人胎脊髓内源性神经营养物质的分离纯化

人胎脊髓存在神经营养物质,在以往的研究中已发现它们对脊髓神经元的存活、生长及分化有营养作用。本文采用Superdex－30、反相C18HPLC以及毛细管电泳对于4月龄和8月龄人胎脊髓的神经营养物质进行了分离纯化,活性检测采用体外培养脊髓神经元-MTT方法。分离过程中发现脊髓中有远不止一种神经营养成分可以提高脊髓神经元的活性,最终分离得到一个P1为8．08的蛋白成分,对脊髓神经元的活性有显著的促进作用。     

人胎脊髓内源性神经营养质的分离纯化

人胎脊髓存在神经营养物质，在以往的研究已发现它们对脊髓神经元的存活，生长及分化有营养作用。本文采用Ｓｕｐｅｒｄｅｘ－３０、反相Ｃ１８ＨＰＬＣ以及毛细管电泳对４月龄和８月龄人胎脊髓的神经营养进行了分离纯化了分离纯，活性检测采用体外培养脊髓神经元－ＭＴＴ方法。     

吗啡备用根大鼠脊髓组织神经营养活性物质的分离纯化和生物活性测定

目的　分离纯化吗啡备用根大鼠脊髓组织提取液 ,以期获得某些神经营养活性物质。　方法　应用SephacrylS 2 0 0HR凝胶层析、高效液相色谱 (HPLC)和组织培养等技术分离和检测神经营养活性物质。　结果　备用根大鼠脊髓组织提取液能够促进体外培养的鸡胚背根节 (DRG)神经突起的生长 ;吗啡作用的大鼠脊髓组织提取液也具有同样的作用 ,但备用根大鼠和吗啡作用的大鼠脊髓组织提取液在促神经突起生长作用方面并没有明显的差异。吗啡备用根大鼠脊髓组织提取液具有明显的神经营养活性作用。吗啡备用根大鼠脊髓组织提取液的SephacrylS 2 0 0HR凝胶层析Ⅱ峰洗脱液和Ⅳ峰洗脱液能够促进DRG神经突起的生长。经SDS PAGE分析 ,Ⅱ峰洗脱液呈现 1条分子量约为 6 5kD的蛋白质主带 ,Ⅳ峰洗脱液的蛋白质成分较为复杂。应用HPLC对凝胶层析Ⅳ峰洗脱液作进一步的分离 ,发现HPLCA峰洗脱液能够促进DRG神经突起的生长。经SDS PAGE显示 ,A峰洗脱液的两条蛋白质主带分子量分别为 30kD和 18kD。　结论　吗啡备用根大鼠脊髓组织提取液中具有神经营养活性作用的物质可能是分子量约为 6 5kD、30kD和 18kD蛋白质     

不同发育期鸡胚脊髓背角提取液的神经营养活性初探

为了探讨在发育过程中鸡胚脊髓背角提取液的神经营养活性作用 ,分别取 Hamburger3 0期和 40期鸡胚的脊髓背角组织制成条件培养液 ,以 Hanks平衡盐溶液代替背角提取液设置对照组。实验组分别培养了两时相的脊神经节及其神经元。培养48h时测量各组每个脊神经节神经元的神经突起平均长度及神经元存活数。结果 :两时相实验组的神经元存活数量均明显大于对照组 (P<0 .0 5 ) ;而神经突起长度仅 40期组者大于对照组 (P<0 .0 5 )。另外 ,40期组脊神经节神经元的神经突起长度及神经元存活数量也明显高于 3 0期者。提示 ,两时相脊髓背角内均存在某些初级感觉神经元诱向因子。但在不同的发育阶段脊髓背角的神经营养活性却有变化。     

备用根大鼠脊髓后角组织提取液对培养的鸡胚背根节神经突起生长的影响

应用组织培养方法，观察备用根大鼠手术侧和非手术侧脊髓后角组织提取液对鸡胚背根节神经突起生长的影响。结果显示：备用根大鼠手术侧脊髓后角组织提取液作用的背根节神经突起生长密度（１５５．２５±１４．２５）明显高于非手术侧提取液作用的背根节神经突起生长密度（８９．１４±９．６０）。提示部份去传入纤维支配的脊髓后角组织可能存在着促进神经突起生长的神经营养活性物质。     

