西南三省一市解剖学会第五届学术会议论文摘要

目的：研究神经生长因子(NGF)在早期人胚神经管发育过程中的定位表达。方法：用35天的人胚进行免疫组织化学ABC法染色。结果：人胚神经管的室带神经元的细胞浆和细胞核NGF免疫反应阳性；在中间带一部分神经元的细胞核NGF、免疫反应阳性，另外一部分神经元的细胞核NGF免疫反应阴性，神经元的突起NGF免疫反应阳性；在边缘带NGF的表达与中间带相似。在神经管的头侧NGF阳性反应较强，神经管的尾侧NGF阳性反应较弱。     

神经生长因子在人胚胎神经管发育的定位表达

目的研究神经生长因子(NGF)在人胚胎神经管发育过程中的定位表达。方法用胚胎龄为30天的人胚胎行免疫细胞化学ABC法染色。结果在人胚胎神经管室带,神经元的细胞浆和细胞核NGF免疫反应阳性;在中间带一部分神经元的胞核NGF免疫反应阳性,另外一部分神经元的胞核NGF免疫反应阴性,神经元的突起NGF免疫反应阳性;在边缘带NGF的表达与中间带相似。神经管的吻侧NGF、蛋白表达较强,神经管的尾侧NGF蛋白表达较弱。中胚层体节之间可见NGF免疫反应阳性的细胞。结论NGF是神经管诱导分化的重要信号分子,它通过神经管中间带的NGF水平信号和神经管与中胚层相互作用的垂直信号诱导神经管的发育分化,在神经管的发育中具有十分重要的作用。     

Fas、FasL和Caspase-3在人胚早期脊髓发育中的分布

目的观察Fas、FasL和Caspase-3在人胚早期脊髓发育的表达。方法收集早期人胚35天,52天和60天标本9例,用4%多聚甲醛固定后制作8μm厚的石蜡切片。间隔取片分组,分别用Fas(效价1:250,Santa Cruz)、FasL(效价1:250,Santa Cruz)、Caspase-3(效价1:500,Sigma)抗体对5例人胚标本的10张切片行免疫组织化学ABC法染色。观察Fas、FasL和Caspase-3在人胚早期脊髓发育中的分布以及亚细胞定位。DAB棕色反应呈色。结果Fas免疫反应阳性的神经元主要分布在室管膜层、中间层和边缘层,可见胞浆和细胞核内染色。FasL免疫反应阳性产物主要分布在室管膜层和套层,可见细胞膜、胞浆和细胞核染色。Caspase-3免疫反应阳性产物主要分布在神经管的室管膜层和中间层,可见细胞核染色。结论在人胚发育早期,Fas、FasL和Caspase-3分布于脊髓,提示这些因子参与人胚早期脊髓的发育,参与神经元凋亡信号的传递。     

神经营养素3和受体在早期人胚脑发育中的定位表达

目的:了解在早期人胚脑的发育过程中,大脑神经元的增殖情况;了解神经营养素3(NT-3)和受体TrkC在人胚脑发育过程中的影响。方法:用6周龄的人胚进行免疫组织化学ABC法染色,以PCNA作为大脑神经元的增殖的指标,NT-3和受体作为了解影响人胚脑发育因素的指标。结果:在人胚胎早期发育阶段,PCNA免疫阳性反应物主要分布于人胚前脑的神经细胞核中,NT-3免疫阳性反应物主要分布于前脑脑室神经上皮层的胞质中和神经细胞的轴突中,其受体TrkC散在分布于人胚前脑神经细胞膜;发育过程中NT-3及其受体在脑室神经上皮层上呈现不同程度的免疫阳性反应。结论:NT-3及其受体TrkC在早期的人胚胎脑发育中呈现表达,提示NT-3可能诱导神经细胞增殖,并协同其它生长因子促进轴突的生长,保证神经元胞体的存活。     

