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目的 探讨人主动脉内皮细胞 (HAEC)韦伯潘力氏小体 (Webel Paladebody ,WPB)释放的信号传导机制。方法 假设小G蛋白Rac1和活性氧自由基 (ROS)介导内皮细胞WPB的释放。通过使用腺病毒介导的Rac1基因的充分表达 ,观察HAEC产生和清除ROS的过程 ,以及对WPB释放的影响。结果 凝血酶 (1单位 ,30min)介导WBP释放增加 (较基础值增加了 3倍 )的同时 ,使Rac GTP结合活性及ROS分别增加了 3 5倍和 2 4倍。与对照组比较 ,阴性突变的腺病毒Rac N17转染使凝血酶处理和未处理组HAEC中WPB的释放分别减少了 77%和 6 4 % ,使ROS分别降低了 83 6 %和6 5 5 %。而且 ,抗氧化剂过氧化氢酶和N 乙酰半胱氨酸分别使WPB的释放较对照组下降了 34%和79 %。同时 ,证实Rac1位于ROS调节WPB释放途径的上游。结论 小G蛋白Rac1介导HAEC凝血酶诱导的WPB的释放。WPB的释放机制是Rac1依赖的ROS调节过程。
Objective To investigate the signal transduction mechanism of Webel Paladebody (WPB) in human aortic endothelial cells (HAEC). Methods We hypothesized that the small G protein Rac1 and reactive oxygen species (ROS) mediate the release of WPB from endothelial cells. By using adenovirus-mediated expression of the Rac1 gene, we observed the process of HAEC production and scavenging ROS and the effect on WPB release. Results Thrombin (1 unit, 30 min) mediated an increase in WBP release (3-fold more than basal) and increased Rac GTP binding activity and ROS by 35- and 24-fold, respectively. Compared with the control group, transfection with the negative mutant adenovirus Rac N17 reduced the release of WPB by 77% and 64%, respectively, in the HAECs of the thrombin treated and untreated groups, and decreased ROS by 83 6% and 655%, respectively . Moreover, the antioxidant catalase and N-acetylcysteine decreased the release of WPB by 34% and 79%, respectively, from the control. At the same time, it was confirmed that Rac1 is located upstream of the ROS-regulated WPB release pathway. Conclusion The small G protein Rac1 mediates HAEC thrombin-induced release of WPB. The release mechanism of WPB is Rac1-dependent ROS regulation.