培养脊髓神经元中微管相关蛋白5的表达

目的　探讨初代培养脊髓神经元中微管相关蛋白 (MAP5 )的表达特征及其调节因素。方法　应用荧光免疫细胞化学检测初代培养脊髓神经元中MAP5的表达。结果　MAP5在培养脊髓神经元中呈网状分布在胞浆及突起中。诺考达唑引起微管解聚及MAP5的结构破坏 ,荧光物质分布不均匀 ,突起呈串珠或断裂。PMA(phorbol 12 myristate 13 acetate)能诱发微管及MAP5聚合 ,胞浆中荧光物质呈疏松的网状结构。结论　MAP5在初代培养脊髓神经元胞浆及突起中明显表达 ,且表达受微管解聚剂诺考达唑及微管聚合剂PMA的调控。     

电镜观察培养脊髓神经元溶酶体的形态及影响因素

目的　探讨初代培养脊髓神经元中线状溶酶体 (NLY)与圆形溶酶体 (SLY)的超微结构及其影响因素。方法　应用电镜酸性磷酸酶 (ACPase)细胞化学方法和透射电镜观察初代培养脊髓神经元中NLY与SLY的超微结构。结果　培养脊髓神经元中存在ACPase阳性的NLY与SLY。诺考达唑处理后NLY明显减少 ,SLY明显增多 ,主要分布在核周。佛波醇酯 (PMA)处理后可见NLY明显增多 ,靠近质膜处比靠近核膜处多 ,在突起中NLY沿着长轴分布 ,SLY明显减少。结论　初代培养脊髓神经元中溶酶体的形态受微管解聚剂诺考达唑及微管聚合剂PMA的调控     

免疫电镜观察培养脊髓神经元中微管相关蛋白-5的表达

目的　研究微管相关蛋白 5在培养脊髓神经元的表达变化。方法　使用ACLAR膜培养脊髓神经元 ,分为四组 ,即正常脊髓神经元组 ,加诺考达唑 (nocodazole)组 ,加乙酸佛波醇 (PMA)组 ,空白对照组。免疫电镜观察四组微管相关蛋白 5的分布。结果　正常脊髓神经元微管相关蛋白 5阳性颗粒在胞浆及突起中均匀分布 ,nocodazole引起微管相关蛋白 5阳性颗粒排列紊乱 ,PMA则使微管相关蛋白 5阳性颗粒在胞浆及突起中密集存在。结论　微管相关蛋白 5在脊髓神经元的胞浆及突起中均有表达 ,并受nocodazole及PMA的调节     

Epothilone D对体外培养的BTBR小鼠皮质神经元 兴奋性突触结构的影响*

目的： 探讨微管稳定剂埃博霉素D（Epo D）对孤独症谱系障碍BTBR小鼠皮质神经元兴奋性突触结构的影 响及机制。方法： 体外培养BTBR小鼠原代大脑皮质神经元,免疫荧光法检测所培养神经元的纯度;培养至相对 成熟的BTBR小鼠皮质神经元随机分为实验组（Epo D 组）、溶剂对照组（0.1% DMSO）干预24 h,再冷处理90 min 后行微管免疫荧光染色,观察微管的形态变化;免疫印迹检测微管稳定的标志蛋白（acetyl-tubulin、α-tubulin）、 微管相关蛋白（MAP2、STOP）的表达,及兴奋性突触结构前、后膜标志蛋白（VGLUT1、PSD95）、兴奋性谷氨 酸受体相关蛋白（GluN2B、mGluR5）的表达水平。结果：与对照组相比,10 nmol/L 的Epo D 可增加BTBR小鼠 皮质神经元微管的冷稳定性及微管稳定的标志蛋白、微管相关蛋白的表达;可以增加BTBR小鼠皮质神经元兴奋 性突触结构、兴奋性谷氨酸受体相关蛋白的表达,差异均有统计学意义。结论：微管稳定剂Epo D能改善体外培 养的BTBR小鼠皮质神经元兴奋性突触结构,其机制可能与增加微管稳定性有关。     

