长春新碱对斑马鱼神经发育和行为的影响

Effects of vincristine on zebrafish neurodevelopment and behavior

目的以斑马鱼胚胎/幼鱼为实验对象,探索长春新碱对斑马鱼神经发育毒性和行为的影响及其毒性的分子机制。方法采用表型观察、触及逃避、幼鱼的自主运动、强光刺激惊恐反射能力等实验及RT-PCR,整体免疫荧光等手段。结果 (1)长春新碱具有斑马鱼胚胎神经和心脏发育毒性;(2)长春新碱具有幼体神经毒性,幼鱼呈浓度依赖性的侧卧体位;自主运动显示幼鱼呈长春新碱浓度依赖性的常规转弯能力下降,运动轨迹紊乱,平均运动速度降低,不活跃运动持续时间增加等神经抑制行为;(3)光刺激实验显示高剂量(30和40μg/ml)长春新碱处理组幼鱼被刺激后应激反应能力变弱,学习记忆能力下降;(4)长春新碱导致多巴胺能神经元相关基因th、dat的转录水平下调,诱导幼鱼间脑及下丘脑多巴胺神经元细胞数量减少。结论长春新碱具有多巴胺能神经元毒性,导致下丘脑多巴胺能神经元细胞丢失。

Objective To explore vincristine-induced neurodevelopmental toxicity and mechanisms in zebrafish embryos and larvae. Methods The neurodevelopment and behavior were assessed by investigating phenotype, touching escape, spontaneous movement, light stimulation panic reflex ability in vincristine-trcated zebrafish embryos and larvae. The expression of th/dat and distribution of TH protein were detected by RT-PCR and Whole Mount in Situ Immunofluorescence in zebrafish larvae. Results （1） Vincristine led toneurodevelopmental and cardiac developmental toxicity in zebrafish embryos; （2） Vincristine induced neurotoxicity in larvae. The neurobehavior of larvae was inhibited in a dose-dependent manner, such as larvae lateral position, ability of routine turn and average velocity decreased, turn trajectory disorders, inactive swimming duration increased; （3） Light stimulation experiments showed that light stress response and learning capability were reduced in high doses （30 and 40 μg/ml） vincristine-treated groups larvae; （4） RT-PCR results showed dopamine transporter gene （dat） and tyrosine hydroxylase gene （th） were inhibited in transcription levels, furthermore, dopaminergic neurons were reduced in larvae diencephalon and hypothalamic by vincristine. Conclusion Vincristine induced dopaminergic neurons toxicity and led to hypothalamic dopaminergic neurons loss.