The zebrafish/tumor xenograft angiogenesis assay
The zebrafish/tumor xenograft angiogenesis assay
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DOI：
10.1038/nprot.2007.412
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发表时间：
2007-01-01
期刊：
影响因子：
14.8
通讯作者：
Presta, Marco
中科院分区：
文献类型：
--
作者：
Nicoli, Stefania;Presta, Marco
在这里，我们描述了一种研究斑马鱼(Danio Rerio)肿瘤血管生成的方法，该方法是在受精后48小时将促血管生成的哺乳动物肿瘤细胞注射到斑马鱼胚胎的卵黄周间隙中。在24-48小时内，原血管生成的肿瘤移植物诱导来自发育中的肠下血管的新生血管反应。对野生型斑马鱼胚胎进行整体碱性磷酸酶染色后，可以在宏观和微观水平上观察到这一现象，或者在VEGFR2：G-RCFP转基因胚胎中，内皮细胞在VEGFR2/KDR启动子控制下表达绿色荧光蛋白的转基因胚胎中可以通过荧光显微镜观察到这一现象。添加到注射细胞悬浮液或鱼水中的血管生成抑制剂可防止肿瘤诱导的新生血管形成。该方法快速、廉价，是研究肿瘤血管生成和抗血管生成药物开发的新工具。此外，在斑马鱼胚胎中注射反义吗啉寡核苷酸使基因失活，可能有助于识别与肿瘤血管生成有关的基因。
Here we describe a method to study tumor angiogenesis in zebrafish ( Danio rerio) based on the injection of proangiogenic mammalian tumor cells into the perivitelline space of zebrafish embryos at 48 h post-fertilization. Within 24-48 h, proangiogenic tumor grafts induce a neovascular response originating from the developing subintestinal vessels. This can be observed at macroscopic and microscopic levels after whole-mount alkaline phosphatase staining of wild-type zebrafish embryos, or by fluorescence microscopy in transgenic VEGFR2:G-RCFP embryos in which endothelial cells express the green fluorescent protein under the control of the VEGFR2/KDR promoter. Angiogenesis inhibitors added to the injected cell suspension or to the fish water prevent tumor-induced neovascularization. The assay is rapid and inexpensive, representing a novel tool for investigating tumor angiogenesis and for antiangiogenic drug discovery. Also, gene inactivation by antisense morpholino oligonucleotides injection in zebrafish embryos may allow the identification of genes involved in tumor angiogenesis.