Tenascin—C分子中重组Fibronectin各片段对胎鼠脊髓培养神经…

用小鼠重组ｔｅｎａｓｃｉｎ－Ｃ（ＴＮ－Ｃ）结构域中类似Ⅲｆｉｂｏｎｅｃｔｉｎ（ＦＮ）的不同重复片段，说明作为基质或培养液成分，研究其对的胚胎小鼠脊髓神经突起生长、粘附力及神经元活性的影响。结果显示，：无论作为基质还是培养液成分，ＦＮ６－８对神经突起生长及神经元活性有促进作用；ＦＮ３－５对神经突起生长及神经元活性均有抑制作用。ＴＮ－Ｃ促进神经突起生长的功能区在ＦＮ６－８，ＦＮ３－５介导了神经元与ＴＮ     

Tenascin-C分子中重组Fibronectin各片段对胎鼠脊髓培养神经元的不同作用

用小鼠重组ｔｅｎａｓｃｉｎ－Ｃ（ＴＮ－Ｃ）结构域中类似Ⅲ型ｆｉｂｒｏｎｅｃｔｉｎ（ＦＮ）的不同重复片段，分别作为基质或培养液成分，研究其对培养的胚胎小鼠脊髓神经突起生长、粘附力及神经元活性的影响。结果显示：无论作为基质还是培养液成分，ＦＮ６－８对神经突起生长及神经元活性均有促进作用；ＦＮ３－５对神经突起生长及神经元活性均有抑制作用。ＴＮ－Ｃ促进神经突起生长的功能区在ＦＮ６－８，ＦＮ３－５介导了神经元与ＴＮ－Ｃ的粘附。     

备用根大鼠脊髓后角组织神经营养活性物质的分离纯化和生物活性测定

目的　进一步分离纯化备用根大鼠部分去传入纤维支配 (手术侧 )的脊髓后角组织提取液 ,以获得某种神经营养活性物质。　方法　用 Superdex G- 75 prep grade凝胶层析、高效液相色谱 (HPL C)层析和细胞培养等技术分离和检测神经营养活性物质。　结果　手术侧脊髓后角组织提取液经 Superdex G- 75 prep grade凝胶层析和 HPL C层析后 ,得到的 A峰洗脱液具有促进体外培养鸡胚背根节 (DRG)分离神经元存活及其神经突起生长的神经营养活性作用。经 SDS-聚丙烯酰胺凝胶电泳 (PAGE)显示 ,A峰洗脱液呈现一条分子量约为 6 0 k D的蛋白质主带。　结论　结合生物活性检测结果 ,分子量约 6 0 k D的蛋白质可能是手术侧脊髓后角组织的神经营养活性物质     

吗啡增强备用根大鼠脊髓后角组织提取液对培养鸡胚背根节的促神经突起生长的作用

用组织培养方法，探讨备用很大鼠手术侧和非手术侧脊髓后角组织提取液对鸡胚背根节（ＤＲＧ）神经突起的促生长作用，以及应用吗啡对此作用的影响。结果显示：备用根大鼠手术侧脊髓后角组织提取液作用的ＤＲＧ神经突起密度（３６．４２±４．６９），比非手术侧的（２３．９６±３．４７）明显增大。提示去初级传入纤维的脊髓后角组织具有促进ＤＲＧ神经突起生长的神经营养活性作用。应用吗啡的备用根大鼠手术侧脊髓后角组织提取液作用的ＤＲＧ神经突起密度（６４．１９±９．２４），又比备用根大鼠手术侧的大。这表明，吗啡具有进一步增强去初级传入纤维支配的脊髓后角组织提取液促进培养的ＤＲＧ神经突起生长的效应。     

备用根猫脊髓背角提取液对鸡胚背根节神经细胞存活的影响

本研究组既往的研究表明，备用根猫脊髓背角组织块和提取液促神经突起生长的活性明显增强。为观察去传入猫脊髓背角提取液对神经元存活的影响，我们采用单侧后肢备用根猫（切除一侧Ll－L。、L，－Sz背报节，保留L。背根为备用根）五只，分别取术后五天T12-S3节段脊髓背角组织，匀浆、离心获得上清液、制成条件培养液后，加入鸡胚背根节神经细胞是液培养。于48h时，以l％台盼蓝染色，计数神经细胞存活率。结果显示：手术侧组神经细胞存活率为0.85±0.04，而非手术侧组神经细胞存活率仅为0．55fo．10。两者相比有显著性差异（P＜0．of）。提示备用根猫脊髓背角提取液对培养中的鸡胚背报节神经元除有促进神经突起生长的作用外还有明显的促神经元存活作用。     

