Laminin促进胚胎中脑多巴胺神经元生长发育的体外培养研究

在体外培养中研究了Laminin(LN)对大鼠胚胎中脑多巴胺神经元生长发育的影响。将胚胎(E15天)腹侧中脑细胞悬液接种于底部涂有LN的24孔培养板中,培养2—6天后取出。用酪氨酸羟化酶免疫组化ABC法观察多巴胺神经元的生长情况。培养48小时后见酪氨酸羟化酶免疫阳性细胞均有突起伸出,以双极细胞为主;有些突起分枝末端有活跃的生长锥,后者又司发出多根丝状伪足。在培养3到6天的标本中除见有单极和双极酪氨酸羟化酶免疫阳性细胞外,每孔均见有多极阳性细胞。这些阳性细胞的突起具有较丰富的分枝,突起长度多数长于胞体直径的6倍,最长的可达400μm以上。每孔酪氨酸羟化酶免疫阳性细胞数可达20到50个,并能见到群聚存在,与不含LN的对照组相比,在酪氨酸羟化酶免疫阳性突起的数量、长度及分枝等方面均有显著差异。实验表明LN可促进体外培养胚胎中脑神经元的生长发育及轴突延伸。     

Laminin促进胚胎中脑多巴胺神经元生长发育的体外培养研究

本文在体外培养中,研究Laminin(LN)对大鼠胚胎中脑多巴胺(DA)神经元生长发育的影响。将胚胎鼠(胎龄15天)腹侧中脑细胞悬液,接种于底部涂有LN的24孔培养板中,培养2～6天后取出,以酪氨酸羟化酶(TH)免疫组织化学ABC法观察DA神经元的生长情况。培养48小时后,几乎所有TH免疫阳性细胞都有突起伸出,以双极细胞为主,有些突起分支末端有活跃生长的生长锥,后者又可发出多根丝状伪足。在培养3～6天的标本中,除见有单极、双极TH免疫阳性细胞外,每孔均见有     

纹状体和中缝核外植块促进体外培养的中脑多巴胺神经元的发育

为了探讨神经元与其靶组织之间的相互作用及作用因素，建立了不同时期的纹状体外植场与１４ｄ大鼠胚胎中脑黑质神经元联合培养；１４ｄ大鼠胚胎中缝核外植块与黑质神经元联合培养。结果表明，生后２周、新生及１４ｄ大鼠胚胎纹状体外植块和１４ｄ大鼠胚胎中缝核外植块，对中脑黑质多巴胺神经元的形态发育有明显促进作用，其中包括长突起神经元数目和神经元突起长度的增加。上述３个时期的纹状体外植块，对中脑黑质多巴胺神经元的作用     

Drebrin在神经元发育过程中的分布变化

目的:探讨肌动蛋白结合蛋白Drebrin在神经元发育过程中的分布情况。方法:采用免疫细胞化学方法检测原代培养大脑皮层神经元内Drebrin的分布。结果:体外培养1天(DIV,day in vitro)时,Drebrin主要分布在神经元胞体;3和7 DIV时,Drebrin分布在神经元胞体、突起末端和分支处;14和21 DIV时,Drebrin主要呈斑点状分布在神经元突起。结论:随着原代培养神经元和树突棘的发育成熟,Drebrin分布从神经元胞体向突起内转移,并逐渐在突起上密集呈斑点状分布。提示在神经元发育过程中,Drebrin表达可能和神经元突起形成及树突棘发育成熟密切相关。     

MPP~+对原代培养SAMP8小鼠中脑神经元的毒性作用

目的:建立MPP+(1-甲基-4-苯基吡啶离子)干预SAMP8(快速老化小鼠P8)小鼠中脑神经元的体外细胞模型。方法:SAMP8新生1 d小鼠的中脑神经元原代混合培养6 d,加入100μmol/L浓度的MPP+,再培养6、9、12 h和24 h后分别对各时间点的中脑原代培养神经元免疫荧光染色或提取蛋白测定TH水平。结果:MPP+导致原代培养的SAMP8小鼠中脑神经元形态学改变。正常对照组神经元形态完整,免疫反应性强,胞体大呈椭圆形,突起多而长且粗壮;MPP+组神经元随时间逐渐出现形态变化,MPP+后6 h即可见突起不完整断续,9 h突起数目减少,缩短;12 h神经元胞体明显变小,24 h突起多消失,神经元胞体小且免疫反应性弱。MPP+导致原代培养的SAMP8小鼠中脑神经元数量在MPP+后9 h开始有显著减少,同时TH蛋白的表达也开始有显著减少,24 h最低。结论:MPP+对原代培养的SAMP8小鼠中脑神经元具有毒性作用。MPP+导致SAMP8小鼠中脑神经元原代混合培养体系的神经元数量下降,同时TH蛋白表达降低,提示MPP+导致其中脑DA能神经元损伤死亡。     

