胶质细胞源性神经营养因子对培养的背根神经节的影响

目的 探讨胶质细胞源性神经营养因子(GDNF)在体外促进正常胚胎大鼠背根神经节(DRGn)的存活及突起生长情况。方法 用原代分离培养法建立体外胚胎大鼠背根神经节单细胞培养体系,通过活体观察、MTT微量比色法、NSE免疫组织化学染色观察不同浓度GDNF对体外培养的正常感觉神经元的影响。结果 GDNF组培养的DRG神经元存活数量增加,神经元突起的长度比对照组明显增长。结论 GDNF能明显促进体外培养的正常大鼠胚胎背根神经节感觉神经元的存活及突起生长,表明GDNF对正常大鼠胚胎发育期感觉神经元具有神经营养作用。     

神经再生素促大鼠背根神经节生长作用的研究

目的研究神经再生素（NRF）对体外培养新生大鼠背根神经节生长及NF—H表达的影响。方法体外大鼠背根神经节（DRG）培养;免疫荧光细胞化学、实时荧光定量PCR和Western blot等方法。结果免疫荧光细胞化学结果提示,NRF能促进背根神经节神经突起的生长,浓度为2．0mg／L时生长状况最佳;实时荧光定量PCR和Western blot结果提示,NRF能增加体外培养的背根神经节NF-H mRNA和蛋白的表达,在浓度为2．0mg／L时表达最高。结论NRF能促进体外培养背根神经节神经突起的生长和NF—H的表达,表明NRF对发育期感觉神经元具有神经营养作用。     

MEF2C调控大鼠脊髓背根节感觉神经元P物质和低分子量神经丝微管蛋白的表达

目的:研究肌细胞增强因子2C(MEF2C)在大鼠脊髓背根神经节神经元细胞内的表达及其与P物质和低分子量神经丝微管蛋白合成的关系。方法:分离培养背根神经节神经元细胞,然后暴露于不同浓度的神经生长因子下24h,最后用实时聚合酶链反应技术检测P物质与低分子量神经丝微管蛋白基因在背根神经节神经元细胞内的表达。通过化学转染方法将合成的3条siRNA-MEF2C分别转染PC12细胞株,并用实时聚合酶链反应技术筛选干扰效率最高的siRNA。采用化学转染方法干扰背根神经节神经元细胞内MEF2C的表达,在高浓度神经生长因子刺激背根神经节神经元细胞后采用实时聚合酶链反应技术检测干扰后背根神经节神经元细胞内P物质与低分子量神经丝微管蛋白基因的表达。结果:P物质及低分子量神经丝微管蛋白基因表达随刺激用神经生长因子浓度的升高而升高。使用化学转染方法成功地干扰背根神经节神经元细胞内MEF2C的表达,MEF2C较对照组下降52%,同时没有检测到对cAMP反应元件结合蛋白表达的影响。实验组背根神经节神经元细胞内P物质在RNA水平较对照组下降了39%,而低分子量神经丝微管蛋白在RNA水平较对照组下降了62%。结论:神经生长因子促进大鼠脊髓背根节感觉神经元内P物质与低分子量神经丝微管蛋白的合成。大鼠背根神经节神经元细胞内P物质及低分子量神经丝微管蛋白基因表达受MEF2C调控。     

神经再生素促大鼠背根神经节神经突起生长显微图像的分形特征

目的:研究分维数与神经再生素(nerve regeneration factor,NRF)剂量促离体培养新生大鼠背根神经节(dorsal root ganglions,DRGs)神经突起生长之间的关系。方法:采用计盒维数方法,计算在不同浓度NRF条件下大鼠DRGs荧光显微图像的分维数。结果:离体培养大鼠DRGs显微图像的分维数随神经突起数量和长度的变化呈现一致的变化规律。结论:分维数可以作为一个重要定量指标表征大鼠DRGs神经元生长与NRF浓度的依赖关系。     