Effects of Reg‐2 on Survival of Spinal Cord Neurons In Vitro

Regeneration gene protein 2 (Reg‐2) is a small secreted protein expressed in motor and sensory neurons of spinal cord during developmental stages and following injury of peripheral nerves. Reg‐2 appears to act as a neurotrophic factor and protects injured neurons from death during regeneration. To illustrate these potential protective effects in vitro, we investigated the blocking effects of Reg‐2 antibodies on the survival of primary cultured spinal cord neurons and astrocytes, as well as on neurite outgrowth. In addition, the effects of Reg‐2 in neuron injury models induced by peroxide and mitochondrial poisoning were assessed. Our results showed that Reg‐2 antibody markedly reduced survival and neurite outgrowth from neurons, whereas astrocyte survival was unaffected. Addition of Reg‐2 into the culture medium had no effect on neuron survival or neurite outgrowth. However, the addition of the Reg‐2 into culture media after peroxide treatment or cellular hypoxia insult induced by mitochondrial poisoning can reduce lactate dehydrogenase release levels and cell death. Thus, the data suggests that Reg‐2 is essential for the survival and neurite outgrowth of developing spinal cord neurons but not the survival of glial cells, and that Reg‐2 plays protective effects on spinal cord neurons against injury in vitro. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

嗅鞘细胞培养上清液对体外培养脊髓神经元存活及活性的影响

目的:通过检测鞘嗅细胞培养上清液,研究其是否能够分泌促进神经元存活及活性的物质,以便为CNS神经损伤修复提供相关移植的理论依据。方法:分离培养嗅神经及嗅球颗粒层(ONGL)细胞,经过传代培养进行纯化嗅鞘细胞,最后收集其培养上清液。浓缩并检测其蛋白浓度,分别以10ug/ml、20ug/ml、40ug/ml、80ug/ml、160ug/ml的浓度加到培养的脊髓神经元的存活及活性指标。结果:嗅鞘细胞培养上清的蛋白浓度为40ug/ml、80ug/ml时对神经元夏活具有促进作用(与对照组相比P<0.01),而80ug/ml时对神经元活性具有促进作用(P<0.01)。结论:嗅鞘细胞在培养状态能够分泌一些活性物质,这些活性物质对神经元的存活和活性有明显的促进作用。     

神经元特异性烯醇化酶酶联免疫吸附分析技术在体外培养神经元中的应用

ＭＴＴ法是用于反映体外培养神经细胞活性的指标，但对检测细胞活性没有特异性。本实验结合神经元特异性烯醇化酶免疫细胞化学技术特异性强、敏感谢性高的优点与酶联免疫吸附试验酶促底物邻苯二胺呈色形成弥散的可溶性有色产物，通过检测有色溶液的ＯＤ值来比较体外培养过程中神经细胞的活性以及相应的数目，在建立此方法的同时并用此方法检测了人胚脊髓提取液对神经细胞活性的影响。结果证明：分子量小于１０ｋＤ的人胚脊髓提取对体     

Piezoelectric Substrates Promote Neurite Growth in Rat Spinal Cord Neurons

We tested the possibility that exogenous electrical activity from a piezoelectric substrate could influence neuronal structure in cultured spinal cord neurons. Oscillating electrical fields were delivered to rat neurons via substrates consisting of poly(vinylidene fluoride) film, both in its piezoelectric (PZ) and non-piezoelectric (PV) forms. To induce oscillating electrical fields at the film surfaces, a 50?Hz mechanical vibration was applied. After 4?days of mechano-electrical stimulation, neuronal densities were increased by 115% and neurons grew 79% more neurites, with more than double the branch points, compared with neurons grown on non-stimulated PZ films (p?<?0.001). The effects were due to electrical field, because vibration applied to non-PZ films did not increase neurite growth. We conclude that the oscillating electric fields produced from PZ polymer substrates can induce plastic changes in neurons of the central nervous system and herein we show its influence on neurite growth and branching.     