纹状体对体外培养中脑多巴胺神经元形态发育的影响

靶细胞在神经细胞的生长发育过程中起着重要的调节作用。本文通过胚胎腹侧中脑和纹状体联合培养研究纹状体对中脑多巴胺(DA)神经元形态发育的影响。纹状体(Str)和腹侧中脑(YMA)细胞悬液取自胚胎14天SD大鼠,联合培养3到14天后取出,用酪氨酸羟化酶(TH)免疫组化ABC法观察不同时期DA神经元的生长。与VMA单独培养相比,联合培养3天,发现细胞聚集程度较低,开始形成单层分布,培养7天TH阳性细胞数增加,多突起阳性细胞较易发现,细胞突起粗短,呈树枝状分枝。培养14天,TH阳性细胞数进一步增加,平均可达49个/25mm~2。本文对纹状体调节DA神经元生长发育的机制进行了讨论。     

20 人胎海马含P物质神经元的发育

<正>本文用ABC法对胎龄15周至新生儿人脑海马吉SP神经元的发育进行研究.15周胎儿其海马相当于CAI区浅层已出现含SP神经元,随后CA2区、CA3区和CA4区始层相继出现含SP神经元.27周后锥体层出现少数含SP神经元.其发育模式似从CAI区依次向CA2区、CA3区和CA4区发育.含SP神经元在人胚出生前主要分布在始层,生前与生后的分布模式有较大的差异.用广州医学院神经科学研究所研制的“计算机自动识别分析神经元系统”对含SP神经元胞体的截面积进行测量和分析.海马含SP神经元的胞体平均截面积:15周为106.7±41.2μm~2,27周为162.9±41.5μm~2;30周为110.8±53.0μm~2,呈负增长.新生儿含SP神经元胞体截面积为129.9±76.3μm~2,比15周略有增大.15周海马含SP神经元为圆形、椭圆形,胞浆成份比例小,胞核多不着色,呈空泡状,未见明显突起.19周含SP神经元以双极神经元为主,27周含SP神经元,双极和多极神经元相间,突起增长,30周和34周含SP神经元胞浆成份比例大,核着色,突起明显增长,突起数目及分支增多,多极神经元比例较前增高.始层含SP神经元纤维走向多与室床平衡.     

NGF和GDNF基因重组逆转录病毒体外感染神经干细胞及其鉴定

目的 探讨神经干细胞（NSC）直接作为基因靶细胞能否被重组逆转录病毒感染以及感染后目的基因的表达。方法 分别用NGF和GDNF基因重组逆转录病毒上清液感染NSC2d;G418筛选后加入bFGF扩增培养;取感染后的NSC培养液（NGF／GDNF条件培养液）培养PC12细胞和中脑腹侧神经元;用免疫组织化学染色方法检测中脑腹侧多巴胺神经元的形态改变以及感染后NSC对目的基因的表达。结果 经G418筛选后,约50％感染NGF和GDNF基因重组逆转录病毒的NSC呈G418抗性;这些感染后的NSC开始分化,分裂球向四周长出放射状突起,部分细胞沿突起向外迁移生长;感染NGF基因的NSC呈星形,胞体较大,突起较粗,感染GDNF基因的NSC呈梭形,胞体较小,突起较长。经NGF条件培养液培养的PC12细胞,数量增多,突起明显变长。经GDNF条件培养液培养的中脑多巴胺神经元（TH阳性细胞）其胞体亦增大,突起伸长。大部分G418抗性的NSC出现NGF和GDNF免疫染色。结论 神经干细胞可直接作为基因靶细胞,能被NGF和GDNF基因重组逆转录病毒有效感染而表达和分泌有生物学活性的NGF和GDNF。     

