Brown Melissa A, Wallace Charles S, Anamelechi Charles C, Clermont Edward, Reichert William M, Truskey George A
Department of Biomedical Engineering, Duke University, 136 Hudson Hall, Campus Box 90281, Durham, NC 27708-0281, USA.
Biomaterials. 2007 Sep;28(27):3928-35. doi: 10.1016/j.biomaterials.2007.05.009. Epub 2007 May 25.
A necessary condition for endothelialization of small diameter grafts is rapid and firm adhesion of endothelial cells upon exposure to flow. To retain integrins on the cell surface, we assessed the effects of trypsin concentration, the duration of trypsin incubation, and trypsin neutralization methods on endothelial cell adhesion. Human umbilical vein endothelial cells which were detached using 0.025% trypsin for 5 min and seeded onto glass pretreated with fibronectin had close to 100% cell retention when shear stresses as high as 200 dyn/cm2 were applied for 2 min. An equivalent level of cell retention was observed on fibronectin coated Teflon-AF for shear stresses up to 60 dyn/cm2 applied for 4h. Using 0.025% trypsin, initial cell spreading and cell surface alpha5beta1 integrins were increased relative to cells treated with 0.5% trypsin. After 1h of attachment, focal adhesions formed when low trypsin concentrations were used but were less evident with high trypsin concentrations. These results showed that low trypsin concentrations produced faster spreading, a higher number of intact integrins, and rapid focal adhesion formation.
小口径移植物内皮化的一个必要条件是内皮细胞在接触血流时能快速且牢固地黏附。为了使整合素保留在细胞表面，我们评估了胰蛋白酶浓度、胰蛋白酶孵育时间以及胰蛋白酶中和方法对内皮细胞黏附的影响。使用0.025%胰蛋白酶处理5分钟后分离得到的人脐静脉内皮细胞，接种到用纤连蛋白预处理的玻璃上，当施加高达200达因/平方厘米的剪切应力2分钟时，细胞保留率接近100%。在纤连蛋白包被的特氟龙 - AF上，当施加高达60达因/平方厘米的剪切应力4小时时，观察到了同等水平的细胞保留率。与用0.5%胰蛋白酶处理的细胞相比，使用0.025%胰蛋白酶处理后，细胞的初始铺展和细胞表面α5β1整合素增加。在附着1小时后，使用低浓度胰蛋白酶时会形成粘着斑，但高浓度胰蛋白酶处理时粘着斑不太明显。这些结果表明，低浓度胰蛋白酶能使细胞更快铺展、产生更多完整的整合素并快速形成粘着斑。