大鼠胎脑细胞脊髓内移植的实验研究

本文是用孕１３天（Ｅ１３）大鼠胎脑细胞悬液，以慢速注入法移植于同种成年大鼠脊髓白质的侧索及后索、中央管及灰质中。移植后分１天、２周、４周、１２周等４组，用光镜检查在脊髓内不同部位移植的胎脑细胞，存活、生长、分化、成熟以及与宿主脊髓嵌合等情况。结果表明：①移植于成鼠脊髓侧索、后索及中央管等不同部位，９０％以上移植成功，胎脑细胞的存活、生长、分化极为良好，术后４周形成神经毡样结构，且多与宿主灰质嵌合；②灰质内移植组的宿主脊髓灰质损伤严重，神经元多变性、坏死消失；③移植于脊髓不同部位的方法将有助于脊髓前、后以及中央等各种类型损伤的修复，探讨是否有可能建立脊髓内不同移植部位的模型。     

脊髓损伤组织匀浆通过甲酰基肽受体2促进神经元突起的生长

文题释义： 甲酰基肽受体2：甲酰基肽受体2在吞噬类细胞中的表达和作用已经进行了广泛的研究，主要是促进吞噬细胞的趋化作用。近年来，甲酰基肽受体2在非吞噬类细胞的表达和作用也有相关的报道，如间充质干细胞、神经细胞等，在这些细胞中也能观察到其趋化作用。作者首次研究观察到甲酰基肽受体2能够促进神经元轴突的生长，并被其他实验室研究证实。那么甲酰基肽受体2对脊髓损伤修复是否起作用及其机制，这是一个十分有意义的问题。 甲酰基肽受体配体：甲酰基肽受体2配体种类繁多，来源不同，不同的配体与甲酰基肽受体2结合可能导致不同、甚至相反的生物学效应，可能与受体不同的结构域发挥作用有关。然而究竟是哪些结构域在起作用，能否在此水平上来调节受体的功能，并使之产生有利作用，避免有害作用，需要具体问题具体研究。 背景：前期研究观察到神经干细胞新分化的神经元表达甲酰基肽受体2，并证实甲酰基肽受体2能促进神经干/祖细胞迁移，诱导向神经元分化。脊髓损伤组织中存在甲酰基肽受体2配体，然而不同的配体与甲酰基肽受体2结合可能导致不同、甚至相反的生物学效应。 目的：探讨脊髓损伤产生的配体与甲酰基肽受体2作用后对神经元突起生长的影响。 方法：采用酶消化法提取胎鼠大脑皮质神经元；制备SD大鼠脊髓损伤模型，提取损伤脊髓组织匀浆。①实验分组1：观察甲酰基肽受体2激活对神经元突起的影响，分组如下：对照组、甲酰基肽受体2阻断剂组(即添加WRW4)、脊髓匀浆组、脊髓匀浆+WRW4组；②实验分组2：观察甲酰基肽受体2激活后AKT和ERK信号通路阻断对神经元突起的影响，分组如下：对照组、AKT和ERK信号通路阻断剂组(即添加Ly294002+PD98059)、脊髓匀浆组、脊髓匀浆+Ly294002+PD98059组。神经元细胞贴壁24 h后，按上述分组处理7 d，免疫荧光染色共聚焦显微镜观察脊髓匀浆激活甲酰基肽受体2对神经元突起的影响；按上述分组处理30 min，Western blotting检测磷酸化蛋白水平；按上述分组处理24 h，Western blotting检测F-actin水平，观察在甲酰基肽受体2特异性阻断剂WRW4存在的情况下，对MAPK和PI3K/Akt通路中关键蛋白磷酸化的影响。 结果与结论：①脊髓损伤组织匀浆液能够使神经元突起长度、初级分枝数、分枝节点数显著增加，这种增加效应大部分被甲酰基肽受体2受体特异性阻断剂WRW4阻断；②脊髓损伤组织匀浆液能够使神经元中ERK1/2和Akt的磷酸化增加，这种效应能够被WRW4阻断；③Akt信号通路阻断剂Ly294002和Erk信号通路阻断剂PD98059能够阻断脊髓匀浆的促进作用；④脊髓损伤组织匀浆能够使F-actin表达量明显增加，这种效应能够被甲酰基肽受体2特异性阻断剂WRW4所阻断；⑤这些实验结果说明，脊髓匀浆液能够通过激活甲酰基肽受体2促进神经元突起生长，这种作用机制可能与ERK1/2和Akt的磷酸化增加相关。 ORCID: 0000-0003-1097-1874(张良) 中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程     

Spinal GABAergic transplants attenuate mechanical allodynia in a rat model of neuropathic pain