人胎视皮质17区血管活性肠肽样神经元的形态、分布及个体发育

用免疫细胞化学技术,研究了人胎(19—38周,共26例)视皮质17区含血管活性肠肽(VIP)神经元的形态、分布及发育。结果证明该区含VIP神经元在受精龄第21周开始出现微弱免疫反应,阳性神经元在皮质Ⅰ—Ⅵ层均可见,但以深层Ⅴ—Ⅵ层明显,以后在Ⅱ—Ⅳ层明显增多。而且主要分布在Ⅱ—Ⅵ层。含VIP神经元有双极和多极两种,大部分是双极神经元。阳性神经元刚出现时突起短,随胎龄增长突起延长并分支。第Ⅵ层阳性神经元刚出现时胞体小,随胎龄增长(从24—38周),胞体体积明显增大,而核的比例变小。除第Ⅳ层阳性双极神经元在单位面积内的数目从36—38周有所下降外,该层阳性多极神经元数目及其他层(Ⅱ、Ⅲ、Ⅴ、Ⅵ层)阳性神经元数从24周到38周变化不明显。     

大鼠胚胎下丘脑神经元体外培养的生长特点

用神经细胞体外培养技术,在光、电镜下进行定量组织化学、免疫组化方法观察17—18天大鼠胚胎下丘脑神经元体外生长发育过程。结果显示:细胞培养3天时,神经元以2个突起为主,占81％;1周时,神经元以3个突起为主,占51％;2周时神经元以3～4个突起为主,分别占37％和31％。定量组化结果显示神经元SDH、AChE含量随培养时间延长而增多,在第3天分别为64.2和3.8,1周时分别为98.2和12.8,2周时分别为127.6和24.6。免疫组化结果显示培养神经元在第3天时,神经元特异烯醇酶(NSE)呈阳性反应。上述结果提示,体外培养胚胎大鼠下丘脑神经元在第3天基本发育成熟。本文还对体外培养神经元的特点进行了详细的描述。     

单唾液酸神经节苷脂对MPTP损害中脑多巴胺神经元的保护作用

本文研究单唾液酸神经节苷脂（ＧＭ１）对大鼠中脑多巴胺（ＤＡ）神经元体外生存及其损伤后的保护作用。取胚胎腹侧中脑制成细胞悬液，常规体外培养。实验分为四组：第一组在培养液中加入ＧＭ１（ＧＭ１组）；第二组在培养过程中使用１－甲基－４－苯基－１，２，３，６－四氢吡啶（ＭＰＴＰ）破坏ＤＡ神经元（ＭＰＴＰ组）；第三组在使用ＭＰＴＰ破坏ＤＡ神经元的同时给予ＧＭ１（ＧＭ１－ＭＰＴＰ组）；第四组为正常对照组。四组培养细胞定期抽出作免疫组化和荧光组化染色。ＧＭ１组培养各阶段，酪氨酸羟化酶（ＴＨ）免疫反应阳性细胞数量均明显多于对照组，至体外培养２周时，每孔阳性细胞数平均可达７２．８个，与对照组相比有显著性差异。ＭＰＴＰ组在加入ＭＰＴＰ１周左右，荧光组化阳性细胞已基本消失，此后ＴＨ免疫反应阳性细胞逐渐减少，至体外培养２周，每孔ＴＨ阳性细胞数平均仅剩１４．５个，与对照组相比有显著性差异。在残存的ＤＡ神经元中，突起变短或消失。ＧＭ１－ＭＰＴＰ组，体外培养期间均可看到荧光组化阳性细胞，ＴＨ免疫反应阳性细胞数量在体外培养２周时平均每孔为３１．７个，明显高于ＭＰＴＰ组。研究结果表明ＧＭ１能提高体外生长的ＤＡ神经元的生存率，并能减轻ＭＰＴＰ对?     

Long-term development of mesencephalic dopaminergic neurons of mouse embryos in dissociated primary cultures: morphological and histochemical characteristics

In 13 and 15 day-old mouse embryos mesencephalic dopaminergic neurons could already be visualized at the level of the mesencephalic flexure by tyrosine hydroxylase immunocytochemistry at day 13. At this time, noradrenergic cells in the locus coeruleus area were not detectable. In most in vitro experiments, dissociated mesencephalic cells of 13 day-old embryos were grown in presence of serum. Four approaches were used to identify the dopaminergic neurons in vitro: fluorescence histochemistry of newly taken up exogenous norepinephrine, radioautography after labelling with (3H) dopamine, tyrosine hydroxylase-like immunoreactivity and fluorescence histochemistry of endogenous stores of catecholamines. Control experiments performed at various times in vitro with selective inhibitors of amine transport into dopaminergic, noradrenergic and serotoninergic neurons indicated that only dopaminergic neurons were detected by these various approaches, noradrenergic neurons being virtually absent from the cultures. The uptake of exogenous norepinephrine was detected already 24 h after plating and preceded the appearance of tyrosine hydroxylase-like immunoreactivity (48 h). The number of neurons revealed by these two techniques increased up to 4 and 10 days, respectively. Endogenous stores of dopamine were only seen after three weeks in vitro by fluorescence histochemistry. At this time, the same number of neurons was revealed whatever the method used. The presence of striatal target cells (co-cultures) affected neither the sequential appearance of the markers nor the number of dopaminergic cells. The two main types of dopaminergic neurons (fusiform and multipolar) described in vivo both in the substantia nigra (A9) and the ventral tegmental area (A10) of adult animals were identified in vitro and their development into well-differentiated neurons can be followed for up to six weeks. This in vitro system seems, therefore, to be particularly suitable for biochemical and electrophysiological studies of these dopaminergic neurons.     