星形胶质细胞和神经生长因子对新生大鼠脑内P物质受体阳性神经元存活与突起生长的影响──体外培养研究

本实验用无血清培养进行神经元与星形胶质细胞的联合培养，观察了星形胶质细胞和神经生长因子对新生大鼠基底神经节和下位脑干内的P物质受体阳性神经元存活与生长的影响。结果表明：（1）含神经生长因子的NeurobasalMedium／N2supplement（N／N2）能支持新生鼠脑神经元的存活和神经元突起的生长，其效果与加血清的Dulbacco改良Eagle培养基（s－DMEM）相似；（2）利用星形胶质细胞作为滋养细胞，种植新生鼠基底神经节和下位脑干细胞于其上，可见星形胶质细胞对神经元的突起生长（生长锥）有明显的促进作用，其中基底神经节神经元神经突的生长优于下位脑干神经元；（3）免疫组织化学反应结果显示，来源于基底神经节的培养神经元有87％为P物质受体阳性神经元，而下位脑干为84％，P物质受体阳性神经元的胞体形态完整，突起生长良好。本实验表明含神经生长因子的N／N2Medium或s－DMEM可作为P物质受体神经元体外培养的培养基，星形胶质细胞可在一定程度上支持新生大鼠脑P物质受体神经元的存活与突起生长，并可保持其分化特性。     

远志对糖尿病周围神经病变大鼠坐骨神经相关神经元胞体凋亡的影响

目的:探讨远志对糖尿病周围神经病变(DPN)大鼠坐骨神经相关神经元胞体的保护作用。方法:雄性Wistar大鼠随机分为空白对照组、DPN模型组和远志治疗组,DPN模型组和远志治疗组均建立DPN模型。待建模后,远志治疗组大鼠给予远志灌胃6周。尼氏染色法观察背根节神经元胞体形态,免疫组织化学显色检测背根节神经元胞体Bcl-2、Bax蛋白的表达,TUNEL法检测背根节神经元凋亡指数。结果:与空白对照组比较,DPN模型组大鼠背根节神经元胞体形态异常,Bcl-2表达降低,Bax表达升高,凋亡指数升高;与DPN模型组比较,远志治疗组大鼠背根节神经元胞体形态接近正常,Bcl-2表达升高,Bax表达降低,凋亡指数降低。结论:远志可通过上调DPN大鼠背根节神经元胞体Bcl-2的表达,减少Bax的表达,抑制背根节神经元凋亡,发挥对DPN大鼠背根节神经元胞体的保护作用。     

维生素D依赖性钙结合蛋白-D25K样阳性神经元在人胎儿和新生儿脊髓和背根神经节内的表达和分布

目的：观察24～27w人胎儿和新生儿的脊髓和背根神经节(DRG)内维生素D依赖性钙结合蛋白-D28K(CB)样阳性神经元的表达和分布。方法：采用免疫细胞化学ABC法对24～27w人胎儿和新生儿进行观察。结果：(1)CB样阳性产物的表达在胎儿DRG和脊髓灰质的神经元胞体及树突内可观察到。在脊髓各段有不同分布；(2)新生儿脊髓和DRG内CB样阳性胞体的数量、大小及染色强度均有所增加，但在空间分布上无大的变化。结论：人胎儿的脊髓和DRG在发育的24～27w至出生时，CB样阳性神经元的表达在定位分布上无差异，但随年龄增长，CB样阳性神经元的数量、大小和染色强度均有所增加。     

维生素D依赖性钙结合蛋白-D28K样阳性神经元在人胎儿和新生儿脊髓和背根神经节内的表达和分布

目的 :观察 2 4～ 2 7w人胎儿和新生儿的脊髓和背根神经节 (DRG)内维生素D依赖性钙结合蛋白 D2 8K(CB)样阳性神经元的表达和分布。方法 :采用免疫细胞化学ABC法对 2 4～ 2 7w人胎儿和新生儿进行观察。结果 :( 1 )CB样阳性产物的表达在胎儿DRG和脊髓灰质的神经元胞体及树突内可观察到。在脊髓各段有不同分布 ;( 2 )新生儿脊髓和DRG内CB样阳性胞体的数量、大小及染色强度均有所增加 ,但在空间分布上无大的变化。结论 :人胎儿的脊髓和DRG在发育的 2 4～ 2 7w至出生时 ,CB样阳性神经元的表达在定位分布上无差异 ,但随年龄增长 ,CB样阳性神经元的数量、大小和染色强度均有所增加。     