脑源性神经营养因子及其受体TrkB在大鼠脊髓中间带外侧核的定位

为了研究脑源性神经营养因子(BDNF)及其特异性受体TrkB在脊髓中间带外侧核的分布,从形态学上探讨BDNF在交感神经通路中发挥调质作用的神经结构基础。本实验中注射逆行示踪剂快蓝至单侧颈上节,然后对脊髓中间带外侧核进行BDNF和TrkB的免疫荧光染色。结果(1)逆行标记细胞广泛分布于注射侧第8颈髓至第5胸髓(C8～T5)的中间带外侧核,其中在T1T3分布相对较集中,成簇排列;(2)部分逆行标记的交感节前神经元胞体和树突周围可见BDNF免疫阳性(BDNFir)终扣,呈点状或串珠状排列;(3)大多数交感节前神经元为TrkB免疫阳性。以上结果提示,BDNF可能作为神经递质样物质或调质在交感神经通路中通过作用节前交感神经元发挥作用     

NGF、BDNF及受体trkA、trkB、trkC在正常猴脊髓的表达

采用免疫组织化学方法观察了神经生长因子 (NGF) ,脑源性神经营养因子 (BDNF)以及 NGF家族因子受体 trk A、trk B、trk C的免疫阳性反应在正常猴脊髓的分布。结果表明 :NGF免疫反应阳性的神经元在脊髓灰质各层中均有分布 ,灰、白质内也可见较多的 NGF免疫反应阳性的胶质细胞。 BDNF在脊髓各型神经无有明显的表达 ,特别是前角运动神经元。 trk A、trk B、trk C的免疫阳性反应产物主要分布在灰质的神经元及胶质细胞。本实验结果揭示了在正常猴脊髓中神经营养因子 (NGF、BDNF )及受体 trk A、trk B、trk C的表达状况 ,提示这些神经营养因子及受体在维持猴脊髓神经元的正常生理功能中具有重要作用。     

人胚胎脊髓发育过程中脑源性神经营养因子的表达及变化

目的:探讨脑源性神经营养因子(BDNF)在不同发育时期人胚胎脊髓中的表达变化及意义.方法:采用免疫组织化学和免疫印迹法对3周～8月人胚脊髓中BDNF蛋白进行定位、定量研究.结果:所检测的15个时段中,神经管上皮和室管膜上皮均可见BDNF阳性细胞,且随胚龄增加呈规律性变化;免疫印迹法显示6～9周,BDNF蛋白含量随胚龄逐渐增加,9周时达到高峰,3月后下降,6月后又有所增加但相对平稳.结论:BDNF在人胚胎脊髓发育特别是胚期脊髓的发育中发挥重要作用,并可能参与神经上皮神经干细胞的分裂增殖.     

BDNF免疫反应阳性神经元在大鼠孤独症动物模型大脑顶叶感觉皮层中的表达

为明确脑源性神经生长因子(BDNF)阳性神经元在孤独症动物模型中的形态的数量变化,探讨BDNF在孤独症发病中的作用,本实验采用Wistar孕鼠妊娠12.5d时腹腔注射丙戊酸钠(VPA,600mg/kg)建立的子代孤独症动物模型,测试和比较了模型组和对照组大鼠的发育状态;用免疫组织化学和图像分析技术,比较观察了生后35d和49d时模型组和对照组动物BDNF免疫反应阳性神经元在大脑感觉皮层中的形态及其变化。结果显示:模型组动物发育迟缓;出生后35d模型组脑内BDNF免疫反应阳性细胞的数目较对照组少,且胞体小,突起细;而生后49d,模型组的BDNF免疫反应阳性细胞的数目则较对照组相对为多,细胞形态也趋正常。这些结果提示BDNF与孤独症的发病密切相关。     

