Use of a monoclonal antibody specific for activated endothelial cells to quantitate angiogenesis in vivo in zebrafish after drug treatment.
Use of a monoclonal antibody specific for activated endothelial cells to quantitate angiogenesis in vivo in zebrafish after drug treatment.
复制标题
使用对活化内皮细胞具有特异性的单克隆抗体来定量药物治疗后斑马鱼体内的血管生成。
DOI：
10.1007/s10456-004-4181-7
复制
发表时间：
2004
期刊：
影响因子：
--
通讯作者：
McGrath,Patricia
中科院分区：
文献类型：
--
作者：
Seng,WenLin;Eng,Kurt;Lee,Jenny;McGrath,Patricia
我们最近产生了一种单克隆抗体（mAb），Phy-V002，它特异性地标记激活的血管内皮细胞（EC）在斑马鱼。在这里，我们表明这种mAb在不染色成熟血管或其他组织的情况下，在体内标记新形成血管中激活的EC。用此mAb，在透明胚胎中通过整体包埋免疫化学染色来视觉评估药物对体内EC迁移和血管形成的影响。此外，我们还开发了一种定量的基于微孔板的ELISA方法，用于测量药物治疗后EC在体内的增殖。我们已经使用几种具有不同作用机制的抗血管生成小分子直接加入到鱼水中，验证了定量体内ELISA。其中一些药物，包括：2-甲氧基紫杉醇，flavopiridol，紫杉醇和染料木素，目前正在临床试验中。我们还注射了大分子药物，包括3 TSR和TSR 2 +KRFK，这是一种天然蛋白质血小板反应蛋白-1的重组人抗血管生成肽。为了证明也可以在斑马鱼中评估促血管生成作用，我们评估了青霉胺和辛伐他汀的作用，这两种促血管生成化合物显示出刺激啮齿动物中的血管形成。使用Phy-V002的整体包埋免疫化学染色，目视观察每种化合物对EC迁移的抑制以及对血管形成的抑制或刺激。接下来，使用定量体内血管生成ELISA，我们生成了每种化合物的剂量反应曲线。与常规测定相比，使用斑马鱼评估药物对血管生成的作用的优点包括：（1）测定时间短;（2）易于动物维护;（3）使用少量药物;（4）单次给药;（5）定量测定形式;以及（6）每次测试使用统计学上显著数量的动物。
We have recently generated a monoclonal antibody (mAb), Phy-V002, which specifically labels activated vascular endothelial cells (EC) in zebrafish. Here, we show that this mAb labels activated EC in newly formed vesselsin vivowithout staining mature vessels or other tissues. Using this mAb, drug effects onin vivoEC migration and vessel formation were visually assessed by whole-mount immunochemical staining in the transparent embryo. In addition, we have developed a quantitative microplate-based ELISA that measures EC proliferationin vivoafter drug treatment. We have validated the quantitativein vivoELISA using several antiangiogenic small molecules with different mechanisms of action which were added directly to the fish water. Some of these drugs, including: 2-methoxyestradiol, flavopiridol, paclitaxel, and genistein, are currently in clinical trials. We also injected large molecule drugs, including 3TSR and TSR2+KRFK, recombinant human antiangiogenic peptides of thrombospondin-1, a natural protein. To demonstrate that proangiogenic effects can also be assessed in zebrafish, we assessed effects of penicillamine and simvastatin, two proangiogenic compounds shown to stimulate vessel formation in rodents. Using whole-mount immunochemical staining with Phy-V002, inhibition of EC migration and inhibition or stimulation of vessel formation were visually observed for each compound. Next, using the quantitativein vivoangiogenesis ELISA, we generated dose-response curves for each compound. Compared to conventional assays, advantages of using zebrafish to assess drug effects on angiogenesis include: (1) a short assay time; (2) easy animal maintenance; (3) use of small quantities of drug; (4) single dosing; (5) a quantitative assay format; and (6) use of statistically significant number of animals per test.