Development and differentiation of neural stem cells co-cultured with epileptic neurons in vitro in rats
首发时间:2011-02-06
Abstract:OBJECTIVE: To model the microenvironment in vivo, and to co-culture the NSCs with normal hippocampal neuron and "epileptic neuron" for the observation of NSCs development. DESIGN: Repeated measurement and observation. METHODS: Rat hippocampal neurons were isolated and cultured, magnesium-free media treatment was applied to establish the model of "epileptic neuron". NSCs were cultured according to regular methods. After labeled by green fluorescence protein, NSCs were co-cultured with normal hippocampal neuron or "epileptic neuron" for 14 days, respectively. Patch clamp was used to record the electrophysiology of NSCs in co-culture; immunocytochemistry was used to demonstrate the expression of synaptophysin antibody of NSCs; patch clamp was also used to record the postsynaptic potential of the neurons differentiated from NSCs in magnesium-free medium. RESULTS: After NSCs was co-cultured with normal hippocampal neuron, 14 beats/5 minutes excitatory postsynaptic potential was recorded in 60% NSCs (6/10) by patch clamp; After co-culture with "epileptic neuron", 12 beats/5 minutes excitatory postsynaptic potential of NSCs was recorded. Immunocytochemistry revealed that 80% NSCs (12/15) was observed to express the synaptophysin in co-culture with normal neuron or "epileptic neuron". In magnesium-free medium, 14 beats/5 minutes excitatory postsynaptic potential with a duration of about 10 seconds was found in 60% neurons differentiated from NSCs (9/15), and no epileptic discharge was recorded. CONCLUSION: Rat hippocampal NSCs can form functional synapse in the co-culture with "epileptic neuron" in vitro. The possibility that NSCs develop into "epileptic neuron" is minimal.
keywords: neural stem cells epileptic co-culture vitro
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大鼠神经干细胞与“癫痫样细胞”体外共培养后的分化发育
摘要:目的:模型模拟体内癫痫微环境,将大鼠海马神经干细胞和正常海马神经元以及“癫痫神经元”体外共培养,观察干细胞的分化发育情况。 方法:①分离大鼠海马神经元,采用“无镁”外液处理神经元建立“癫痫神经元”模型。常规方法培养大鼠海马神经干细胞,将绿色荧光蛋白标记的神经干细胞分别与正常海马神经元、“癫痫神经元”共培养14 d。②应用膜片钳记录与两种神经元共培养后细胞突触后电位;利用免疫荧光检测神经干细胞突触素抗体染色情况;将神经干细胞分化的神经元放入“无镁”外液,应用膜片钳记录其突触后电位。主要观察指标:①海马神经干细胞与两种神经元共培养14 d 后突触后电位、突触素抗体染色结果。②分化后神经元在“无镁”外液中突触后电位及“癫痫样放电”情况。 结果:①神经干细胞与正常海马神经元共培养后,膜片钳记录到60%(6/10)神经干细胞14 次/5 min 兴奋性突触后电位;与“癫痫神经元”共培养后记录到12 次/5 min 兴奋性突触后电位。②神经干细胞分别与正常海马神经元及“癫痫神经元”共培养后,免疫荧光检测均显示80%(12/15)表达绿色荧光蛋白的干细胞突触素抗体染色阳性。③60%(9/15)干细胞分化的神经元在“无镁”外液中出现14 次/5 min 时程约10 s 的兴奋性突触后电位,未记录到“癫痫样放电”。 结论: 大鼠海马神经干细胞与“癫痫神经元”体外共培养后可形成功能性突触,未转变成“癫痫神经元”。
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