细胞骨架F-actin在层流剪切应力诱导EPCs内皮分化中的作用

Role of F-actin cytoskeleton in differentiation of endothelial progenitor cells induced by laminar shear stress

目的探讨细胞骨架F-actin在层流剪切应力促内皮祖细胞(endothelial progenitor cells,EPCs)向内皮分化中的作用。方法对大鼠骨髓来源的EPCs施以层流剪切应力(1.2 Pa),以荧光定量RT-PCR及流式细胞术检测特异性内皮细胞标记分子vWF、CD31 mRNA及蛋白的表达来反映EPCs分化程度;以免疫荧光染色观测F-actin的排列情况;应用Ras GTPase Pull-Down方法检测Ras活性。结果层流剪切力处理后,EPCs分化标记vWF及CD31的基因及蛋白表达较静止组明显升高(P<0.05),细胞骨架F-actin发生重排,Ras活性明显增高。细胞骨架稳定剂Jasplakinolide(JAS)及细胞骨架松弛剂Cytochalasin D(CytoD)预处理均不同程度地抑制了层流剪切应力所致的细胞骨架的重排、Ras活性的上调及EPCs分化效应(P<0.05),而过表达Ras则对层流剪切应力诱导的EPCs分化有明显促进作用(P<0.05)。结论一定大小的层流剪切应力可促进EPCs向内皮细胞分化,其机制可能与层流剪切应力重塑细胞骨架F-actin,进而影响Ras活性有关;这对于揭示受损血管内皮修复的具体机制、阐明动脉粥样硬化等心血管疾病的发病机理及临床防治此类疾病具有一定的意义。

Objective To investigate effects of F-actin cytoskeleton on differentiation of endothelial progenitor cells（EPCs） under laminar shear stress.Methods EPCs isolated from rat bone marrow were treated with laminar shear stress（1.2 Pa）.Then the gene and protein expressions of the endothelial cell differentiation markers,such as vWF and CD31,were assayed with real time RT-PCR and Flow Cytometry.The effects of laminar shear stress on F-actin cytoskeleton and Ras activity were investigated by immunofluorescence technique and Pull-down assay.Results Compared with the untreated group,the expressions of vWF and CD31 were obviously increased in the group treated with laminar shear stress（P0.05）.Moreover,exposure of EPCs to laminar shear stress led to the reorganization of cytoskeleton and enhanced the activity of Ras in EPCs.The treatment to EPCs with either F-actin stabilizer jasplakinolide or depolymerizers cytochalasin D inhibited the cytoskeleton reorganization induced with laminar shear stress,the activity of Ras and the up-regulation of the vWF and CD31 genes.However,over-expression of Ras augmented the up-regulation of the vWF and CD31 genes induced by laminar shear stress in EPCs.Conclusions The mechanism that laminar shear stress accelerates the differentiation of EPCs may be related with the laminar shear stress-induced cytoskeleton rearrangement and Ras activation.This study is of significance in revealing the mechanism of vascular endothelial repair which could be useful for the prevention and treatment of atherosclerosis.