-forming cells are morphologically and functionally heterogeneous. Confocal microscopy reveals differences in cell size, viability, cytoplasmic content and in the presence and distribution of active mitochondria. By electron microscopy, neurospheres appear as complex structures in which biological events such as mitosis, apoptosis and even phagocytosis are influenced by NSCs localization within the architecture of the neurosphere. NSCs derived from neurospheres are not synchronized and are represented in all phases of the cell cycle. Cytofluorimetric studies demonstrate NSCs' heterogeneity in cell size by forward scatter (FSC) analysis, and in cytoplasmic granularity by side scatter (SSC) profiling. These findings may contribute to our understanding of the morphogenesis of the neurospheres, particularly as this process relates to the high environmental adaptability of the NSCs and the reported existence of different subpopulations of neural stem cells.     

Silver nanoparticles exhibit coating and dose-dependent neurotoxicity in glutamatergic neurons derived from human embryonic stem cells

Silver nanoparticles (AgNPs) are used extensively as anti-microbial agents in various products, but little is known about their potential neurotoxic effects. In this study, we used glutamatergic neurons derived from human embryonic stem cells as a cellular model to study 20 nm citrate-coated AgNPs (AgSCs) and Polyvinylpyrrolidone-coated AgNPs (AgSPs) induced neurotoxicity. AgSCs significantly damaged neurite outgrowths; increased the production of reactive oxygen species and Ca²⁺ influxes; reduced the expression of MAP2, PSD95, vGlut1 and NMDA receptor proteins at concentrations as low as 0.1 μg/ml. In contrast, AgSPs exhibited neurotoxicity only at higher concentration. Furthermore, our results showed that AgSCs induced glutamate excitotoxicity by the activation of calmodulin and the induction of nitric oxide synthase; increased the phosphorylation of glycogen synthase kinase-3 α/β at Tyr²¹⁶ and Tau at Ser³⁹⁶ and reduced the expression of Tau46, which are typically observed in Alzheimer’s disease. This study indicated that stem cells can provide an excellent platform for studying nanoparticle induced neurotoxicity.     

Efficient generation of mature cerebellar Purkinje cells from mouse embryonic stem cells

Mouse embryonic stem cells (ESCs) can generate cerebellar neurons, including Purkinje cells (PCs) and their precursor cells, in a floating culture system called serum‐free culture of embryoid body‐like aggregates (SFEB) treated with BMP4, Fgf8b, and Wnt3a. Here we successfully established a coculture system that induced the maturation of PCs in ESC‐derived Purkinje cell (EDPC) precursors in SFEB, using as a feeder layer a cerebellum dissociation culture prepared from mice at postnatal day (P) 6–8. PC maturation was incomplete or abnormal when the adherent culture did not include feeder cells or when the feeder layer was from neonatal cerebellum. In contrast, EDPCs exhibited the morphology of mature PCs and synaptogenesis with other cerebellar neurons when grown for 4 weeks in coculture system with the postnatal cerebellar feeder. Furthermore, the electrophysiological properties of these EDPCs were compatible with those of native mature PCs in vitro, such as Na⁺ or Ca²⁺ spikes elicited by current injections and excitatory or inhibitory postsynaptic currents, which were assessed by whole‐cell patch‐clamp recordings. Thus, EDPC precursors in SFEB can mature into PCs whose properties are comparable with those of native PCs in vitro. © 2009 Wiley‐Liss, Inc.     

Neural precursor cells derived from human embryonic brain retain regional specificity

Recent studies have revealed that neural precursor cells can be expanded not only from the subventricular zone and hippocampus but also from other regions of the human embryonic brain. To determine the regional differences of these precursor cells, we divided the brain of a 9-week-old human embryo into four parts, i.e., telencephalon, diencephalon, mesencephalon, and rhombencephalon. All cultures of the tissues yielded neurospheres, and these spheres gave rise to neurons, astrocytes, and oligodendrocytes. An analysis of clonal populations revealed that these precursor cells were multipotent, and two region-specific differences in neural precursor cells were revealed: 1) The precursor cells from the rostral part of the brain tended to proliferate faster than those from the caudal part, and 2) the precursor cells from the diencephalon and mesencephalon gave rise to more tyrosine hydoxylase (TH)-positive neurons than those from the telencephalon and rhombencephalon. When 50-day-cultured spheres were caused to differentiate, the percentage of TH-positive cells per total cell population was 1.2% for diencephalic and mesencephalic precursors, whereas it was 0.4% for telencephalic and rhombencephalic ones. Furthermore, the TH-positive cells from diencephalic and mesencephalic precursors were large, multipolar, and gamma-aminobutyric acid (GABA)-negative, which suggested that these cells were midbrain dopaminergic neurons. In contrast, TH-positive cells from telencephalic and rhombencephalic precursors were small, bipolar, and GABA-positive. These results suggest that human neural precursor cells might have the potential to differentiate into a variety of cells but retain regional specificity.     

