Navigate this Article


 

High Throughput Chemical Screens Using Zebrafish Embryos   

张倩倩张倩倩*陈漪陈漪*袁浩袁浩  (*contributed equally to this work)
How to cite Favorites Q&A Share your feedback Cited by

摘要:传统药物筛选大都是在分子和细胞水平进行,具有快速、灵敏度高、成本低等优点,但也有明显缺点,例如不能真实反应化合物在复杂生理环境下的生物学活性及安全性等。因此,在生理或病理水平下进行活体药物筛选的重要性与优势日益突显。斑马鱼作为一种新兴的脊椎动物模式生物,具有众多优势,如饲养成本低、产卵多、胚胎体外发育、早期透明、组织器官发育及分子调控机制与哺乳动物高度保守等(Haffter et al.,1996; Howe et al.,2013)。此外,许多人类疾病模型也已在斑马鱼中成功建立。因此,斑马鱼作为一种比较理想的活体药物筛选模型,将在新药研发中具有广阔的应用前景(MacRae and Peterson, 2015; Rennekamp and Peterson, 2015)。中性粒细胞是固有免疫系统中的主要效应细胞,在炎症、抗感染和免疫等方面发挥重要作用(Burn et al., 2021)。其功能和数量异常与人类许多重要疾病密切相关,例如中性粒细胞减少症或炎症性肠病等。我们以标记粒细胞的转基因斑马鱼系(Tg(mpx:EGFP))为模型,在胚胎发育过程中加入不同小分子化合物处理,通过活体-高通量、高内涵图像分析系统(Opera Phenix)来寻找影响中性粒细胞增殖、分化或凋亡的小分子化合物。

关键词: 斑马鱼, 高通量药物筛选, 中性粒细胞

材料与试剂

  1. CellCarrier-96 孔板(PerkinElmer, catalog number: 6055302),常温保存
  2. Pronase (Roche, catalog number: 11459643001),4 °C保存
  3. Tricaine (Sigma, catalog number: A5040),常温保存
  4. 亚甲基蓝 (Sigma, catalog number: 1592700010),常温保存
  5. FDA-approved Drug Library (Selleck, catalog number: L1300),-80 °C保存
  6. Pronase (500 mg/ml) (见溶液配方)
  7. 0.2%亚甲基蓝溶液 (见溶液配方)
  8. E3培养基 (见溶液配方)
  9. Tricaine (4mg/ml) (见溶液配方)

仪器设备

  1. 移液枪
  2. 生化培养箱 (Haier, model: HRSP-生化培养箱)
  3. 自动化液体工作站 (含微孔板振荡器) (PerkinElmer, model: JANUS液体工作站)
  4. 高内涵图像分析仪 (PerkinElmer, model: Opera Phenix)及配套分析软件Harmony4.1

实验步骤

  1. 收集受精后48h斑马鱼胚胎,Pronase (2mg/ml)处理5min,去掉胚胎绒毛膜(或人工使用镊子去掉绒毛膜);
  2. 将胚胎移入CellCarrier-96孔板中,每孔5枚胚胎,加入200 μl E3培养基;
  3. 通过液体工作站将待筛化合物加入上述96孔板中,微孔板振荡器震荡1 min,充分混匀;
  4. 胚胎放置28.5 °C培养箱中孵育24 h;
  5. 每孔加入2 μl Tricaine麻醉胚胎,微孔板振荡器震荡1 min;
  6. Opera Phenix高内涵图像分析仪进行扫描拍照,每孔一个视野,设置见表1:

    表1 Opera Phenix高内涵图像分析仪器参数设置


  7. 建立分析序列,计算荧光点数,面积,强度等。
    7.1
    选定分析区域:以EGFP通道为背景,采用“common threshold”方法,将整个视野图片作为分析区域;
    7.2
    选定荧光点:以EGFP通道为背景,选择“Method A”圈定分析区域中的荧光点(也可根据具体情况选择其他方法),并调整二级参数使选定的荧光点最符合实际情况;
    7.3
    计算上述分析区域中荧光点的强度均值;
    7.4
    设置输出结果,包括荧光点数,荧光点平均强度及面积;
    7.5
    在“Evaluation”中选择全部孔,计算所有孔的结果。

结果与分析

使用Opera Phenix机器完成每块96孔板扫描仅需3-5 min,大大缩短了筛选时间。并且,扫描结果通过Harmony4.1软件分析,能较真实反应实际情况。如图1所示,软件能够计算每孔胚胎中EGFP+细胞数量,荧光强度等参数,为化合物的作用效果提供参考。


1. Opera Phenix机器扫描结果

注意事项

  1. 实验操作过程中,勿触碰CellCarrier-96孔板底部,否则会干扰Opera Phenix机器扫描结果;
  2. 在Opera Phenix机器扫描之前应充分麻醉胚胎,避免在扫描过程中因胚胎游动而造成实验误差。

溶液配方

  1. Pronase (500 mg/ml): 称取25 g Pronase溶解于50 ml去离子水,并分装到1.5 ml离心管中,-20 °C保存。
  2. 0.2%亚甲基蓝溶液:称取0.1 g亚甲基蓝粉末溶解于50 ml去离子水。
  3. E3培养基:称取1.2 g海盐溶解于20 L去离子水,并加入2.5 ml的0.2%亚甲基蓝溶液。
  4. Tricaine (4mg/ml):称取2 g Tricaine溶解于100 ml去离子水,加入10 ml Tris-HCl(pH 9.5),再次加入去离子水至500 ml。

致谢

感谢国家自然科学基金(31371479)和广慈卓越青年培养计划对本工作的支持。

参考文献

  1. Burn, G.L., Foti, A., Marsman, G., Patel, D.F., Zychlinsky, A.(2021). The Neutrophil. Immunity 54(7):1377-1391.
  2. Haffter, P., Granato, M., Brand, M., Mullins, MC., Hammerschmidt, M., Kane, D.A., Odenthal, J., van Eeden, F.J., Jiang, Y.J., Heisenberg, C.P., Kelsh, R.N., Furutani-Seiki, M., Vogelsang, E., Beuchle, D., Schach, U., Fabian, C., Nüsslein-Volhard, C.(1996). The identification of genes with unique and essential functions in the development of the zebrafish, Danio rerio. Development 123: 1-36.
  3. Howe, K., Clark, M., Torroja, C.et al. (2013) .The zebrafish reference genome sequence and its relationship to the human genome. Nature 496, 498–503.
  4. MacRae, C.A., Peterson, R.T. (2015). Zebrafish as tools for drug discovery. Nat Rev Drug Discov14(10):721-731.
  5. Rennekamp, A.J., Peterson, R.T. (2015).15 years of zebrafish chemical screening. Curr Opin Chem Biol 24:58-70.
Please login or register for free to view full text
Copyright: © 2021 The Authors; exclusive licensee Bio-protocol LLC.
引用格式:张倩倩, 陈漪, 袁浩. (2021). 基于斑马鱼胚胎的活体高通量药物筛选. Bio-101: e1010858. DOI: 10.21769/BioProtoc.1010858.
How to cite: Zhang, Q.Q., Chen, Y. and Yuan, H. (2021). High Throughput Chemical Screens Using Zebrafish Embryos. Bio-101: e1010858. DOI: 10.21769/BioProtoc.1010858.
Q&A
By submitting a question/comment you agree to abide by our Terms of Service. If you find something abusive or that does not comply with our terms please contact us at eb@bio-protocol.org.

If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.

If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.