成年大鼠嗅球成鞘细胞的原代培养

本实验取材于成年 Wistar大鼠嗅球的最外两层 ,进行原代培养嗅球成鞘细胞 ,用胶质纤维酸性蛋白抗体免疫组化方法确认。结果显示 :( 1)培养至第 10 d,原代培养物多见两种形态的细胞 :一种为单极、双极或多极细胞 ,带有细长的突起 ;另一种为所谓“煎蛋样”细胞 ,扁平状 ,胞质少极化 ,边缘不规则 ;( 2 )胶质纤维酸性蛋白反应显示这两种细胞均呈阳性 ,Nissl法复染后 ,可计数其纯度为 70 %～ 85 %。     

Neuronal cell types in the anterior ventral thalamic nucleus of the camel

The anterior ventral nucleus neurons in of the camel brain were morphologically studied by Golgi impregnation method. Two neuronal types of were found in the camel anterior ventral thalamic nucleus, namely, Golgi-type I neurons and Golgi-type II neurons. Those neurons were generally similar to their counterparts in the human thalamus. The Golgi-type I neurons exhibited medium to large cell body (mean diameter = 25 μm) which was either multipolar or triangular in shape. They had from 3 to 10 primary dendrites with many branches but with no spines or appendages. The Golgi-type II neurons had small to medium size (somatic mean diameter = 17.5 μm), their cell bodies were variable in shape, some were round, and others were multipolar or fusiform. These cells bodies had two to six primary dendrites with few branches that may have spines and/or grape-like appendages. Our findings shed some light on the anterior ventral thalamic nucleus structure of the camel as one of the strongest adaptive mammals to the hard climatic conditions.     

体外诱导人脐带间充质干细胞分化为许旺细胞

背景：人脐带Wharton’s Jelly源间充质干细胞避免了伦理的限制,来源丰富,可以作为种子细胞进行组织修复。 目的：观察体外诱导脐带Wharton’s Jelly中间充质干细胞向许旺细胞分化的可行性。  方法：分离、培养脐带Wharton’s Jelly中间充质干细胞,流式细胞术鉴定细胞表面标志。利用神经细胞培养基、碱性成纤维生长因子、表皮生长因子、维甲酸、血小板源性生长因子等采用两步法将脐带间充质干细胞诱导分化为许旺细胞,倒置显微镜下观察细胞形态变化。利用免疫细胞化学染色法检测巢蛋白、S-100、纤维酸性蛋白的表达,反转录-聚合酶链反应、免疫印记技术检测许旺细胞特异性蛋白产物表达。 结果与结论：脐带细胞培养第7天形态发生变化,部分细胞变成梭形。原代细胞培养10 d左右可达80%~90%融合,细胞呈梭形。分离培养的细胞表达具有间充质干细胞表面特有标志：CD44(91.4%),CD29(91.3%),CD105(99.2%),不表达CD34(0.2%),CD45(0.9%),CD14(0.6%)。脐带Wharton’s Jelly中间充质干细胞经第一阶段诱导后,细胞由短梭形变成长梭形或纺锤形,并出现聚集现象,由形状规则、表面圆滑的球形细胞团形成。第二阶段诱导后,有长梭形细胞从球形细胞团爬出,96 h后细胞形态多为长梭形,伴有多极现象。免疫细胞化学染色结果示：长梭形多极细胞具有许旺细胞特异的纤维酸性蛋白、S100蛋白染色。结果表明脐带Wharton’s Jelly中间充质干细胞可在体外诱导分化为许旺细胞。     