体外切割损伤脊髓神经元对其Fos、ChAT及GAD表达的影响

目的:研究切割损伤对体外培养脊髓神经元Fos、胆碱乙酰转移酶(ChAT)及谷氨酸脱羧酶(GAD)表达的影响。方法:脊髓神经元纯化培养后分别计数碘化丙啶(PI)阳性细胞数和微管相关蛋白2(MAP2)阳性细胞数,决定脊髓神经元纯度。根据本实验室已建立的损伤模型,对原代纯化培养脊髓神经元进行切割损伤。通过免疫组化及WesternBlot方法比较神经元损伤前后Fos、ChAT和GAD表达的变化情况。结果:脊髓神经元纯化培养后纯度达到92%。免疫组化及WesternBlot显示,切割损伤组与正常组相比,Fos、GAD表达上调,而ChAT表达降低。结论:Fos、GAD和ChAT可能参与脊髓神经元急性损伤及随后的修复过程,其生物学意义有待进一步探讨。     

超极化激活环核苷酸门控阳离子通道亚型在大鼠脊髓背角浅层的特异性表达

目的:探讨超极化激活环核苷酸门控阳离子通道(hyperpolarization-activated cyclic nucleotide-gated cation channels,HCN通道)4种亚型在大鼠脊髓背角浅层的表达与分布特点。方法:选取3~5周龄SD大鼠,雌雄不拘,制作L4~L5段脊髓横切片,应用免疫组织化学技术及激光共聚焦成像技术,观察HCN通道的4种亚型在脊髓背角浅层神经元、胶质细胞及神经元亚细胞结构中的分布。结果:HCN通道不同亚型在正常SD大鼠脊髓背角中的表达和分布具有特异性:(1)HCN1主要与神经元标志物[神经元核抗原(neuronal nuclei,NeuN)]和星形胶质细胞标志物[胶质细胞原纤维酸性蛋白(glial fibrillary acidic protein,GFAP)]共存,HCN2和HCN3主要与NeuN共定位;(2)HCN2主要与肽能初级传入神经末梢标志物[降钙素基因相关肽(calcitonin gene-related peptide,CGRP)]共存,HCN4主要与非肽能初级传入神经末梢标志物[异凝集素B4(isolectin B4,IB4)]共存;(3)HCN1和HCN4主要与神经元树突标志物[微管相关蛋白2(microtubule-associated protein 2,MAP2)]共存;(4)HCN4主要与抑制性中间神经元轴突末梢标志物[囊泡γ-氨基丁酸转运体(vesicularγ-aminobutyric acid transporter,VGAT)]共存。结论:HCN1~4主要分布在大鼠脊髓背角浅层,且在神经元、胶质细胞及神经元亚细胞结构中呈特异性分布。     

豚鼠脊髓腹角神经元线状溶酶体酶细胞化学研究及其电子探针X射线能谱分析

为探讨豚鼠脊髓腹角神经元是否存在线状溶酶体及其酶细胞化学活性分布特点，用偏磷酸酶（Ｍｅｔａｐｈｏｓｐｈａｔａｓｅ，ＭＰａｓｅ）和酸性磷酸酶（Ａｃｉｄｐｈｏｓｐｈａｔａｓｅ，ＡＣＰａｓｅ）电镜细胞化学方法和电子探针Ｘ射线能谱分析技术，证实豚鼠脊髓腹角神经元存在线状溶酶体（Ｎｅｍａｔｏｌｙｓｏｓｏｍｅ，ＮＬＹ），同时于原位测定ＮＬＹ内的铅含量以反映酶活性强弱。ＭＰａｓｅ和ＡＣＰａｓｅ反应产物分布于圆形溶酶体和ＮＬＹ，同时在高尔基复合体的部分扁囊也有酶活性，表明该酶是在高尔基复合体上加工后输送至溶酶体。在神经元胞体、突起及突触前成分中均有呈ＭＰａｓｅ阳性和ＡＣＰａｓｅ阳性的ＮＬＹ的分布，并和线粒体紧密相贴，提示酶是由线粒体提供能量的ＮＬＹ从胞体输送到神经终末，可能参与神经递质的降解及神经元代谢物质的处理。电子探针Ｘ射线能谱分析测定结果，ＭＰａｓｅ活性强于ＡＣＰａｓｅ活性，提示ＭＰａｓｅ是比ＡＣ－Ｐａｓｅ更为敏感的溶酶体标志酶     

