斑马鱼侧线神经丘再生模型的构建

Establishment of Lateral Line Neuromast Regeneration Model in Zebrafish

目的建立斑马鱼侧线神经丘再生模型。方法通过碱性磷酸酶催化底物氯化硝基四氮唑兰(NBT)及5-溴-4-氯-3-吲哚基-磷酸盐(BCIP)反应显色方法观察受精后2~5 d斑马鱼侧线神经丘的发育状态;运用新霉素或硫酸铜对受精后2~5 d斑马鱼侧线神经丘进行杀伤并用NBT/BCIP显色分析杀伤情况及再生情况。结果斑马鱼侧线神经丘在受精后2 d出现,受精后2~4 d斑马鱼头部和躯干部位神经丘不断增加及长大;受精后2~3 d,斑马鱼侧线神经丘对新霉素或硫酸铜处理不敏感。受精后4 d,新霉素或硫酸铜可对斑马鱼头部及躯干部位神经丘进行杀伤,但新霉素杀伤效果较好;在神经丘杀伤后再生过程中,斑马鱼侧线神经丘经125μmol/L新霉素处理24 h后可完全恢复,但经250、500μmol/L新霉素处理后恢复所需时间较长;而25、50μmol/L硫酸铜处理后斑马鱼侧线神经丘的再生均较慢。结论斑马鱼侧线神经丘再生模型在受精后4 d被成功构建,此时利用新霉素或硫酸铜可对神经丘进行有效杀伤,经125μmol/L新霉素杀伤后斑马鱼侧线神经丘可迅速恢复。

Objective To establish the regeneration model for neuromasts in zebrafish lateral line neuromast.Methods The development of zebrafish lateral line neuromast was observed by using alkaline phosphatase staining method (NBT/BCIP staining) during 2~5 days.Neomycin or CuSO_(4) was used to delete the neuromasts at different stages during 2~5 days,and NBT/BCIP staining was used to analyze the regeneration of the neuromasts at different stages.Results The experiments showed that the neuromasts appeared on 2dpf (day post-fertilization),and from 2dpf to 4dpf,the size and the number of neuromasts clusters in the head and body were increased.From 2dpf to 3dpf,the neuromasts were insensitive to Neomycin or CuSO_(4) treatment.From 4dpf onwards,Neomycin or CuSO_(4) could effectively kill neuromasts in head and body,but the efficiency of Neomycin treatment was better.During the regeneration process,the neuromasts treated with 125μmol/L Neomycin could fully recover on 24 hours after killing,but the recovery time of the neuromasts was longer if the embryos were treated with 250 umol/L or 500 umol/L of Neomycin.In addition,compared with Neomycin treatment,the regeneration of the neuromasts treated with 25 umol/L or 50 umol/L of CuSO_(4) was slower.Conclusion The development of zebrafish neuromasts tends to be mature on 4dpf,and Neomycin or CuSO_(4) can effectively delete the neuromasts at this time.In addition,Neomycin is a good chemical for the deletion of the neuromasts,and the neuromasts can fully recover within 24 hours after chemical treatment.Based on this,we successfully established the regeneration model of neuromast in zebrafish.