二脱氧肌苷引发培养的背根神经节神经元的形态学改变

为探讨二脱氧肌苷(ddI)对培养的背根神经节(DRG)神经元的影响,我们对分散培养的胎鼠DRG神经元培养3d后,再分别以不同浓度的ddI(1μg/ml,5μg/ml,10μg/mll和20μg/ml)孵育3d。终止培养后,行微管相关蛋白2(MAP2)标记,用共聚焦激光扫描显微镜观察神经元胞体和突起的改变。结果表明,DRG神经元用ddI孵育3d,神经元突起的数目减少和长度变短,呈剂量依赖性,而神经元的直径则没有变化。本研究的结果表明,ddI可影响培养的DRG神经元突起的再生和生长。     

细胞外基质对培养神经元细胞存活和轴突生长的影响

目的：探讨不同的细胞外基质对体外培养胚胎大鼠脊髓运动神经元和背根神经节感觉神经元生长的影响。方法：取大鼠胚胎脊髓腹侧组织和背根神经节体外分离培养，选取不同生长底物包括多聚赖氮酸(PLL)、Ⅰ型胶原(CoⅠ)、层粘连蛋白(LN)、PLL联合LN进行包被培养板，观察运动神经元和感觉神经元的体外生长状况。结果：PLL和LN联合包被时，神经元存活率高，细胞分散良好。CoⅠ包被时细胞聚集成团．突起粗长。结论：不同的细胞外基质影响神经元的生长方式，PLL联合LN包被是体外研究单一神经元胞体和突起的较好方法。     

Functional involvement of synaptotagmin I/II C2A domain in neurite outgrowth of chick dorsal root ganglion neuron

Synaptotagmin I or II (Syt I/II) is involved in Ca²⁺-regulated exocytosis of secretory vesicles, probably serving as a Ca²⁺-sensor via its C2A domain. Synaptotagmin is also known to be expressed in neuronal growth cone vesicles, but its functional involvement in neurite outgrowth remains largely unknown. In this study, we examined the function of Syt I/II in neurite outgrowth in cultured chick dorsal root ganglion neurons using an anti-synaptotagmin I and II C2A domain (anti-STI/II-C2A) antibody that inhibits Ca²⁺-regulated exocytosis. Immunoblots confirmed the high specificity of the anti-STI/II-C2A antibody and showed the expression of synaptotagmin I or II in chick dorsal root ganglion neurons. Immunocytochemistry revealed that synaptotagmin I or II is enriched at the growth cone region of chick dorsal root ganglion neurons, in both lamellipodia and filopodia. Whole or Fab-fragment of the anti-STI/II-C2A antibody loaded into dorsal root ganglion neurons by trituration significantly inhibited neurite outgrowth, whereas preimmune IgG had no effect. These results showed that the C2A domain of synaptotagmin I or II plays a crucial role in neurite outgrowth.     

Regulation of neurite outgrowth mediated by neuronal calcium sensor-1 and inositol 1,4,5-trisphosphate receptor in nerve growth cones

Calcium acts as an important second messenger in the intracellular signal pathways in a variety of cell functions. Strictly controlled intracellular calcium is required for proper neurite outgrowth of developing neurons. However, the molecular mechanisms of this process are still largely unknown. Neuronal calcium sensor-1 (NCS-1) is a high-affinity and low-capacity calcium binding protein, which is specifically expressed in the nervous system. NCS-1 was distributed throughout the entire region of growth cones located at a distal tip of neurite in cultured chick dorsal root ganglion neurons. In the central domain of the growth cone, however, NCS-1 was distributed in a clustered specific pattern and co-localized with the type 1 inositol 1,4,5-trisphosphate receptor (InsP₃R1). The pharmacological inhibition of InsP₃ receptors decreased the clustered specific distribution of NCS-1 in the growth cones and inhibited neurite outgrowth but did not change the growth cone morphology. The acute and localized loss of NCS-1 function in the growth cone induced by chromophore-assisted laser inactivation (CALI) resulted in the growth arrest of neurites and lamellipodial and filopodial retractions. These findings suggest that NCS-1 is involved in the regulation of both neurite outgrowth and growth cone morphology. In addition, NCS-1 is functionally linked to InsP₃R1, which may play an important role in the regulation of neurite outgrowth.     

