Ito Y, Iwamoto Y, Tanaka K, Okuyama K, Sugioka Y
Department of Orthopaedic Surgery, Faculty of Medicine, Kyushu University, Fukuoka City, Japan.
Int J Cancer. 1996 Jul 3;67(1):148-52. doi: 10.1002/(SICI)1097-0215(19960703)67:1<148::AID-IJC24>3.0.CO;2-9.
We describe a quantitative assay for assessing tumor angiogenesis in vivo using basement membrane extracts (Matrigel). Nude mice were injected s.c. with liquid Matrigel mixed with HT1080 human fibrosarcoma cells. Since Matrigel rapidly forms a solid gel at body temperature, the gel containing tumor cells can be removed immediately and then processed for histological studies. Tumor angiogenesis was monitored quantitatively by measuring both the number and the total area of neovessels present in the gels using an image analyzer, which could be achieved approximately 72 hr later. Furthermore, HT1080 cell-conditioned medium, which may contain various tumor-derived factors, promoted the basement membrane degradation, migration, proliferation and tube formation of endothelial cells in vitro, as did Matrigel, although to a lesser extent. In addition, Northern blot analysis demonstrated that the amount of vascular endothelial growth factor (VEGF) mRNA in HT1080 cells was much higher than that in human fibroblasts or NIH3T3 cells. Our results suggest that angiogenesis observed in our assay may be due to the synergic effects of tumor angiogenic factors such as VEGF, and Matrigel. The advantages of our assay are: 1) it is possible to assess early angiogenesis quantitatively; and 2) this assay may be applicable for screening anti-angiogenic therapeutic agents to be used against human neoplasms.
我们描述了一种使用基底膜提取物（基质胶）在体内评估肿瘤血管生成的定量测定方法。将裸鼠皮下注射与HT1080人纤维肉瘤细胞混合的液态基质胶。由于基质胶在体温下迅速形成固体凝胶，含有肿瘤细胞的凝胶可立即取出，然后进行组织学研究。使用图像分析仪通过测量凝胶中存在的新血管数量和总面积来定量监测肿瘤血管生成，这大约在72小时后可以实现。此外，可能含有各种肿瘤衍生因子的HT1080细胞条件培养基，与基质胶一样，促进了体外内皮细胞的基底膜降解、迁移、增殖和管形成，尽管程度较小。此外，Northern印迹分析表明，HT1080细胞中血管内皮生长因子（VEGF）mRNA的量远高于人成纤维细胞或NIH3T3细胞。我们的结果表明，在我们的测定中观察到的血管生成可能是由于VEGF等肿瘤血管生成因子与基质胶的协同作用。我们测定方法的优点是：1）可以定量评估早期血管生成；2）该测定方法可能适用于筛选用于治疗人类肿瘤的抗血管生成治疗药物。