Distribution of calretinin immunoreactivity in the mouse dentate gyrus

Calretinin containing elements were visualized with immunocytochemistry in the adult mouse dentate gyrus (DG). In the ventral DG calretinin immunoreactive (CR-IR) large multipolar cells were clustered; they extended between two and four thick cylindrical dendrites which further branched into several thinner processes. Characteristic grape like spiny appendages were occasionally observed on these thick and thinner dendritic processes. On the basis of these structural features these large CR-IR cells were identified as hilar mossy cells. At the supragranular zone a dense CR-IR band was seen, where numerous CR-IR punctae and fibers were packed tightly among putative granule cell dendrites. In the granule cell layer, especially at the dorsal DG, numerous faintly CR-IR cells were located at the interface with the hilus. They were triangular in shape and neither calbindin D28k nor GABA positive, but were immunoreactive for highly polysialylated neural cell adhesion molecule (NCAM-H) and thus considered as newly generated neurons. In the molecular layer CR-IR cells were also scattered; they were mainly located near the pial surface and the hippocampal fissure, small in size, ovoid in shape and usually gave rise to one very thin axon like and one thin cylindrical dendritic process. These cells were assumed to be Cajal-Retzius cells. Throughout the layers, that is, the molecular layer, the granule cell layer and the hilus, CR-IR multipolar and/or fusiform cells were encountered. They resembled those reported in the rat DG in their structural features and usually extended smooth or varicose or sparsely spiny dendritic processes; some of them were confirmed to be GABA-like immunoreactive and/or glutamic acid decarboxylase immunoreactive. The present study showed that CR immunoreactivity in the mouse DG differed significantly from that in the rat and monkey dentate gyri reported previously.     

Human fetal dopamine neurons grafted in a rat model of Parkinson's disease: ultrastructural evidence for synapse formation using tyrosine hydroxylase immunocytochemistry

Summary Human fetal mesencephalic dopamine (DA) neurons, obtained from 6.5–9 week old aborted fetuses, were grafted to the striatum of immunosuppressed rats with 6-hydroxydopamine lesions of the ascending mesostriatal DA pathway. The effects on amphetamine-induced motor asymmetry were studied at various timepoints after grafting. At eight weeks, functional graft effects were not evident but after 11 weeks small effects on motor asymmetry could be monitored and rats tested 19–21 weeks after grafting exhibited full reversal of the lesion-induced rotational behaviour. Four rats were sacrificed at different timepoints between 8 and 20 weeks and the grafted DA neurons were studied in tyrosine hydroxylase (TH) immunocytochemically stained sections at the light and electronmicroscopic level. The grafts contained a total of 500–700 TH-positive neurons in each rat. In one rat sacrificed 8 weeks after grafting the grafted neurons were TH-positive but exhibited virtually no fiber outgrowth. In another rat, sacrificed after 11 weeks, a sparse TH-positive fiber plexus was seen to extend into the adjacent host neostriatum. Two rats sacrificed after 20 weeks both contained TH-positive neurons that gave rise to a rich fiber network throughout the entire host neostriatum, and this fiber network was also seen to extend into the globus pallidus and nucleus accumbens. Very coarse TH-positive processes, identified as dendrites in the electron microscope, projected up to 1.5–2.0 mm from the graft into the host striatum. Ultrastructural analysis revealed that the grafted neurons had formed no TH-positive synaptic contacts with host striatal neurons after 8 weeks, and at 11 weeks some few TH-positive synapses were identified. Twenty weeks after transplantation, abundant TH-positive synaptic contacts with host neurons were seen throughout the neostriatum, and such contacts were identified in the globus pallidus as well. Thus, the present study provides tentative evidence for a time-link between the development of synaptic contacts and the appearance of functional graft effects. Similar to the normal mesostriatal DA pathway, ingrowing TH-positive axons formed symmetric synapses and were mainly seen to contact dendritic shafts and spines. However, in comparison to the normal rat striatum there was a higher incidence of TH-immunoreactive boutons forming synapses onto neuronal perikarya. The TH-positive dendrites that extended into the host striatum were seen to receive non-TH-immunoreactive synaptic contacts, presumably arising from the host neurons. These results suggest that human fetal DA neurons are able to develop a reciprocal synaptic connectivity with the host rat when grafted to the adult brain. Grafting of human fetal DA neurons may therefore be expected to provide a means of restoring regulated synaptic DA release in patients with Parkinson's disease.Some of the results of this study were presented at the Schmitt Neurological Sciences Symposium on Transplantation into the Mammalian CNS, Rochester, NY, USA, June 30, 1987