RNA干扰CRMP 5抑制大鼠海马神经元突起生长

目的探讨CRMP5对大鼠海马神经元突起生长的影响。方法将带FAM标记的CRMP5的特异性干扰片段及阴性对照转染培养成熟的海马神经元,用免疫荧光的方法验证干扰片段对神经元内源性CRMP5的干扰效果,并利用共聚焦显微镜观察神经元突起以及侧枝的形成。结果携带FAM的si RNA可以成功的进入细胞,分布于神经元的胞体以及树突;免疫荧光证实CRMP5 si RNA可以有效的沉默CRMP5蛋白的表达;沉默CRMP5基因表达后的海马神经元突起短小,而且缺少分支,而对照细胞突起长,分支多;定量分析显示,导入CRMP5 si RNA的细胞突起的长度较对照细胞缩短,差异显著(P0.05);突起的数目比较,一级突起数目无显著差异,而二级及其以上突起的数目明显减少,差异显著(P0.05)。结论沉默CRMP5可抑制海马神经元突起的生长和侧枝形成。     

Distinct and overlapping alterations in motor and sensory neurons in a mouse model of spinal muscular atrophy

Motor neuron degeneration is the predominant pathological feature of spinal muscular atrophy (SMA). In patients with severe forms of the disease, additional sensory abnormalities have been reported. However, it is not clear whether the loss of sensory neurons is a common feature in severe forms of the disease, how many neurons are lost and how loss of sensory neurons compares with motor neuron degeneration. We have analysed dorsal root ganglionic sensory neurons in Smn-/-;SMN2 mice, a model of type I SMA. In contrast to lumbar motor neurons, no loss of sensory neurons in the L5 dorsal root ganglia is found at post-natal days 3-5 when these mice are severely paralyzed and die from motor defects. Survival of cultured sensory neurons in the presence of NGF and other neurotrophic factors is not reduced in comparison to wild-type controls. However, isolated sensory neurons have shorter neurites and smaller growth cones, and beta-actin protein and beta-actin mRNA are reduced in sensory neurite terminals. In footpads of Smn-deficient mouse embryos, sensory nerve terminals are smaller, suggesting that Smn deficiency reduces neurite outgrowth during embryogenesis. These data indicate that pathological alterations in severe forms of SMA are not restricted to motor neurons, but the defects in the sensory neurons are milder than those in the motor neurons.     

Synergistic effects of physical and chemical guidance cues on neurite alignment and outgrowth on biodegradable polymer substrates

This article demonstrates that directional outgrowth of neurites is promoted by applying a combination of physical and chemical cues to biodegradable polymer substrates. Films of poly-D,L-lactic acid and poly(lactide-co-glycolide) were micropatterned to form grooves on substrate surfaces, using novel indirect transfer techniques developed specifically for biodegradable polymers that cannot be micropatterned directly. Laminin was selectively adsorbed in the grooves. Whole and dissociated dorsal root ganglia were seeded on the substrates and neurite outgrowth and alignment along the microgrooves were measured. The microgrooves provide physical guidance, whereas laminin provides chemical cues to the neurons. The groove depth and spacing were found to significantly influence neurite alignment. The presence of laminin was found to promote neurite adhesion and outgrowth along the grooves. Using a combination of optimized physical and chemical cues, excellent spatial control of directional neurite outgrowth, with up to 95% alignment of neurites, was obtained. The synergistic effect of physical and chemical guidance cues was found to be more effective than individual cues in promoting directional outgrowth of neurites.     

Conditioned medium promotes neurite growth from both central and peripheral neurons

Explants or dissociated neurons of spinal cord and other parts from chick embryos and rat fetuses were cultured for 1–4 days. Extensive outgrowth of neurites from the spinal cord was observed in the medium which had been conditioned by skeletal and heart muscle cells. In the fresh medium, the neurites were short and accompanied by the migrating non-neuronal cells. The conditioned medium promoted also the neurite outgrowth from retina, optic tectum, ciliary ganglion, superior cervical ganglion and dorsal root ganglion.     

体外培养海马神经元生长过程中Nogo-A分布的变化

目的 探讨Nogo-A在神经元中的表达与神经元发育分化的关系,以推测Nogo-A在中枢神经系统发育分化过程中的意义。方法 采用怀孕18d大鼠胚胎海马神经元体外高密度和低密度培养方法,应用免疫荧光细胞化学染色和Western blot,观察Nogo-A在体外培养的大鼠海马神经元的分布模式及其在不同生长阶段的变化。结果 Nogo-A在海马神经元生长过程中均表达,主要分布在胞浆、胞膜和突起上。神经元突起形成过程中,Nogo-A主要表达于突起的近端,在轴突上随着轴突的伸长逐渐表达于轴突远端和生长锥。Nogo-A在成熟神经元网状的突起上呈串珠样分布。结论 Nogo-A在中枢神经系统中具有不同于抑制作用的其他功能,可能参与神经元突起生长、轴突投射等过程。     

