重组人胶质细胞源性神经营养因子的生物学活性研究

从人星形胶质细胞瘤BT-325细胞中克隆胶质细胞源性神经营养因子(GDNF) cDNA序列.以大肠杆菌作为表达系统,GDNF蛋白在大肠杆菌JM103中获得了高效表达;表达产物经纯化、复性后,以8日龄鸡胚背根节（DRG）、14日龄胎鼠脊髓前角运动神经元以及新生大鼠大脑皮层胶质细胞作为实验材料,研究了GDNF的生物学活性,结果表明： rhGDNF可有效地促进DRG突起的生长,rhGDNF对体外培养的运动神经元表现出明显的促突起生长作用,并可显著提高体外培养运动神经元的存活率,rhGDNF 对体外培养的胶质细胞具有促增殖作用.     

人酸性成纤维细胞生长因子神经营养作用的初步研究

本实验研究了人酸性成纤维细胞生长因子(haFGF)的体外神经营养作用。结果表明,haFGF在体外能明显促进鸡胚(E-8)脊髓组织神经突起的生长,并能明显改变新生大鼠脑星形胶质细胞的形态,使扁平、多角形紧密联接的细胞转化为具有纤维样突起的胶质细胞,同时对胶质细胞DNA合成也有一定促进作用。实验还证明,haFGF可增加体外培养新生大鼠海马神经元的存活,且大大增加神经元胞体体积及突起长度。     

人睫状神经营养因子的原核高效表达

睫状神经营养因子(ciliary neurotrophic factor,CNTF)是神经生长因子家族之外的一种神经营养因子,由200个氨基酸组成,分子量约22.86kD,等电点6.00。CNTF对睫状副交感神经元、交感神经元、感觉神经元、视网膜神经节细胞、脊髓运动神经元、海马神经元等多种中枢及外周神经元有促存活作用。CNTF也是第一个被发现的能维持在体和离体脊髓运动神经元的存活及突起生长的神经营养因子,因此在神经创伤和神经退行性病变的诊断与治疗中有巨大的临床价值。由于CNTF在天然组织中含量甚微,故用基因工程     

肝细胞生长因子在正常大鼠腰段脊髓和背根神经节的表达

目的:观察肝细胞生长因子(hepatocyte growth factor,HGF)在大鼠脊髓和背根神经节(dorsal root ganglion,DRG)的表达。方法:取健康成年6只SD大鼠运用免疫组织化学染色技术检测HGF在腰段脊髓、背根神经节内的表达和分布。结果:在L4-6段脊髓,HGF免疫阳性产物可见于各板层神经元,尤以脊髓前角运动神经元明显;在DRG中,HGF免疫阳性物质可见于以大、中型为主的神经元的胞浆及突起中。结论:脊髓和背根神经节内的HGF通过与受体c-Met结合可能在神经再生及突触可塑性方面起一定作用。     

胶质细胞源性神经营养因子对疼痛的调节作用

胶质细胞源性神经营养因子(glial cell derived neurotrophic factor,GDNF)属转化生长因子β超家族成员,其成熟蛋白由134个氨基酸残基组成,而GDNF受体广泛分布于外周和中枢神经系统。GDNF不仅可以促进多巴胺能神经元、运动神经元的存活,对交感、副交感以及感觉神经元具有营养作用,还能够影响神经元的发育、分化并对非神经系统的发育也具有重要作用。近年来随着人们对疼痛认识的深入,疼痛的机制也不再限于神经元功能的改变,还受胶质细胞活化、多种营养因子、细胞因子及相应受体、离子通道等多方面因素的影响。为此,本文就近年来GDNF参与疼痛调节的相关研究进展做一简要综述。     

蛋白激酶A介导慢性压迫背根节神经元的 肾上腺素能兴奋反应

本实验在背根节(DRG)慢性压迫模型上, 采用离体灌流DRG和单纤维记录神经元自发放电的方法研究了初级感觉神经元的交感-感觉耦联作用及其细胞内机制。 用外源性去甲肾上腺素(NE, 10 μmol/L)浸浴损伤的DRG时, 在95个DRG神经元中有85个有自发放电的神经元产生明显反应。 其中, 44个呈现单纯兴奋效应, 21个表现先兴奋后抑制效应, 6个出现兴奋-抑制交替振荡现象, 14个表现抑制效应。 NE对损伤神经元的兴奋作用可分别被哌唑嗪(5 μmol/L)和育亨宾(10 μmol/L)明显阻断。 蛋白激酶A(PKA)选择性抑制剂 Rp-cAMPS(50～250 μmol/L)和PKA催化亚单位抑制剂H-89(10 μmol/L)可以明显减弱NE的兴奋作用。 此外, 腺苷酸环化酶抑制剂SQ22, 536(1 mmol/L)亦明显减弱NE的兴奋作用。 结果显示 在DRG慢性压迫模型上, 损伤的DRG神经元存在肾上腺素能敏感性, α     

周围神经损伤后外源性GDNF对神经元的保护作用

采用硅管套接大鼠切断的坐骨神经模型 ,局部给予胶质细胞源性神经营养因子 (GDNF) ,应用尼氏染色、酶组织化学染色方法 ,观察到外源性GDNF能减少脊髓修复侧前角运动神经元死亡的数目 ,降低脊髓前角运动神经元及脊神经节感觉神经元中胆碱酯酶 (CHE)及酸性磷酸酶 (ACP)变化的幅度。这表明外源性GDNF能保护周围神经切断后引起的神经元损伤。     

胶质细胞原神经营养因子研究进展

叙述了新近纯化的胶质细胞源神经营养因子（GDNF）的生物功能及其在鼠胚中的分布,着重介绍了该因子对损伤的多巴胺能神经元及运动神经元促进存活、修复损伤及再生的活性.     