Injury to the spinal cord or peripheral nerves can lead to the development of allodynia due to the loss of inhibitory tone involved in spinal sensory function. The potential of intraspinal transplants of GABAergic cells to restore inhibitory tone and thus decrease pain behaviors in a rat model of neuropathic pain was investigated. Allodynia of the left hind paw was induced in rats by unilateral L5- 6 spinal nerve root ligation. Mechanical sensitivity was assessed using von Frey filaments. Postinjury, transgenic fetal green fluorescent protein mouse GABAergic cells or human neural precursor cells (HNPCs) expanded in suspension bioreactors and differentiated into a GABAergic phenotype were transplanted into the spinal cord. Control rats received undifferentiated HNPCs or cell suspension medium only. Animals that received either fetal mouse GABAergic cell or differentiated GABAergic HNPC intraspinal transplants demonstrated a significant increase in paw withdrawal thresholds at 1 week post-transplantation that was sustained for 6 weeks. Transplanted fetal mouse GABAergic cells demonstrated immunoreactivity for glutamic acid decarboxylase and GABA that colocalized with green fluorescent protein. Intraspinally transplanted differentiated GABAergic HNPCs demonstrated immunoreactivity for GABA and beta-III tubulin. In contrast, intraspinal transplantation of undifferentiated HNPCs, which predominantly differentiated into astrocytes, or cell suspension medium did not affect any behavioral recovery. Intraspinally transplanted GABAergic cells can reduce allodynia in a rat model of neuropathic pain. In addition, HNPCs expanded in a standardized fashion in suspension bioreactors and differentiated into a GABAergic phenotype may be an alternative to fetal cells for cell-based therapies to treat chronic pain syndromes.     

Spinal cord contains neurotrophic activity for spinal nerve sensory neurons. Late developmental appearance of a survival factor distinct from nerve growth factor

Several tissues of the developing chick embryo have been reported to contain neurotrophic activity which can sustain the survival of sensory neurons maintained in culture. In a previous study, however, we noted that such nerve growth promoting activity was exceptionally low, if not absent, from extracts of spinal cord from chick embryos of up to 16 days incubation. Since then the combined results from a number of tissue culture studies have suggested that the central nervous system may be the source of a neurotrophic growth factor essential during the late development of sensory neurons. We have therefore carried out an extended range study of the neurotrophic properties of avian spinal cord. Extracts of spinal cord tissue prepared from chicks at stages between the last wk of embryogenesis and 12 wks after hatching were tested for their ability to promote survival and neurite outgrowth from both explant and dissociated neuron-enriched cultures of dorsal root, trigeminal, nodose and paravertebral chain sympathetic ganglia from chick embryos between 8 and 16 days old. We conclude from our results that spinal cord is a potent source of neurotrophic activity for sensory neurons, although this activity appears relatively late in development of the spinal cord. The predominant ontogenic increase in spinal cord neurotrophic activity was seen to occur during the first week after hatching. Sensory neurons from both spinal and cranial nerve ganglia were sustained in culture by spinal cord extracts, whereas sympathetic neurons did not respond. Neurons from older sensory ganglia (12-16 day old embryos) were much more responsive than similar neurons from young embryos (8 day).(ABSTRACT TRUNCATED AT 250 WORDS)     

大鼠胎脑移植对急性脊髓损伤的组织修复作用

目的　为了减轻宿主急性脊髓损伤的继发性病理改变 ,使移植的胎脑细胞能更好地存活、分化 ,与宿主脊髓组织整合 ,以替代受损或变性坏死的神经元。方法　用妊娠 13天的大鼠胎脑为移植物 ,采用细胞悬液立体慢速注射法 ,依脊髓损伤类型移植于脊髓白质不同部位。进行光电镜观察。结果　①单纯脊髓损伤后 ,损伤脊髓发生出血 ,变性坏死 ,液化呈囊腔 ,最终形成胶质瘢痕 ;②损伤后早期移植胎脑悬液后明显减轻了继发性病理改变 ,减轻胶质瘢痕的形成 ;③依造成的后脊髓损伤类型进行的按损伤部位移植 ,胎脑细胞80 %存活 ,分化 ,并与宿主脊髓组织整合 ,形成了多种类型的突触及器官样分化 ,构成“中继”的形态学所见。结论　按损伤部位移植的方法能较大程度地保留损伤区残存的神经组织 ,避免损伤脊髓遭受第二次损伤 ,尤其是脊髓灰质