Preparation of aniti-tumor tissue endothelium antibodies and its application of cancer-missle therapy

用小鼠黑色素瘤B16制备的条件培养液培养牛主动脉内皮细胞(BAEC)5天后，FITC标记的大分子右旋糖苷(Mw=70,000)对BAEC的通透性增加。B16条件培养液(B16-CM)处理BAEC 30min后，通透性无明显增加。B16-CM还能增加牛静脉和人脐静脉内皮单分子层的通透性，但不增加上皮单分子层的通透性。B16-CM不改变BAEC上F-肌动蛋白的分布或含量。在B16-CM存在下培养5天的BAEC从培养皿中分离出来，然后以融合细胞密度的五分之一接种到培养箱中。在正常培养液中培养5d后，BAEC生长为融合，通透性增加。这些结果表明B16-CM通过…不可逆转地增加内皮细胞的通透性。不仅F-肌动蛋白的减少，而且肿瘤细胞分泌的可溶性因子(S)参与了体内肿瘤血管高通透性结构的构建。采用Percoll梯度离心法结合贴壁快速分离技术分离大鼠KMT-17纤维肉瘤来源的内皮细胞，并对其培养特性进行检测。原代培养的肿瘤源性内皮细胞(TEC)显示血管紧张素转换酶活性，第八因子相关抗原染色阳性，Matrigel上有典型的毛细血管样形成。原代培养的TEC单层对FITC-葡聚糖扩散(相对分子质量70,000)的通透性高于正常组织源内皮细胞(主动脉、腔静脉和附睾脂毛细血管)单层。与脂肪来源的毛细血管内皮细胞相比，白细胞与TEC的粘附性降低。这些特征与肿瘤血管内皮细胞相似，在原代和第一代细胞培养中均可观察到，但在第四代细胞培养中未观察到。我们的研究结果表明，原代或传代培养的TEC适用于肿瘤内皮细胞的生理特性研究。较少

The permeation of macromolecular FITC-labeled dextran (Mw=70,000) through bovine aortic endothelial cells (BAEC) monolayr, which were cultured for 5 days with conditioned medium prepared from mouse melanoma B16, was increased. However, when BAEC,which were cultured with normal medium until confluent, were treated with B16 conditioned medium (B16-CM) for 30 min, the permeability did not increase. The B16-CM also increased the permeability of the endothelial monolayrs of bovine veins and the human umbilical vein, but did not increase that of the epithelial monolayr. The B16-CM did not alter the distribution or content of F-actin on the BAEC.BAEC cultured in the presence of B16-CM for 5 days were detached from the dish, and then seeded into a chamber at one-fifth of confluent cell density. After 5 days of culture in nomal medium, the BAEC were grown to confluence and their permeability was increased. These findings suggest that B16-CM increased the endothelial permeability irreversibly wi … More thout the decrease of F-actin, and that soluble factor (s) which were secreted from the tumor cells participate in the construction of the hyperpermeable structure of tumor vessels in vivo.Rat KMT-17 fibrosarcoma-derived endothelial cells were isolated by Percoll gradient centrifugation with an attaching-speed separation technique, and their properties in culture were examined. The primary cultured tumor-derived endothelial cells (TEC) showed angiotensin-converting enzyme activity, positivity for Factor VIII-related antigen staining, and typical capillary-like formation on Matrigel. The primary cultured TEC monolayr showed greater permeability than normal tissue-derived endothelial cell (aorta, vena cava and epididymal fat capillary) monolayrs on FITC-dextran diffusion (molecular weight 70,000). Leukocyte adhesion to TEC was reduced compared to that to fat-derived capillary endothelial cells. These characteristics resembled those of tumor vascular endothelium, and were observed both in the primary and first-passage cell cultures, but not in the fourth-passage cell cultures. Our findings indicate that primary or subcultured TEC are applicable for studies of the physiological characteristics of tumor endothelial cells. Less