Chakravorty Srabasti J, McGettrick Helen M, Butler Lynn M, Buckley Christopher D, Rainger G Ed, Nash Gerard B
Centre for Cardiovascular Sciences, The Medical School, The University of Birmingham, Birmingham, UK.
Biorheology. 2006;43(1):71-82.
To model the later stages of neutrophil migration into tissue, we developed an assay in which human umbilical vein endothelial cells (HUVEC) were cultured on porous filters, treated with the inflammatory cytokine tumour necrosis factor-alpha (TNF), and then incorporated in a flow chamber. Video-microscopic observations were made of neutrophils as they were perfused over the HUVEC. When 3 microm pore filters were used (as opposed to 0.4 microm pore filters), neutrophils could be observed to migrate not only through the endothelial monolayer but also through the filter within minutes. The proportion of adherent neutrophils migrating through the endothelial monolayer and velocity of migration underneath it, were similar on the different filters, and also when neutrophils were perfused over cultures in glass capillaries, or settled on HUVEC cultured in standard plastic dishes. However, neutrophils migrated through HUVEC/filter constructs more rapidly in the flow chamber than in a standard, static, Transwell system, even though the velocities of migration under HUVEC were similar when directly observed under flow or static conditions. A function-blocking antibody against CD31 did not alter movement through the endothelial monolayer or the filter in the new flow system, but did reduce the migration velocity of neutrophils underneath the HUVEC (by 24%). Thus, we have developed a method for following each stage of neutrophil migration, including exit from the sub-endothelial space, and shown how they may be modified by applied fluid shear stress and blockade of a regulatory adhesion molecule.
为了模拟中性粒细胞向组织迁移的后期阶段，我们开发了一种实验方法，即先将人脐静脉内皮细胞（HUVEC）培养在多孔滤膜上，用炎性细胞因子肿瘤坏死因子-α（TNF）处理，然后将其置于流动腔室中。当向流动腔室中灌注中性粒细胞时，利用视频显微镜对其进行观察。使用3微米孔径的滤膜（与0.4微米孔径的滤膜相对比）时，可观察到中性粒细胞不仅能在数分钟内穿过内皮单层，还能穿过滤膜。在不同滤膜上，以及当向玻璃毛细管中的培养物灌注中性粒细胞，或者将中性粒细胞置于标准塑料培养皿中培养的HUVEC上时，贴壁中性粒细胞穿过内皮单层的比例及其在内皮单层下方的迁移速度均相似。然而，尽管在流动或静态条件下直接观察时，中性粒细胞在HUVEC下方的迁移速度相似，但在流动腔室中，中性粒细胞穿过HUVEC/滤膜构建体的速度比在标准静态Transwell系统中更快。在新的流动系统中，抗CD31功能阻断抗体不会改变中性粒细胞穿过内皮单层或滤膜的运动，但会降低中性粒细胞在HUVEC下方的迁移速度（降低24%）。因此，我们开发了一种跟踪中性粒细胞迁移各个阶段的方法，包括从内皮下间隙穿出，并展示了它们如何受到施加的流体剪切应力和调节性粘附分子阻断的影响。
