斑马鱼幼体癫痫模型的建立及应用  

Establish and use of an epilepsy model in larval zebrafish

作　　者：郑仰民[1] 张靖溥[1] 唐胜[1] 宋丹青[1] 

机构地区：[1]中国医学科学院北京协和医学院医药生物技术研究所,北京100050

出　　处：《药学学报》2016年第4期580-587,共8页Acta Pharmaceutica Sinica

基　　金：十二五"重大新药创制"科技重大专项课题-药物毒理研究(2012ZX09301-002-001-021);国家自然科学基金面上项目资助(81173043);"创新研究群体科学基金"项目资助(81321004)

摘　　要：本研究用戊四氮(pentylenetetrazole,PTZ)方法建立了一种斑马鱼幼体癫痫模型。结果显示,PTZ可导致斑马鱼幼体癫痫样异常兴奋行为,如游动速度加快、抽搐等异常现象;癫痫标志基因c-fos信号在斑马鱼幼体脑区增强并范围扩大,而lgi1基因表达则被抑制。这些表型与癫痫临床病例和文献报道相符。给予癫痫治疗药丙戊酸钠(VPA)可使斑马鱼癫痫症状减轻或消失:异常兴奋游动、c-fos和lgi1基因表达,以及神经核蛋白Neu N均恢复到近正常组水平。利用所建模型,检测了两种先导化合物Y53和BMT(均为小檗碱结构类似物)的抗癫痫作用,Y53抑制光刺激下的发作更有效;BMT对无刺激的发作有较好的抑制作用。综上,所建斑马鱼癫痫模型对于化合物有无刺激因素条件下的差异作用也具有较好的辨识力,可作为抗癫痫先导化合物的简便实用的药效筛选平台及用于癫痫发病机制研究。Epilepsy is a kind of neurogenic diseases with high prevalence and characterized by seizure, brain paradoxical discharge and convulsion in spontaneous, transient, recurrent and uncontrolled manner. Development of new anti-epilepsy drugs requires a new reliable and high-performance animal models in screening of leading compounds. In this study, an epilepsy model in larval zebrafish was established using pentylenetetrazole（PTZ） compound. The results show that PTZ induced epilepsy-like seizure behavior such as irregular circular swimming, exciting locomotion, high swim velocity and convulsion in zebrafish. Expression patterns of two epilepsy-related gene c-fos and lgi1 were analyzed using RT-PCR and in situ hybridization; c-fos was enhanced and extended and lgi1 expression was reduced in PTZ concentration-dependent in the larval brain. When the model larvae exposed to anticonvulsant valproate（VPA）, the epilepsy-like symptom decreased or disappeared, the marker genes c-fos and lgi1, as well as Neu N protein recovered to the normal levels. These responses to PTZ and to antiepileptic drug VPA are consistent with the observations in clinical studies and mouse models. Using this model, we evaluated anti-epilepsy activity of compounds Y53 and BMT, two homolog of berberine. The results show that the model larvae seizure triggered by lighting was partly remedied by Y53; and the larval exciting locomotion under the condition of no stimulation was suppressed by BMT. The findings indicate that the zebrafish larval epilepsy model is able to distinguish compounds with different activities in eleptiform seizure. We conclude that the zebrafish epilepsy model may be as a reliable and useful platform in screening of new anti-epilepsy candidates, which is suitable for basic research in epilepsy pathogenesis.

关 键 词：癫痫 斑马鱼幼体模型 戊四氮 丙戊酸钠 c-fos LGI1 标志基因 兴奋性运动 

分 类 号：R965[医药卫生—药理学] R966[医药卫生—药学]