HUMAN VASCULAR ENDOTHELIAL CELLS IN CULTURE
HUMAN VASCULAR ENDOTHELIAL CELLS IN CULTURE
复制标题
培养中的人类血管内皮细胞
作者：
M. Gimbrone;R. Cotran;J. Folkman
用胶原酶处理足月脐静脉获得的人脐静脉内皮细胞，在添加20%胎牛血清的199培养液中培养。小的细胞簇最初散布在塑料或玻璃上，7天后结合并生长形成融合的多边形细胞单层。原代和传代培养的细胞经电子显微镜鉴定为内皮细胞。对一些原代培养的内皮细胞进行选择性传代培养，并根据超微结构标准鉴定为血管平滑肌。内皮细胞经[~3H]胸腺嘧啶核苷作用后的放射自显影显示，在生长中的培养细胞中，标记指数从24小时开始逐渐增加。在最近融合的培养中，标记指数在中央密集区域为2.4%，在外围生长区域为53.2%。融合后3d，中心区和周边区标记均匀，分别为3.5%和3.9%。当融合培养的小区域被实验性地“剥离”时，[~3H]胸腺嘧啶核苷标记的局部性增加和最终单层的重建。微孔板原代和继代培养的内皮细胞[~3H]胸腺嘧啶核苷掺入的液体闪烁测量显示，DNA合成与细胞密度的相关性相似。这些数据表明，内皮细胞培养可能为研究内皮再生的病理生理因素提供了一种有用的体外模型。
Human endothelial cells, obtained by collagenase treatment of term umbilical cord veins, were cultured using Medium 199 supplemented with 20% fetal calf serum. Small clusters of cells initially spread on plastic or glass, coalesced and grew to form confluent monolayers of polygonal cells by 7 days. Cells in primary and subcultures were identified as endothelium by the presence of Weibel-Palade bodies by electron microscopy. A morphologically distinct subpopulation of cells contaminating some primary endothelial cultures was selectively subcultured, and identified by ultrastructural criteria as vascular smooth muscle. Autoradiography of endothelial cells after exposure to [3H]thymidine showed progressive increases in labeling in growing cultures beginning at 24 h. In recently confluent cultures, labeling indices were 2.4% in central closely packed regions, and 53.2% in peripheral growing regions. 3 days after confluence, labeling was uniform, being 3.5 and 3.9% in central and peripheral areas, respectively. When small areas of confluent cultures were experimentally "denuded," there were localized increases in [3H]thymidine labeling and eventual reconstitution of the monolayer. Liquid scintillation measurements of [3H]thymidine incorporation in primary and secondary endothelial cultures in microwell trays showed a similar correlation of DNA synthesis with cell density. These data indicate that endothelial cell cultures may provide a useful in vitro model for studying pathophysiologic factors in endothelial regeneration.