大鼠神经干细胞与"癫痫样细胞"体外共培养后的分化发育料

背景:在癫痫微环境神经干细胞能否被诱导分化为异常放电的"癫痫神经元"?癫痫微环境包括两种情况:一是"无镁"细胞外液,二是与癫痫细胞共培养.其中前者比后者的致癫痫作用强.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.设计:重复测量观察.单位:哈尔滨医科大学附属第一医院.材料:实验于2005-08/2007-04在哈尔滨医科大学病原学教研室及药理学教研室完成,选用150只新生Wistar大鼠,雌雄不拘,由哈尔滨医科大学附属第二医院实验动物中心提供,实验过程中对动物的处置符合动物伦理学标准.兔抗鼠突触素抗体购自美国LabVision公司.携带增强型绿色荧光蛋白标记基因的血清型2型腺相关病毒购自北京本元正阳公司.Axopatch 200B放大器为美国Axon公司产品.5111A示波器为美国Tektronix公司产品.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/5min兴奋性突触后电位;与"瘴痫神经元"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索抗体染色阳性.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".

Development and differentiation of neural stem cells co-cultured with epileptic neurons in vitro in rats

BACKGROUND: Can neural stem cells (NSCs) differentiate into "epileptic neurons" in epileptic microenvironment in vitro? Epileptic microenvironmcut includes magnesium-free media and co-culture with "epileptic neuron", the former is stronger than the latter to induce epilepsy.OBJECTIVE: To model the rnicroenvironment 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.SETTING: The First Affiliated Hospital of Harbin Medical University (Harbin, Heilongjiang Province, China).MATERIALS: Experiment was done in Department of Microorganism and Department of Pharmacology in Harbin Medical University from August 2005 to April 2007. A total of 150 neonatal Wistar rats, irrespective of genders, were applied by the Experimental Animal Center of the Second Affiliated Hospital of Harbin Medical University. All the procedures were in line with ethical standards of animal. Rabbit anti-rat synaptophysin antibody was purchased from Lab Vision Company (USA). Adeno-associated virus containing enhanced green fluorescent protein gone was applied by Beijing Vector Geue Technology Company., Ltd (China). Axopatch 200B magnification was.the product of Axon (USA). 5111A oscillograph was the product of Tektronix (USA).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 me, thods. 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; immunccytochemistry was used to demonswate 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.MAIN OUTCOME MEASURES: Postsynaptic potential and the expression of synaptophysin antibody of NSCs in co-cultore with "epileptic neuron" and normal neuron for 14 days. The postsynaptic potential and epileptic discharge 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-flee 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.