论文部分内容阅读
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。Axopatch200B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14d。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14d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“癫痫神经元”共培养后记录到12次/5min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/5min时程约10s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”。
Background: In epilepsy microenvironment neural stem cells can be induced to differentiate into abnormally discharged “epileptic neurons”. The epileptic microenvironment includes two conditions: one is “magnesium-free” extracellular fluid, and the other is associated with epilepsy cells Co-culture. The former is more epileptic than the latter. OBJECTIVE: To model the epileptic microenvironment in vivo and to co-culture rat hippocampal neural stem cells, normal hippocampal neurons and epileptic neurons in vitro to observe the differentiation and development of stem cells. Design: Repeat measurement observation. Unit: First Affiliated Hospital of Harbin Medical University. MATERIALS: The experiment was performed at Department of Pathology and Department of Pharmacology, Harbin Medical University from August 2005 to April 2007 with 150 neonatal Wistar rats, both male and female, provided by Experimental Animal Center of the Second Affiliated Hospital of Harbin Medical University. The experimental process In the disposal of animals in line with animal ethical standards. Rabbit anti-mouse synaptophysin antibody was purchased from the United States LabVision company. Serotype 2 adeno-associated virus carrying enhanced green fluorescent protein marker gene was purchased from Beijing BenYuan Zhengyang Company. Axopatch200B amplifier for the United States Axon products. 5111A oscilloscope for the United States Tektronix company products. Methods: ① The hippocampal neurons were isolated and the “epileptic neurons” model was established by using “MgO free” external fluid to process neurons. Conventional methods were used to culture rat hippocampal neural stem cells, and green fluorescent protein-labeled neural stem cells were co-cultured with normal hippocampal neurons and epileptic neurons for 14 days respectively. ② Patch clamp was used to record the postsynaptic potential of the cells co-cultured with the two neurons. The immunostaining was used to detect the synaptophysin antibody staining of neural stem cells. The neurons differentiated from neural stem cells were placed in the “MgO free” , The use of patch clamp record of its postsynaptic potential. MAIN OUTCOME MEASURES: ①Synapse potential and synaptophysin staining after co-cultured with hippocampal neural stem cells and two neurons for 14 days. Differentiated neurons in the “magnesium-free ” extra-synaptic potential and “epileptiform discharge ” situation. RESULTS: ① After co-culture of neural stem cells and normal hippocampal neurons, 60% (6/10) neural stem cells were excised for 14 times / 5 minutes after patch-clamp. After co-cultured with “epileptic neurons” Record 12 times / 5min excitatory postsynaptic potential. ② After co-culture of neural stem cells with normal hippocampal neurons and epileptic neurons respectively, immunofluorescence showed that 80% (12/15) green fluorescent protein-positive stem cell synaptophysin staining was positive. ③60% (9/15) of the differentiated neurons in the “magnesium-free” extra-aqueous 14 times / 5min duration of about 10s excitatory postsynaptic potential, not recorded “epileptiform discharge”. Conclusion: Rat hippocampal neural stem cells can form functional synapses after co-cultured with “epileptic neurons” in vitro, but not into “epileptic neurons”.