Using Zebrafish for high-throughput screening of drugs regulating neutrophil migration by live imaging in vivo
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目的 建立半自动化高通量药物筛选系统，探索影响中性粒细胞迁移能力的药物，并探究药物对中性粒细胞免疫能力的作用，以期高效寻找可以用于治疗炎症和自身免疫性疾病的新型药物。 方法 应用荧光延时追踪技术记录斑马鱼卵黄囊内中性粒细胞的迁移，结合TrackMate软件建立半自动化检测与G蛋白偶联受体作用相关的不同药物处理后对中性粒细胞速度影响情况，从而进行高通量药物筛选，通过三轮重复独立筛选试验得到影响中性粒细胞迁移能力的药物；结合斑马鱼尾鳍横断实验及大肠杆菌感染实验进一步探讨该药物对中性粒细胞免疫反应能力的作用。 结果 建立了可靠、稳定、高效的半自动化高通量药物筛选系统，通过系统筛选得到丁香酚能够促进中性粒细胞迁移能力(0.08 μm/s比0.14 μm/s，t＝6.01，P<0.05)，氯化腾喜龙则抑制其迁移能力(0.08 μm/s比0.05 μm/s，t＝3.93，P<0.05)。斑马鱼尾尾鳍横断损伤和斑马鱼耳蜗内大肠杆菌感染实验表明，丁香酚可抑制损伤部位中性粒细胞的趋化迁移(1、2、3 h的t值分别为1.65、1.45、1.36，P值均<0.05)，而氯化腾喜龙在损伤/感染情况下对中性粒细胞免疫能力的影响差异无统计学意义(P>0.05)。 结论 高通量药物筛选系统可用于开发调控中性粒细胞迁移相关作用药物的研究，进而发现可用于治疗炎症和自身免疫性疾病相关的新型药物，或可以用于其他免疫相关疾病的研究和筛选任务。 Objective To establish a semi-automated high-throughput drug screening system to explore drugs that affect neutrophil migration ability, and to investigate the effects of drugs on neutrophil immune function, with the aim of efficiently identifying novel drugs for the treatment of inflammation and autoimmune diseases. Methods Taking advantage of the fluorescence time-lapse tracking technology to record the migration of neutrophils on the yolk sac of Zebrafish, a semi-automated high-throughput drug screening system for detecting of neutrophils velocity related to the action of G protein-coupled receptors was established by TrackMate cell tracking software. The drugs affecting the migration ability of neutrophils were obtained through 3 repeated independent screening tests. Zebrafish tail transection and Ecoli: RFP bacteria infection experi ments were conducted to further explore the effects of these drugs on neutrophil immune response. Results The reliable, stable and efficient semi-automated high-throughput drug screening system was established. Through the screening process, Eugenol was found to promote neutrophil migration (0.08 μm/s vs 0.14 μm/s, t=6.01, P<0.05), while Edrophonium chloride inhibits neutrophil migration (0.08 μm/s vs 0.05 μm/s,t=3.93, P<0.05). Zebrafish tail fin transection andEcoli: RFP infection experiments showed that Eugenol inhibited the chemotactic migration of neutrophils in the case of infection(t1h , 2h, 3h values are 1.65, 1.45, 1.36, respectively, all P values <0.05), while edrophonium chloride did not exhibit significant statistical differences under injury/infection conditions. Conclusion The high-throughput drug screening system can be used to develop drugs that regulate neutrophil migration and discover novel drugs for the treatment of inflammation and autoimmune diseases, or other immune-related disease research and screening tasks.
Objective To establish a semi-automated high-throughput drug screening system to explore drugs that affect neutrophil migration ability, and to investigate the effects of drugs on neutrophil immune function, with the aim of efficiently identifying novel drugs for the treatment of inflammation and autoimmune diseases. Methods Taking advantage of the fluorescence time-lapse tracking technology to record the migration of neutrophils on the yolk sac of Zebrafish, a semi-automated high-throughput drug screening system for detecting of neutrophils velocity related to the action of G protein-coupled receptors was established by TrackMate cell tracking software. The drugs affecting the migration ability of neutrophils were obtained through 3 repeated independent screening tests. Zebrafish tail transection and Ecoli: RFP bacteria infection experi ments were conducted to further explore the effects of these drugs on neutrophil immune response. Results The reliable, stable and efficient semi-automated high-throughput drug screening system was established. Through the screening process, Eugenol was found to promote neutrophil migration (0.08 μm/s vs 0.14 μm/s, t=6.01, P<0.05), while Edrophonium chloride inhibits neutrophil migration (0.08 μm/s vs 0.05 μm/s,t=3.93, P<0.05). Zebrafish tail fin transection andEcoli: RFP infection experiments showed that Eugenol inhibited the chemotactic migration of neutrophils in the case of infection(t1h , 2h, 3h values are 1.65, 1.45, 1.36, respectively, all P values <0.05), while edrophonium chloride did not exhibit significant statistical differences under injury/infection conditions. Conclusion The high-throughput drug screening system can be used to develop drugs that regulate neutrophil migration and discover novel drugs for the treatment of inflammation and autoimmune diseases, or other immune-related disease research and screening tasks.
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