背侧抑制性轴突导向蛋白对鸡胚脊髓背侧中间神经元发育的影响

目的:探讨在鸡胚脊髓发育早期背侧抑制性轴突导向蛋白(draxin)对脊髓背侧中间神经元(dorsal in-terneurons,dI)迁移特性的影响。方法:应用免疫组织化学的方法观察鸡胚脊髓dI的发育特性;应用电穿孔的方法在鸡胚一侧脊髓内过表达draxin,观察draxin过表达后对鸡胚脊髓内dI神经元发育特性的影响。结果:随着胚胎的发育,鸡胚脊髓内dI3中间神经元首先在脊髓背侧区形成并逐渐向腹侧迁移,而dI2、dI4和dI6中间神经元没有形成明显的腹侧迁移特性;鸡胚脊髓内分别过表达分泌型和跨膜型draxin时,dI3中间神经元的腹侧迁移延迟,且在跨膜型draxin过表达时其受影响程度较高;而dI2、dI4和dI6中间神经元的迁移未受到明显影响。结论:Draxin参与鸡胚脊髓内dI3中间神经元腹侧迁移的调节。     

BDNF、NGF对体外培养的胚胆碱能神经元生长发育的影响

本文用AChE组化方法，研究了BDNF、NGF对培养的胚鼠基底前脑胆碱能神经元的作用及BDNF和NGF的协同作用。结果表明BDNF和NGF都具有增加AChE阳性神经元数量的作用，二者的不同在于BDNF作用出现的时间较早、强度较小；而NGF作用出现的时间较迟但强度较大。并发现BDNF对体外培养的胚胆碱能神经元胞体早期的生长发育作用比较明显，而NGF的作用则不甚显著。BDNF对胚胆碱能神经元发出突起和突起的延伸作用较NGF强。BDNF和NGF的联合作用较单独使用BDNF或NGF为好。本文的结果提示在体外培养中两种营养因子联合应用较只用一种因子有益。     

波形蛋白与层粘连蛋白在人胚早期神经管中的表达

目的观察层粘连蛋白（LN）和波形蛋白（Vim）在人胚早期神经管发育中的表达变化,探讨人胚神经管发育早期细胞微环境的特点。方法收集早期人胚23天和45天标本8例,分别用LN和Vim抗体对人胚标本的组织切片行免疫组织化学ABC法染色。图像分析不同观测指标阳性细胞的积分光密度值,结果用t检验进行统计分析。结果人胚发育早期,LN和Vim在神经管的神经上皮层、中间层和边缘层的神经细胞胞中表达。在神经管发育到23天时,LN的积分光密度值（1258．17±635）比Vim的积分光密度值（2611．34±502）低（P〈0．05）。结论在人胚早期,神经管的细胞基质成分以Vim为主,LN为辅,可能对神经干细胞的发育有重要作用。     

脊髓半横断损伤后腹角神经元神经生长因子、脑源性神经营养因子、神经营养因子-3和神经营养因子-4表达的变化

目的:探讨大鼠脊髓半横断损伤(htSCI)后脑源性神经营养因子(BDNF)、神经生长因子(NGF)、神经营养因子(NT-3、NT-4)在脊髓腹角神经元表达的早期变化。方法:免疫组织化学ABC法分别染4种神经因子并作阳性细胞计数。结果:NGF主要分布于脊髓腹角神经元的胞核,BDNF、NT-4与NT-3主要分布于胞浆。htSCI前后它们在细胞内的分布范围没有变化。BDNF、NGF与NT-3的3 d在损伤尾侧段脊髓双侧腹角阳性神经元数与对照组相比显著减少。BDNF与NGF的14 d的双侧腹角阳性神经元数量均较正常组明显增多,NT-3与NT-4的14 d~21 d的双侧腹角阳性神经元数量均较正常组明显增多,BDNF7~21 d以及NGF14 d的健侧的阳性神经元数量均分别多于相应的伤侧。结论:内源性BDNF、NGF、NT-3、NT-4增加对脊髓损伤修复具有重要作用,BDNF和NGF在健侧表达的增加说明健侧代偿功能的活跃。     