Synergistic effects of corticosterone and kainic acid on neurite outgrowth in axotomized dorsal root ganglion

Corticosterone is the main adrenal glucocorticoids induced by stress in rats. Therapeutic use of high concentration of synthetic glucocorticoids in clinical treatment of spinal cord injury suggests that pharmacological action of glucocorticoids might be beneficial for nerve repair. In this article we cultured axotomized rat dorsal root ganglion neurons to investigate the effects of corticosterone and a glutamate receptor agonist kainic acid on neurite outgrowth. Our results revealed a synergistic effect of corticosterone and kainic acid in promoting neurite outgrowth when applied as early as one and two days in vitro, but not effective at three and four days in vitro. In addition, applied corticosterone and kainic acid were neurotoxic at three and four days in vitro but not at one and two days in vitro. The minimal concentrations of corticosterone and kainic acid to be effective were 10 microM and 1 mM, respectively. The neurotrophic effect of corticosterone and kainic acid was attenuated by the receptor tyrosine kinase A (TrkA) inhibitor AG-879. Western blot analysis and immunocytochemical studies revealed an increase of expressions of both TrkA and growth-associated protein GAP-43 in dorsal root ganglion neurons with combined treatment of corticosterone and kainic acid. Immunocytochemistry showed that corticosterone+kainic acid increase nerve growth factor immunoreactivity in dorsal root ganglion neurites and enhance GAP-43 immunointensity in dorsal root ganglion neurons. These results suggest that the neurotrophic effect of glucocorticoids on axonal regeneration might require facilitation of excitatory stimulation at an early stage of nerve injury, and nerve growth factor may mediate a growth signaling to accomplish the effect.     

PKAc促进成年大鼠背根神经节神经元突起生长及其机制的研究

观察慢病毒载体介导PKA催化亚单位PKAc转染促进成年大鼠原代培养的背根神经节(DRG)神经元突起生长及其机制的研究。我们运用三质粒系统共转染293T细胞合成慢病毒载体LV/PKAc-IRES-GFP和对照病毒LV/GFP,原代分离培养成年大鼠DRG神经元,应用免疫组织化学染色和Western blot等方法检测cAMP/PKA信号通路下游关键转录因子cAMP反应元件连接蛋白(CREB)磷酸化水平,图像分析经组织化学染色后的神经元突起长度和有突起神经元的百分比。结果观察到,慢病毒能介导外源基因在哺乳动物原代培养的神经元中表达,转染LV/PKAc-IRES-GFP的PC12细胞和DRG神经元均能表达外源基因并激活CREB,克服CNS髓鞘蛋白的抑制,促进突起生长。以上实验结果表明拯救成年大鼠神经元cAMP/PKA水平可有效改变神经元内在生长能力,从而改变它们对抑制环境的敏感性,进而促进突起生长。     

Optimal Micropattern Dimensions Enhance Neurite Outgrowth Rates,Lengths, and Orientations

Micropattern dimensions can significantly influence neurite outgrowth orientation, rate, and length. Laminin micropatterns of various widths from 10 to 50 μm at 10 μm intervals separated by 40 μm spaces were generated on poly(methyl methacrylate) surfaces using microscale plasma-initiated patterning (μPIP). Dissociated dorsal root ganglion (DRG) neurons were seeded on the micropatterned surfaces and cultured for 24 h in serum-free media. Neurite outgrowth numbers, lengths, rates, and orientations were measured on all micropatterned substrates. The results indicated that the dimension of the laminin pattern influenced the neurite outgrowth length, rate, and orientation, but not the numbers of neurite outgrowth. Neurons on more than 30 μm wide laminin pattern showed faster neurite outgrowth compared to other dimensions, and relatively low orientation at 50 μm pattern dimensions. Neurites at 40 μm laminin pattern widths demonstrated the fastest outgrowth rates and were highly oriented. The 40 μm laminin dimension is wide enough to provide sufficient laminin amounts for neuron growth and narrow enough to efficiently guide neurites. Based on these results, adhesive protein micropatterns of 40 μm dimensions are recommended when investigating DRG neurons.