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该研究探讨了趋化因子CX3CL1对人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)骨架的影响及其作用机制。CX3CL1刺激HUVECs后,采用免疫荧光染色技术检测细胞骨架蛋白纤维状肌动蛋白(F-actin)的分布和形态改变,采用Western blot技术检测胞质内F-actin和磷酸化促分裂原活化蛋白激酶(mitogen activated protein kinases,MAPKs)的三种亚型[p38、细胞外调节蛋白激酶1/2(extracellular regulated protein kinase 1/2,ERK1/2)和c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)]的表达水平。结果显示,10 nmol/L CX3CL1刺激HUVECs 30 min后,细胞的致密外周带逐渐被破坏,胞质内有应力纤维形成;120 min后,外周带消失,胞质内有大量的致密应力纤维形成;180 min后,胞质内应力纤维减少,少数细胞可见致密外周带。10 nmol/L CX3CL1刺激HUVECs30 min后,F-actin的表达水平逐渐升高,并于120 min后达峰值;10 nmol/L CX3CL1刺激HUVECs 1 min后,磷酸化p38、ERK1/2和JNK表达水平升高,5 min后三者的表达水平达峰值;5μg/m L抗CX3CR1抗体抑制10 nmol/L CX3CL1刺激HUVECs后,磷酸化p38、ERK1/2和JNK的表达水平降低;30μmol/L p38的特异性抑制剂SB203580和ERK1/2的特异性抑制剂PD98059抑制10 nmol/L CX3CL1刺激HUVECs后,胞质内应力纤维减少,应力纤维变短,F-actin的表达水平降低。以上研究结果表明,CX3CL1能通过p38和ERK1/2信号通路以时间依赖方式介导HUVECs细胞骨架的重构。
This study explored the effect of chemokine CX3CL1 on the skeleton of human umbilical vein endothelial cells (HUVECs) and its mechanism of action. CX3CL1 stimulation of HUVECs, the use of immunofluorescence staining technique to detect the distribution of cytoskeletal F-actin (F-actin) and morphological changes by Western blot detection of cytoplasmic F-actin and phospho-mitogen activated protein kinase Three subtypes of mitogen activated protein kinases (MAPKs) [p38, extracellular regulated protein kinase 1/2 (ERK1 / 2) and c-Jun N-terminal N- terminal kinase, JNK)] expression level. The results showed that the dense peripheral zone of HUVECs was gradually destroyed after treated with 10 nmol / L CX3CL1 for 30 min, and there was stress fiber in the cytoplasm. After 120 min, the peripheral zone disappeared and a large amount of dense stress fibers formed in the cytoplasm. After 180 min, the cytoplasmic stress fibers decreased, and few cells showed dense peripheral zone. The expression of F-actin increased gradually after 30 min stimulated by 10 nmol / L CX3CL1 for 30 min and peaked at 120 min. The phosphorylation levels of p38, ERK1 / 2 and JNK were up-regulated after 10 nmol / L CX3CL1 stimulation for 1 min 5 μm / L anti-CX3CR1 antibody inhibited the expression of phosphorylated p38, ERK1 / 2 and JNK after stimulating HUVECs with 10 nmol / L CX3CL1. The expression of p38, 30 μmol / L p38 PD98059, a specific inhibitor of specific inhibitors SB203580 and ERK1 / 2, reduced the cytoplasmic stress fibers, reduced the stress fibers and decreased the expression of F-actin in HUVECs stimulated with 10 nmol / L CX3CL1. The above results show that CX3CL1 can mediate the remodeling of HUVECs cytoskeleton through p38 and ERK1 / 2 signaling in a time-dependent manner.