Enforced expression of the transcription factor HOXD3 under the control of the Wnt1 regulatory element modulates cell adhesion properties in the developing mouse neural tube

HOX genes expressed in a specific spatial and temporal manner play a crucial role in determining the body plan during the early development of vertebrates. In adult tissues, many HOX genes participate in normal hematopoiesis and carcinogenesis. We previously found that overexpression of the homeobox gene HOXD3 alters expression levels of cell adhesion molecules in human cancer cell lines. Here, we have investigated whether HOXD3 expression is related to the cell adhesion processes during mouse development focusing on dorsal midline cells or roof-plate cells of the neural tube and neural crest cells. We created transgenic mouse embryos, in which HOXD3 is expressed in the dorsal midline under the control of the Wnt1 regulatory element, and analyzed these embryos at embryonic day 10.5-13.5. In HOXD3-expressing transgenic embryos, although neural crest-derived structures in the trunk region appeared to be normal, striking abnormalities were found in the neural tube. In transgenic embryos expressing the lacZ gene under the control of the Wnt1 regulatory element, expression of lacZ was restricted to roof-plate cells within the neural tube. By contrast, in HOXD3-expressing transgenic embryos, expression of HOXD3 was not only located in the dorsal neural tube, but also had spread inside the ventricular zone in more ventral regions of the neural tube. These findings show that the HOXD3 transgene is expressed more broadly than the Wnt1 gene is normally expressed. Expression of both Wnt1 and Msx1, marker genes in the roof plate, was further extended ventrally in HOXD3-expressing embryos than in normal embryos, suggesting that expression of the HOXD3 transgene expands the roof plate ventrally within the neural tube. In the ventricular zone of HOXD3-expressing embryos at embryonic day 10.5, we observed an increase in the number of mitotic cells and failure of interkinetic nuclear migration of progenitor cells. Furthermore, in HOXD3-expressing embryos at embryonic day 12.5, the ventricular zone, in which progenitor cells became more loosely connected to each other, was composed of a large number of cells that did not express N-cadherin. Our results indicate that expression of HOXD3 is closely associated with modulation of cell-adhesive properties during embryonic development.     

脑源性神经营养因子过表达促进大鼠神经干细胞向神经元分化

Objective To construct eukaryotic expression vector of brain-derived neurotrophic factor (BDNF) and detect its effect of overexpression on differentiation of rat neural stem cells (NSCs) into neurons. Methods The RT-PCR was used to amplify rat BDNF gene from RNA of rat hippocampus. The BDNF gene was inserted into eukaryotic expression vector pEGFP-N1 to construct recombinant expression vector pEGFP-N1-BDNF. The recombinant vector was transfected into NSCs by Lipofectamine 2000.The expression of BDNF mRNA in NSCs was detected by RT-PCR. The differentiation of rat NSCs into neurons was detected by immunohistochemistry staining. Results The sequence of the cloned BDNF was confirmed to be correct by DNA sequencing. The NSCs transfected with pEGFP-N1-BDNF expressed BDNF efficiently. The pEGFP-N1-BDNF transfected NSCs differentiated into more neurons than the pEGFP-N1 transfected ones (EM>P/EM>0.01). Conclusion All these results indicate that BDNF overexpression significantly promotes the differentiation of rat NSCs     

Neuronal growth factors and development of respiratory control

Neurotrophic molecules, released by neurons and neural target tissues, play a pivotal role in regulating neuronal development and plasticity. This article reviews recent work demonstrating the pivotal role of two such molecules, brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF), in the growth and maturation of respiratory neurons and the expression of normal ventilatory behavior. Although BDNF and GDNF are structurally dissimilar and signal through wholly distinct receptors, they are both required for development of peripheral chemoafferent neurons that provide hypoxic drive to the brainstem respiratory network. Studies of genetically engineered mice carrying targeted deletions in the genes encoding BDNF and GDNF, as well as genetic linkage analysis in humans, indicate that these trophic molecules may be candidate genes for human developmental disorders of breathing.     

