Quantitative single-cell motility analysis of platelet-rich plasma-treated endothelial cells in vitro.
Quantitative single-cell motility analysis of platelet-rich plasma-treated endothelial cells in vitro.
复制标题
体外富含血小板血浆处理的内皮细胞的定量单细胞运动分析。
作者：
Kawase T;Tanaka T;Okuda K;Tsuchimochi M;Oda M;Hara T.
富血小板血浆（PRP）因其具有高浓度的生长因子而在再生治疗中得到广泛应用。以前的体外和体内研究已经提供了支持PRP血管生成活性的证据。为了更直接地证明PRP是如何作用于内皮细胞的，我们通过检测VEGF的参与来检测PRP诱导的人脐静脉内皮细胞运动性的变化。延时定量成像显示，在伤口愈合试验中，在治疗的初始阶段（~ 2小时），PRP实质性地刺激了细胞迁移。然而，在初始阶段之后，PRP的这种效果并没有持续到显著水平。在对照组和PRP刺激的细胞中，10 h细胞迁移的平均净距离分别为0.45±0.16 mm和0.82±0.23 mm。这种效应在重组人VEGF中也得到证实，并被中和性抗VEGF抗体显著减弱。免疫荧光检测paxillin和肌动蛋白纤维显示PRP同时上调局灶黏附和细胞骨架的形成。磷酸化VEGFR2的Western blotting分析表明，PRP主要以剂量和时间依赖的方式刺激未成熟VEGFR2的磷酸化，这一作用被中和抗体完全阻断。综上所述，这些数据表明PRP通过激活VEGFR2直接作用于内皮细胞，短暂上调其运动。因此，在设计PRP成分的控释系统时，应考虑PRP在短期激活后对靶内皮细胞脱敏时间较长的可能性。©2015 Wiley期刊公司
Platelet‐rich plasma (PRP) has been widely applied in regenerative therapy due to its high concentration of growth factors. Previousin vitroandin vivostudies have provided evidence supporting the angiogenic activity of PRP. To more directly demonstrate how PRP acts on endothelial cells, we examined the PRP‐induced changes in the motility of human umbilical vein endothelial cells by examining the involvement of VEGF. Time‐lapse quantitative imaging demonstrated that in the initial phase (∼2 h) of treatment, PRP substantially stimulated cell migration in a wound‐healing assay. However, this effect of PRP was not sustained at significant levels beyond the initial phase. The average net distance of cell migration at 10 h was 0.45 ± 0.16 mm and 0.82 ± 0.23 mm in control and PRP‐stimulated cells, respectively. This effect was also demonstrated with recombinant human VEGF and was significantly attenuated by a neutralizing anti‐VEGF antibody. Immunofluorescent examination of paxillin and actin fibers demonstrated that PRP concomitantly up‐regulated focal adhesion and cytoskeletal formation. Western blotting analysis of phosphorylated VEGFR2 demonstrated that PRP mainly stimulated the phosphorylation of immature VEGFR2 in a dose‐ and time‐dependent manner, an action that was completely blocked by the neutralizing antibody. Taken together, these data suggest that PRP acts directly on endothelial cells via the activation of VEGFR2 to transiently up‐regulate their motility. Thus, the possibility that PRP desensitizes target endothelial cells for a relatively long period of time after short‐term activation should be considered when the controlled release system of PRP components is designed. © 2015 Wiley Periodicals, Inc.