GDNFR-〆在体外培养的大鼠脊髓和背根节神经元中分布的免疫组织化学研究

目的　观察胶质细胞源性神经营养因子受体 - α(GDNFR- α)在体外培养的大鼠脊髓和背根节神经元中的分布 ,探讨 GDNF对脊髓运动神经元和感觉神经元的作用。　方法　原代培养脊髓和背根节神经元 ,5 d后行抗 GDNFR- α多克隆抗体免疫组织化学 SP法染色。　结果　 GDNF免疫反应存在于体外培养的脊髓神经元、背根节神经元以及胶质细胞中。　结论　 GDNF可能对脊髓神经元、背根节神经元和胶质细胞的生理功能具有一定的调节作用     

GDNFR-α在体外培养的大鼠脊髓和背根节神经元中分布的免疫组织化学研究

目的观察胶质细胞源性神经营养因子受体-α(GDNFR-α)在体外培养的大鼠脊髓和背根节神经元中的分布,探讨GDNF对脊髓运动神经元和感觉神经元的作用. 方法原代培养脊髓和背根节神经元,5d后行抗GDNFR-α多克隆抗体免疫组织化学SP法染色. 结果 GDNF免疫反应存在于体外培养的脊髓神经元、背根节神经元以及胶质细胞中. 结论 GDNF可能对脊髓神经元、背根节神经元和胶质细胞的生理功能具有一定的调节作用.     

微管相关蛋白2与脊髓发育和损伤的修复

背景：微管相关蛋白2作为神经元特异性标记蛋白之一,其表达活性在一定程度上反映了脊髓神经细胞增殖、分化、迁移的演变规律。 目的：综述微管相关蛋白2的分子结构、生物学功能及其在脊髓发育及损伤修复中的表达分布规律,进而为脊髓损伤后神经干细胞的移植治疗提供前期研究基础。 方法：应用计算机检索1984/2012 维普、万方数据库相关文章,检索词为“脊髓,微管相关蛋白2”。同时计算机检索1984/2012 PubMed和Springer外文电子期刊及电子图书(全文)数据库相关文章,检索词为“spinal cord,Microtubule-associated protein 2(MAP-2)”。共检索到文献325篇,最终纳入符合标准的文献27篇。 结果与结论：微管相关蛋白2不仅是神经细胞的骨架成分,更是一种活跃的功能蛋白,参与神经元的生长和损伤后的修复过程,它的表达强弱可以反映脊髓神经元的发育情况。微管相关蛋白2免疫活性下降在脊髓损伤中的作用日益受到重视,而它的高表达可能在脊髓损伤后突触结构重建及受损神经功能代偿和修复中发挥重要作用。通过检测微管相关蛋白2的含量和定位,可以用来研究人类多种神经干细胞增殖、分化、迁移规律,进而探索脊髓等中枢神经系统的发育和损伤修复状况。     

MAP5 in cultured hippocampal neurons: Expression diminishes with time and growth cones are not immunostained