Pure astrocyte cultures derived from cells isolated from mature brain

Enriched preparations of oligodendrocytes, isolated either from adult bovine brain or from 30-day-old rat brain, eventually yield cultures in MEM-15% calf serum that contain, in addition to oligodendrocytes, proliferating astrocytes and variable numbers of fibroblast-like cells. If these cultures are switched to a serum-free defined medium during the 1st week, mixed cultures containing only oligodendrocytes and astrocytes are obtained. Bovine cultures can be replated and purified by selective adhesion to yield cultures that are greater than 99% astrocytes; similar procedures were not successful with rat cultures. Cytoskeletal preparations of the purified astrocyte cultures from mature bovine brain contain both vimentin and glial fibrillary acidic protein (GFAP), but vimentin is by far the major intermediate filament protein. Thus, the intermediate filament composition of these astrocytes is similar to that of astrocytes in primary cultures obtained from neonatal rat brain. Immunofluorescent studies of these cultures at 24 hr in vitro show that there are no GFAP+ cells in cultures of either species; the bovine cultures contain greater than 95% GC+ cells; and the rat cultures contain 90% GC+ cells. After a few days in vitro flat cells appear that are vimentin+/GFAP-/GC-. In serum-free medium these cells eventually become vimentin+/GFAP+. We propose that the astrocytes that grow in these cultures arise from a population of glial precursor cells, which are present even in adult brain and are isolated together with oligodendroglia, and that they do not derive from contaminating mature astrocytes. Thus, the astrocytes in our cultures may have the same origin as astrocytes grown in culture from dissociated neonatal brain.     

Molecular forms and solubility of acetylcholinesterase during the embryonic development of rat and human brain

Acetylcholinesterase (EC 3.1.1.7) and butyrylcholinesterase (EC 3.1.1.8) form homologous sets of multiple molecular forms. The central nervous system of mammals contains mostly tetramers (G4) and monomers (G1). Their proportions have been shown to vary during maturation in rat brain. In order to examine whether a similar evolution occurs in the human, we performed parallel studies of the activity, solubility and molecular forms of acetylcholinesterase in rat and human brains at various stages. We find both similarities and differences: in rat brain, the enzyme increases mostly postnatally but in human brain acetylcholinesterase reaches a maximum at birth. There is an increase in the proportion of G4 and a decrease in the solubility of this from in the absence of detergent in human as well as in rat brain. These changes occur around birth in rat, but during early pregnancy, before 11 weeks in human brain. In both species, the solubility of the enzyme in detergent-free buffers decreases progressively from more than 50% before birth to about 10-20% in the adult. In addition we analyzed butyrylcholinesterase as well as the levels of the neuron-specific enolase and of the glial S-100 protein. In human, gamma gamma-enolase rises to its adult level after birth, but before the S-100 protein.     

Maternal embryonic leucine zipper kinase is a key regulator of the proliferation of malignant brain tumors, including brain tumor stem cells

Emerging evidence suggests that neural stem cells and brain tumors regulate their proliferation via similar pathways. In a previous study, we demonstrated that maternal embryonic leucine zipper kinase (Melk) is highly expressed in murine neural stem cells and regulates their proliferation. Here we describe how MELK expression is correlated with pathologic grade of brain tumors, and its expression levels are significantly correlated with shorter survival, particularly in younger glioblastoma patients. In normal human astrocytes, MELK is only faintly expressed, and MELK knockdown does not significantly influence their growth, whereas Ras and Akt overexpressing astrocytes have up-regulated MELK expression, and the effect of MELK knockdown is more prominent in these transformed astrocytes. In primary cultures from human glioblastoma and medulloblastoma, MELK knockdown by siRNA results in inhibition of the proliferation and survival of these tumors. Furthermore, we show that MELK siRNA dramatically inhibits proliferation and, to some extent, survival of stem cells isolated from glioblastoma in vitro. These results demonstrate a critical role for MELK in the proliferation of brain tumors, including their stem cells, and suggest that MELK may be a compelling molecular target for treatment of high-grade brain tumors.     

The selective viability of human foetal brain cells

The influence of various factors upon the survival of human foetal neurones has been examined The viability of several brain structures was assessed using ethidium bromide and acridine orange fluorescence in both ‘intact’ and mechanically dispersed tissue. Striatum was least vulnerable to dissociation while cortex, mesencephalon, pons, cerebellum and cord were more vulnerable to a greater or lesser extent. Material can be preserved in vitro with greater viability in the undissociated rather than dissociated state. The effects of other factors including foetal age upon viability are discussed.     

Accelerated differentiation of oligodendrocytes in neuronal-rich embryonic mouse brain cell cultures

The expression of two oligodendroglial markers, galactocerebroside (GC) and myelin basic protein (MBP), was studied in brain cell cultures (BCC) from 14-day-old mouse embryos by immunocytochemical methods. The presence of neurons and astrocytes was also investigated. Results show that oligodendrocytes simultaneously express both GC and MBP already at 7 days in vitro. These cultures are rich in neurons, and the astrocyte layer is also well represented. A comparison is made between these data and those previously obtained by the use of newborn mouse brain cell cultures, which are very poor in neurons. The differentiation of oligodendrocytes, as reflected in the expression of MBP, is accelerated in embryonic mouse BCC when compared to neonatal mouse BCC. We therefore speculate that neurons are involved in the enhancement of the ability of oligodendrocytes to express myelin related components in culture.