中文摘要：

背景：外源性刺激引起血管屏障功能损伤的分子机制是血管病理生理学尚未阐明的热点问题之一。目的：探讨炎症递质脂多糖诱导的人脐静脉内皮细胞单层通透性改变的效应分子,寻找有效治疗靶点。方法：应用脂多糖刺激并观察人脐静脉内皮细胞骨架蛋白F-actin和细胞单层通透性的改变。应用荧光免疫组化和Westernblot方法检测脂多糖刺激前后细胞中RhoA和SRF等信号分子的改变。并通过阻断实验证实RhoA-SRF信号通路的作用。结果与结论：100μg/L脂多糖刺激6h可引起人脐静脉内皮细胞中F-actin快速重构并形成大量应力纤维,细胞单层通透性明显增强。细胞中活化RhoA的表达明显增加,SRF发生明显的入核转位现象。应用特异性分子抑制剂Y27632抑制RhoA的活化后,细胞中F-actin重构现象消失,细胞单层通透性增加也受到明显抑制,SRF蛋白发生明显的出现转位。而应用LatrunculinB抑制脂多糖刺激的人脐静脉内皮细胞中F-actin应力纤维形成,对抗通透性增加,但RhoA活化未受到干扰,SRF入核现象则受到抑制。提示RhoA-SRF通路的活化介导了脂多糖诱导的人脐静脉内皮细胞中F-actin重构和内皮单层通透性增加,特异性抑制F-actin也可以阻断脂多糖引起的血管内皮单层通透性增加,同时反馈抑制SRF的入核活化现象。

英文摘要：

BACKGROUND：The molecular mechanism underlying exogenous stimulation-caused vascular barrier dysfunction remains poorly understood in the field of vascular pathophysiology.OBJECTIVE：To investigate the effective factors for regulating monolayer permeability of human umbilical vein endothelial cells and search for the effective treatment target.METHODS：Human umbilical vein endothelial cells（HUVECs） were exposed to lipopolysaccharide（LPS） with and without specific inhibitors of RhoA and F-actin stress fibre.Changes in RhoA/SRF/F-actin activity in association with cell permeability and F-actin rearrangement signaling were investigated by immunostaining,transwell assay and western blot analysis.RESULTS AND CONCLUSIONS：When HUVECs were treated with LPS,F-actin cytoskeleton rearrangement was detected by rhodamine-phalloidin staining in a dose and time-dependent manner,meanwhile the HUVEC hyperpermeability was investigated.Furthermore,the RhoA activity and SRF translocalization into nuclei were identified to be associated with elevation of permeability in HUVECs.The effects were blocked when cells were pretreated with Y27632 or Latrunculin B,respectively.Moreover,Latrunculin B added also inhibited the translocalization of SRF into the nuclei mediated by RhoA activity.Our results have identified the RhoA/SRF and their substrates F-actin rearrangement mediated LPS-induced changes in HUVECs permeability.