以斑马鱼模型评价6种药物的神经毒性

Zebrafish Model for Assessing Neurotoxicity of Six Drugs

目的以斑马鱼为模式生物,评价6种已知对人类具有神经毒药物吗替麦考酚酯、异烟肼、环孢素A、卡马西平、苯妥英钠和乙醇对斑马鱼的神经毒性。方法挑选受精后5 d(5 dpf)的野生型AB系斑马鱼在含吗替麦考酚酯(4.5、15、45和51μmol/L)、异烟肼(1 460、4 866、14 599和15 328μmol/L)、卡马西平(17.5、58.3、175和187μmol/L)、苯妥英钠(16.7、55.7、167和185μmol/L)、乙醇(0.18%、0.63%、1.81%和2.0%)、环孢素A(5、16.7和50μmol/L)的养鱼用水中暴露,实验过程中不做换液处理,24 h后,利用斑马鱼行为分析仪检测斑马鱼的总运动距离,评价6种药物对斑马鱼运动活力的影响;受精后1 d(1 dpf)的运动神经元转基因斑马鱼胚胎在含吗替麦考酚酯(0.15、0.5、1.5和5.5μmol/L)、异烟肼(10 971、36 596、109 708和111 825μmol/L)、卡马西平(45.9、153、459和484μmol/L)、苯妥英钠(200、667和2000μmol/L)、乙醇(0.19%、0.62%、1.86%和1.98%)、环孢素A(5、16.7和50μmol/L)的养鱼用水中暴露,实验过程中不做换液处理,48 h后,在荧光显微镜下拍照,分析统计斑马鱼次级运动神经元轴突的长度,评价6种药物对斑马鱼运动神经元轴突生长的影响。结果吗替麦考酚酯、乙醇、卡马西平暴露后,随着浓度增加,斑马鱼运动距离先增加后减少,产生典型剂量依赖性倒"U"型运动;异烟肼和苯妥英钠暴露后,随着浓度增加,对斑马鱼运动活力抑制作用逐渐增强;环孢素A暴露后,随着浓度增加,斑马鱼产生兴奋性,斑马鱼总运动距离逐渐增加;6种药物作用后,斑马鱼运动神经元轴突长度均变短,表明6种药物在实验剂量下均能抑制运动神经元轴突的生长。结论斑马鱼模型可用于初步评价药物神经毒性。

Objective To explore the neurotoxicity of six kinds of known positive drugs,Mycophenolate Mofetil,Isoniazid,Ciclosporin A, Carbamazepine, Dilantin, Ethyl Alcohol, using Zebrafish embryos. Method We selected normally developed 5 dpf Wild-type AB line Zebrafish,and then Mycophenolate Mofetil（ 4. 5,15,45 and 51 μmol/L）,Isoniazid（ 1 460,4 866,14 599 and 15 328 μmol/L）,Carbamazepine（ 17. 5,58. 3、175 and187 μmol/L）,Dilantin（ 16. 7,55. 7,167 and 185 μmol/L）,Ethyl Alcohol（ 0. 18%,0. 63%,1. 81% and2. 0%） and Ciclosporin A（ 5,16. 7 and 50 μmol/L） were added to the artificial seawater. After 24 h,Zebrafish total movement distance was acquired by using the Zebralab larvae behavior video tracking system,and then 6 kinds of drug’s effect on Zebrafish movement dynamic was evaluated according to the research results. We selected normally developed 1 dpf motor neurons transgenic zebrafish,and then Mycophenolate Mofetil（ 0. 15、0. 5、1. 5 and5. 5 μmol/L）,Isoniazid（ 10 949、36 496、109 708 and 111 825 μmol/L）,Carbamazepine（ 45. 9、153、459 and484 μmol/L）,Dilantin（ 200、667 and 2 000 μmol/L）,Ethyl Alcohol（ 0. 19% 、0. 62% 、1. 86% and 1. 98%） and Ciclosporin A（ 5、16. 7 and 50 μmol/L） were added to the artificial seawater. After 48 h,Zebrafish ’s motor neuron axons length was acquired by analyzing motor neurons transgenic Zebrafish fluorescent images,and then 6kinds of drug’s effect on Zebrafish’s motor neuron axons growth was evaluated according to the Zebrafish’s axon length. Result After exposed by Mycophenolate Mofetil,Ethyl Alcohol and Carbamazepine,With the increase of concentration,Zebrafish movement distance increased after decreased,produce a typical dose dependent reverse＂U＂type; After exposed by Isoniazid and Dilantin,with the increase of concentration,the inhibiting effect on Zebrafish increased; After exposed by Ciclosporin A,with the increase of concentration,Zebrafish movement distance increased gradually,produce a excited effect; the inhibiting effect on Zebrafish increased; 6 kinds of drug all can inhibit the growth of motor neuron axons under the experimental doses. Conclusion Zebrafish model can be used to evaluate nerve toxicity of drugs preliminary.