Brain derived neurotrophic factor and neurotrophic factor 3 modulate neurotransmitter receptor expressions on developing spiral ganglion neurons

Cochlear spiral ganglion neurons (SGN) provide the only pathway for transmitting sound evoked activity from the hair cells to the central auditory system. Neurotrophic factor 3 (NT-3) and brain derived neurotrophic factor (BDNF) released from hair cells and supporting cells exert a profound effect on SGN survival and neural firing patterns; however, it is unclear what the effects NT-3 and BDNF have on the type of neurotransmitter receptors expressed on SGN. To address this question, the whole-cell patch clamp recording technique was used to determine what effect NT-3 and BDNF had on the function and expression of glutamate, GABA and glycine receptors (GlyR) on SGN of cochlea from postnatal C57 mouse. Receptor currents induced by the agonist of each receptor were recorded from SGN cultured with or without BDNF or NT-3. NT-3 and BDNF exerted different effects. NT-3, and to a lesser extent BDNF, enhanced the expression of GABA receptors and had comparatively little effect on glutamate receptors. Absence of BDNF and NT-3 resulted in the emergence of glycine-induced currents; however, GlyR currents were absent from the short term cultured SGN. In contrast, NT-3 and BDNF suppressed GlyR expression on SGN. These results indicate that NT-3 and BDNF exert a profound effect on the types of neurotransmitter receptors expressed on postnatal SGN, results that may have important implications for neural development and plasticity.     

核受体相关因子1基因转染骨髓源性神经干细胞体外诱导向多巴胺能神经元的分化

背景：核受体相关因子1基因修饰是否促进骨髓源性神经干细胞向多巴胺能神经元分化少见报道。 目的：观察核受体相关因子1基因修饰骨髓源性神经干细胞在体外诱导分化为多巴胺能神经元的作用。 方法：体外培养和纯化大鼠骨髓源性神经干细胞,将骨髓源性神经干细胞分为4组,将未转染的骨髓源性神经干细胞随机分为对照组,脑源性神经营养因子组;将筛选出的重组质粒转染阳性的神经干细胞分为核受体相关因子1组和核受体相关因子1+脑源性神经营养因子组。 结果与结论：RT-PCR显示转染的骨髓源性神经干细胞4 d后核受体相关因子1高表达,分化结果显示：核受体相关因子1+脑源性神经营养因子组细胞内酪氨酸羟化酶在mRNA水平上的表达量最高,神经干细胞贴壁分化后各组酪氨酸羟化酶阳性细胞比例均明显高于对照组,其中以核受体相关因子1+脑源性神经营养因子组分化比例最高,为(52.44±15.9)%。提示核受体相关因子1基因修饰可促进骨髓源性神经干细胞向多巴胺能神经元分化,并通过脑源性神经营养因子的诱导作用,在体外可获得大量的多巴胺能神经元。     

Activation of expression of brain-derived neurotrophic factor at the site of implantation of allogenic and xenogenic neural stem (progenitor) cells in rats with ischemic cortical stroke

Ischemic stroke was modeled in the sensorimotor zone of the brain cortex in adult rats. Rat embryonic nervous tissue, neural stem cells from human olfactory epithelium, and rat fibroblasts (cell control) were implanted into the peri-infarction area of rats of different groups immediately after stroke modeling. Expression of BDNF mRNA was analyzed 7 days after surgery by real-time PCR. BDNF expression in cell preparation before their implantation was minimum. The expression of BDNF mRNA increased by 5–6 times in the areas of implantation of rat fibroblasts and human olfactory epithelium and by 23 times in the area of implantation of rat embryonic nervous tissue compared to peri-infarction areas without cell implantation. These findings confirm the possibility of realization of the therapeutic effects of neural stem cells via expression of trophic factors.