1-苯基-2-硫脲抑制flk1型斑马鱼黑色素生成的最佳浓度

Optimal Concentration of 1-Phenyl 2-thiourea for Inhibiting Melanogenesis in flk1 Zebrafish

【目的】探讨1-苯基-2-硫脲（PTU）对抑制血管荧光转基因型flk1斑马鱼黑色素生成的能力,探索在不影响斑马鱼正常生长发育条件下,能保持斑马鱼透明性的最佳浓度。【方法】用0.0125～1.0 mmol/L浓度区间的PTU与24 hpf（hours post fertilization）斑马鱼胚胎共同孵育,观测PTU抑制黑色素生成情况,通过观察斑马鱼形态学变化、记录80 hpf后的孵化率和死亡率、测量心率和静脉窦-动脉球间距等生理学变化,评价PTU处理对斑马鱼生长发育的影响。【结果】随着PTU浓度的增加,黑色素抑制能力增强,毒性逐渐增加,表现为斑马鱼死亡率增加、孵化率下降、心率下降、静脉窦-动脉球之间的距离增大。浓度为0.0125-0.025 mmol/L的PTU对斑马鱼活性基本无影响,浓度≥0.05 mmol/L的PTU能完全抑制黑色素的生成。当PTU浓度为0.025 mmol/L时,生成的黑色素不影响斑马鱼血管观察,当PTU浓度为0.05 mmol/L时,斑马鱼的心率和静脉窦-动脉球间距基本不受影响。【结论】对于72 hpf前的flk1型斑马鱼实验,应根据具体实验要求,在浓度≤0.05 mmol/L范围内选择适当的PTU浓度,使黑色素的生成既不影响实验观察和信号检测,同时对生长发育的影响减至最小。

[Objective] To investigate the ability of 1-phenyl 2-thiourea （PTU） to inhibit melanogenesis in flkl zebrafish, and to explore the optimal concentration of PTU for generating transparent zebrafish without affecting their growth and development. [Methods] Zebrafish embryos at 24 hpf （hours post fertilization） were incubated with 0.0125-1.0 mmol/L PTU for some time. Then at designated time points, the effects of PTU with different concentrations on the melanogenesis, the growth and development of zebrafish embryos were evaluated through the changes in melanin area, the morphology and physiology at 80 hpf including the mortality, hatching rate, heart rate and SV-BA distance of zebrafish embryos. [Results] With increasing concentrations of PTU, the inhibition on melanogenesis enhances with an increase in the toxicity, which is characterized by the increased mortality and SV-BA distance, the reduced hatching rate and heart rate, 0.0125-0.025 mmol/L PTU has no impact on zebrafish, and PTU at a concentration higher than 0.05 mmol/L can completely suppress melanogenesis in zebrafish. When treated with 0.025 mmol/L of PTU, the detection of zebrafish vessel was not affected by generated melanin. And 0.05 mmol/L of PTU has little impact on the heart rate and SV-BA distance of zebrafish. [Conclusion] For flkl zebrafish experiment, before 72 hpf, the concentration of PTU is suggested to be less than 0.05 mmol/L. And then in this concentration range, such a PTU concentration is optimal, that is the melanogenesis does not affect the viviperception and signal detection required by a specific experiment, while the influence on the development and growth of zebrafish is minimum.