Rat embryonic motoneurons in long-term co-culture with Schwann cells - a system to investigate motoneuron diseases on a cellular level in vitro
Rat embryonic motoneurons in long-term co-culture with Schwann cells - a system to investigate motoneuron diseases on a cellular level in vitro
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DOI：
10.1016/j.jneumeth.2004.09.003
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发表时间：
2005-03-30
影响因子：
3
通讯作者：
Claus, P
中科院分区：
文献类型：
--
作者：
Haastert, K;Grosskreutz, J;Claus, P
在细胞和分子水平上研究运动神经元疾病需要长期培养原代细胞。在这里，我们提出了一个新的培养系统，其中成熟的运动神经元与它们的生理伙伴，如中间神经元，星形胶质细胞和外周胶质细胞相互作用。这使得运动神经元成熟长达3周，而运动神经元始终达到30-45 μ m，偶尔超过80 μ m的胞体的大直径。分离的大鼠胚胎腹侧脊髓细胞通过密度梯度离心富集运动神经元，并接种在高度富集的新生大鼠雪旺细胞的非融合单层上。分别对所有神经元中的神经元特异性β III-微管蛋白和运动神经元特异性非磷酸化神经丝H/M进行免疫细胞化学显像，结果显示在体外3天后，>70%的所有神经元是运动神经元。在体外培养20天后，运动神经元分数保持在12%。当脂质体转染和增强剂物质相结合时，运动神经元对绿色荧光蛋白cDNA的瞬时转染敏感。突触连接能够形成自发活动的神经元网络，这为在分子水平上研究谷氨酸兴奋毒性和钙调节失调提供了培养模型。这两种机制都隐含在肌萎缩侧索硬化症的病理生理学中，这是一种神经退行性运动神经元疾病。(C)2004 Elsevier B. V.保留所有权利。
Investigations of motoneuron diseases on a cellular and molecular level require long-term cultivation of primary cells. Here we present a new culture system in which matured motoneurons interact with their physiological partners like interneurons, astroglia and peripheral glia cells. This enables motoneuron-maturation for up to 3 weeks, while motoneurons consistently reached large diameters of their somata of 30-45 mum, occasionally more than 80 mum. Dissociated rat embryonic ventral spinal cord cells were enriched for motoneurons by density gradient centrifugation and seeded on a non-confluent mono-layer of highly enriched neonatal rat Schwann cells. Immunocytochemical visualization of neuron specific betaIII-tubulin in all neurons and of motoneuron specific non-phosphorylated neurofilament H/M, respectively, revealed that after 3 days in vitro >70% of all neurons were motoneurons. After 20 days in vitro, a motoneuron fraction of 12% was maintained. Motoneurons were susceptible to transient transfection with green fluorescent protein cDNA when liposomal transfection and an enhancer substance were combined. Synaptic connections enabled formation of spontaneously active neuronal networks which provide a culture model to study glutamate excitotoxicity and calcium deregulation on a molecular level. Both mechanisms are implied in the pathophysiology of amyotrophic lateral sclerosis, a neurodegenerative motoneuron disorder. (C) 2004 Elsevier B.V. All rights reserved.