CX3CL1介导人脐静脉内皮细胞骨架改变的功能研究

Functional Characterization of CX3CL1 in Altering the Cytoskeleton of Human Umbilical Vein Endothelial Cells

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 research investigated the effect of CX3CL1 on cytoskeleton in human umbilical vein endothelial cells（HUVECs）, and evaluated some possible mechanisms. HUVECs were stimulated with CX3CL1. Immunofluorescence staining technique was used to test changes in distribution and formation of F-actin in HUVECs. Western blot was used to detect expressions of cytoplasmic F-actin and phosphorylated mitogen-activated protein kinases（MAPKs）： p38, extracellular regulated protein kinase（ERK1/2） and c-Jun N-terminal kinase（JNK）. After 30 min stimulation with 10 nmol/L of CX3CL1 in HUVECs, dense peripheral band began to be destroyed and cytoplasmic stress fiber began to form. After 120 min stimulation with 10 nmol/L of CX3CL1 in HUVECs, peripheral band disappeared and massive cytoplasmic intense stress fiber formed. After 180 min stimulation with 10 nmol/L of CX3CL1 in HUVECs, cytoplasmic stress fiber decreased, and peripheral band could be seen in some cells. The prominent enhancement of cytoplasmic F-actin was detected starting after 30 min and peaking after 120 min of stimulation with 10 nmol/L of CX3CL1 in HUVECs. The prominent enhancements of phosphorylated p38, ERK1/2 and JNK were detected starting after 1 min and peaking after 5 min of stimulation with 10 nmol/L of CX3CL1 in HUVECs. The up-regulation of phosphorylated p38, ERK1/2 and JNK induced by 10 nmol/L of CX3CL1 could be decreased by 5 μg/m L of anti-CX3CR1 antibody in HUVECs. The reconstruction of F-actin, the formation of stree fiber and the up-regulation of cytoplasmic F-actin induced by CX3CL1 could be decreased by 30 μmol/L of SB203580 and PD98059 in HUVECs. SB203580 was used as standard inhibitor for p38. PD98059 was used as standard inhibitor for ERK1/2. In conclusion, CX3CL1 might induce a time-dependent reconstruction of the cytoskeleton through p38 and ERK1/2 signaling pathways in HUVECs.