Summary A monoclonal antibody was used to determine both the expression of the microtubule-associated protein MAP5 in cultured foetal rat hippocampal neurons as a function of culture age and the cellular distribution of the protein. When cultures at days 2 and 3 were examined by fluorescence microscopy, MAP5 immunostaining was localized intensely in neuronal cell bodies and neurites but not in growth cones. Extensive labelling of axons was seen at days 4 and 5. MAP5 staining was still prominent in neurons after 16 days in culture, and neuntes at this time had grown over astrocytes but had completely avoided islands of non-astrocytic cells. MAP5 immunostaining was almost undetectable in cells that had been in culture for 20 days. The decreasing expression of MAP5 in cultured neurons as a function of time parallels that previously shown for MAP5 in intact neonatal rat brain. The effect of elevated temperature on MAP5 expression was also examined. Neurons grown for 9 days at 40 ° C showed the same cellular distribution of MAP5 as cells grown at 37 ° C. In particular, growth cones were again negative for MAP5 immunostaining. The absence of MAP5 in growth cones appears consistent with the fact that these structures contain labile microtubules. MAP5 has been shown to be a component of microtubule crossbridges and its absence might thus be expected to contribute to microtubule lability.     

BDNF及其受体TrkB在成年恒河猴脊髓的表达

目的观察脑源性神经营养因子BDNF及其高亲合力受体TrkB在成年恒河猴胸髓的表达分布,为成年恒河猴脊髓BDNF、TrkB的功能研究提供形态学依据。方法灌注固定成年雄性恒河猴,取其胸段脊髓制作冰冻切片,用BDNF、TrkB特异性抗体行免疫组织化学SP法染色,观察BDNF、TrkB免疫阳性物质在胸段脊髓的分布。结果成年恒河猴胸髓内均可见BDNF、TrkB免疫反应阳性神经元,反应产物呈棕黄色颗粒。BDNF和TrkB免疫反应阳性细胞遍及胸髓灰质各板层,在腹角、侧角以及中央管邻近区域均可见阳性神经元,它们的阳性产物主要定位于神经元胞浆中,胞浆着色深,胞浆与胞核的界线清楚。BDNF免疫反应细胞的胞核未见染色,而不同的TrkB阳性细胞其胞核染色不一。结论成年恒河猴胸髓存在BDNF、TrkB的表达,其作用可能与猴脊髓的正常生理功能和损伤后的修复有关。     

Pramipexole has astrocyte-mediated neuroprotective effects against lactacystin toxicity

Pramipexole, a dopamine D2/D3 receptor agonist used in the treatment of Parkinson's disease, has been reported to have neuroprotective potential. We investigated the effect of pramipexole against cell death induced by a proteasome inhibitor, lactacystin, using primary mecencephalic neuronal cultures and SH-SY5Y cells. In E14 rat primary mesencephalic cultures, the number of surviving tyrosine hydroxylase (TH)-positive neurons and microtubule associated protein 2 (MAP2)-positive neurons was decreased by exposure to 1-5 microM lactacystin in a dose-dependent manner. Pretreatment with 100 microM pramipexole rescued TH-positive neurons and MAP2-positive neurons from the toxicity of lactacystin. The protective effect of pramipexole was not selective for TH-positive dopaminergic neurons. However, the treatment with 100 microM pramipexole did not protect SH-SY5Y cells against lactacystin-induced cell toxicity and proteasome dysfunction. We hypothesized that the protective effect of pramipexole against the lactacystin-toxicity was not direct but a secondary effect mediated by astrocytes. Therefore, we investigated the efficacy of conditioned medium collected from mecencephalic astrocytes treated with pramipexole. The conditioned medium increased the viability of SH-SY5Y cells against the toxicity of lactacystin. Pramipexole increased the levels of brain derived neurotrophic factor (BDNF) in the conditioned medium of astrocyte cultures. These protective effects were not significantly inhibited by dopamine D2 or D3 receptor antagonists. We demonstrated that pramipexole had the protective effect against lactacystin toxicity, mediated by a neurotrophic effect of astrocyte-produced factors including BDNF.     