尼氟灭酸对γ-氨基丁酸激活DRG神经元电流的作用

目的:观察尼氟灭酸(NFA)对大鼠背根神经节(DRG)神经元γ-氨基丁酸(GABA)激活电流的调制作用。方法:在新鲜分离的大鼠DRG神经元,应用全细胞膜片钳技术记录NFA和GABA激活电流。结果:部分DRG神经元(21/48,43.75%)外加NFA(0.1～100μmol/L)能引起浓度依赖性的外向电流,而大多数DRG神经元(150/159,94.32%)外加GABA(0.1～100μmol/L)则引起明显的浓度依赖性的内相电流。NFA-(100μmol/L)和GABA-(100μmol/L)激活电流的幅值分别是(0.27±0.06)nA(n=12)和(1.29±0.72)nA(n=53)。然而,预使用NFA(0.1～100μmol/L)能明显的抑制GABAA受体介导的内向电流。NFA的这一抑制作用也具有明显的浓度依赖性。但NFA没有改变GABA激活内向电流的EC50(大约30μmol/L)和翻转电位(大约-10mV)(P>0.05)。结论:预加NFA对GABA激活电流的峰值有明显的浓度依赖性的抑制作用。     

GDNF对原代培养脊髓神经元的影响

本文研究了GDNF对体外培养各个时期的脊髓神经元的作用。通过MTT法检测GDNF对脊髓神经元存活率的影响,发现GDNF能促进培养7天及14天的神经元存活。 通过活体观察、尼氏染色、NSE免疫细胞化学染色观察GDNF对脊髓神经元生长锥数目、胞体大小、突起长度及分枝、侧棘形成的影响,发现GDNF对体外培养1—3周的脊髓神经元有明显的营养作用。     

Regenerating neurons

We have been studying the phosphorylation of proteins of both normal and regenerating superior cervical ganglia of the rat. Here we report the incorporation of radioactive phosphate into proteins of ganglia homogenates incubated with³²P-labeled ATP under various conditions at day 3 after postganglionic axotomy. The proteins were analyzed by two-dimensional electrophoresis followed by autoradiography. Incubation in the presence of Ca²⁺ or Ca²⁺ plus cyclic AMP produced only about 20 spots corresponding to distinctly labeled proteins. This number was reduced to about five under EGTA plus cyclic AMP conditions, whereas the presence of EGTA alone suppressed the phosphorylation reaction almost totally. All these proteins fell within the narrow pI range of 4–6, whereby no qualitative differences between regenerating and control cases were observed. However, the growth-associated protein, variously designated GAP-43, B-50, F-1, and pp-46, had enhanced levels of phosphate incorporation in regenerating ganglia compared to controls. Injury also caused consistently higher levels of phosphorylation of proteins running in the position of α- and β-tubulin. Since these three proteins are major constitutents of regenerating axons, these results suggest that the changes in their phosphorylation induced by injury may be involved in the regulation of their transport.     

Balanced neurons: analysis of leaky integrate-and-fire neurons with reversal potentials

 A new technique is presented for analyzing leaky integrate-and-fire neurons that incorporates reversal potentials, which impose a biologically realistic lower bound to the membrane potential. The time distribution of the synaptic inputs is modeled as a Poisson process. The analysis is carried out in the Gaussian approximation, which comparison with numerical simulations confirms is most accurate in the limit of a large number of inputs. The hypothesis that the observed variability in the spike times of cortical neurons is caused by a balance of excitatory and inhibitory synaptic inputs is supported by the results for the coefficient of variation of the interspike intervals. Its value decreases with both increasing numbers and amplitude of inputs, and is consistently lower than 1.0 over a wide range of realistic parameter values. The dependence of the output spike rate upon the rate, number, and amplitude of the synaptic inputs, as well as upon the value of the inhibitory reversal potential, is given. Received: 15 February 2001 / Accepted in revised form: 27 March 2001     

Lipids of isolated neurons

1. Lipids were extracted from neurons isolated from the lateral vestibular nucleus of ox (Bos taurus L.) and the ganglia of Aplysia punctata Cuvier. 2. Thin-layer chromatography of ox-neuron lipid revealed three major fractions corresponding to neutral lipid, phosphatidylethanolamine and phosphatidylserine. Part of the phosphatidylethanolamine was present as the plasmalogen. 3. Aplysia-neuron lipid contained neutral lipid, phosphatidylethanolamine and phosphatidylserine. Both phospholipids appeared to be present predominantly as the plasmalogen form. 4. The fatty acids of alkali-labile lipids of ox neurons were examined by gas–liquid chromatography. The major fatty acids were oleic acid, stearic acid and palmitic acid.