Neurons containing retrogradely transported Fluoro-Gold exhibit a variety of lysosomal profiles: A combined brightfield, fluorescence, and electron microscopic study

Summary The advantages of axonally transported Fluoro-Gold as a retrograde fluorescent marker are numerous. The objective of the present study was to determine whether transported Fluoro-Gold is visible in either semi-thin sections for light microscopy or thin sections for electron microscopy. Rats received injections of Fluoro-Gold into either the striatum or thoracic spinal cord. After appropriate survival times, labelled neurons were observed with the fluorescence microscope in brain regions that are known to project to the injected areas.Sections that contained labelled cells were embedded in plastic and examined with a fluorescence microscope. Semi-thin sections of unosmicated tissue displayed high-resolution fluorescent labelling of somata and dendrites. In contrast, osmicated tissue did not fluoresce, but numerous dark granules were observed in the dendritic and perikaryal cytoplasm of labelled neurons in toluidine blue stained sections that were examined with brightfield optics. The unosmicated tissue did not display these granules, and this finding suggested that the granules are composed of membranes. Neurons in other brain regions that are known not to project to the injection sites did not contain these dark granules.Adjacent thin sections examined with the electron microscope displayed numerous electron-dense, lysosome-like organelles in the cytoplasm of labelled neurons. The electron density of these organelles was greater than that of lysosomes in unlabelled neurons. Three types of distinctive organelles were observed in these preparations: (1) relatively dense concentric lamellar bodies of various sizes; (2) heterogeneous or lipofuscin-like lysosomes; and (3) coarse grained lysosomes. Control sections and unlabelled neurons did not display these organelles. Therefore, these organelles appear to correlate with Fluoro-Gold localized within the somata and dendrites of retrogradely labelled neurons. It is not known if they are the Fluoro-Gold itself, or represent a physiological effect on membranes. The results of this study indicate that Fluoro-Gold may be useful for tract tracing at the electron microscopic level.     

Microtubule-associated protein 2 and tubulin are differently distributed in the dendrites of developing neurons

We have followed the appearance of two microtubule proteins, tubulin and microtubule-associated protein 2, in rat hippocampal neurons differentiating in cell culture. Double-label immunofluorescence staining showed that from day 1 in vitro onward tubulin appeared as filaments but that microtubule-associated protein 2 remained distributed throughout the cytoplasm. This difference persisted throughout development and was also detectable in cells that had reached morphological maturity. When cells were treated with the microtubule-depolymerizing agent nocodazole, the depolymerized tubulin became spread throughout the cytoplasm so that its distribution was then identical to microtubule associated protein 2. At the same time, multiple side branches began to emerge along the dendrites. When cells which had been exposed to nocodazole were allowed to recover before staining, the tubulin was again present as filaments but the microtubule-associated protein 2 remained distributed throughout the dendritic cytoplasm. Under these conditions the previously extended proximal side branches were resorbed into the main process. These results suggest that cellular microtubule-associated protein 2 is not necessarily exclusively associated with microtubules. Neuronal dendrites in particular appear to contain this protein at levels in excess of the capacity of microtubular microtubule-associated protein 2 binding sites. In view of the known effectiveness of microtubule-associated protein 2 as a promoter of tubulin polymerization, its abundance in dendrites suggests that it acts to ensure total polymerization of dendritic microtubules. In this way it would contribute both to the support of the growing process and the suppression of adventitious sidebranching.     

信号转导子与转录激活子3和细胞因子信号转导抑制因子-3蛋白在成年大鼠脊髓内的表达与定位

目的分析JAK-STAT信号转导途径重要成员STAT3蛋白和该途径抑制蛋白SOCS-3在大鼠脊髓内的表达和细胞内定位分布。方法免疫细胞化学技术和形态计量学方法。结果STAT3免疫阳性产物主要分布于脊髓前角运动神经元的胞浆内；SOCS-3免疫阳性产物广泛分布于脊髓前角及后角神经元内、部分胶质细胞及神经纤维内。在前角运动神经元内，SOCS-3免疫阳性产物主要分布于核内，有时也可分布于胞浆中。结论STAT3和SOCS-3广泛表达于正常大鼠脊髓内，其中SOCS-3具有核蛋白或胞浆蛋白两种存在形式。