基质细胞衍生因子-1对人脐静脉血管内皮细胞迁移的影响

目的:利用Boyden chamber体外迁移体系初步探索基质细胞衍生因子-1α(SDF-1α)在人脐静脉血管内皮细胞(HUVEC)迁移中的作用及其信号转导机制。方法:以HUVEC为实验材料,首先利用免疫荧光技术及蛋白印迹方法检测HUVEC表达SDF-1α受体CXCR4,其次利用Boyden chamber体外迁移体系,先观察不同浓度的SDF-1α对HUVEC迁移的影响,最后以磷脂酰肌醇-3激酶(PI-3K)阻断剂wortmannin(50 M)或LY294002(10μM)、丝裂酶原活化蛋白激酶(MAPK)阻断剂PD98059(50μM)、磷脂酰肌醇特异性磷脂酶C(PI-PLC)阻断剂U73122(10μM)、CXCR4特异性阻断剂AMD3100(0.1 mg/ml)等不同预处理HUVEC 30 min,观察分析SDF-1α对HUVEC迁移影响的信号转导通路。结果:培养的HUVEC表达SDF-1α受体CXCR4,HUVEC体外迁移能力随着SDF-1α浓度的递增而逐渐增强,并且SDF-1α浓度在100 ng/ml时,HUVEC迁移到滤膜上的细胞数最多;Wort-mannin、LY294002、PD98059、U73122、AMD3100对HUVEC迁移均有影响,其中U73122、AMD3100对HUVEC迁移阻断的效应最显著。结论:SDF-1α/CXCR4所介导的HUVEC迁移与MAPK)、PI-PLC和蛋白激酶C(PKC)等信号途径有关,且PKC途径可能处于中心环节。

Effect of SDF-1αon Human Umbilical Vein Endothelial Cell Migration

Objective To explore the signal transduction mechanism of stromal derived factor-1α(SDF-1α) on migration of human umbilical vein endothelial cell(HUVEC).Methods Firstly,expression of SDF-1α receptor CXCR4 in cultured HUVECs was analyzed with immunofluorescent microscopy and western Blotting.Secondly,with Boyden chamber in vitro migration assay system,the effect of SDF-1α on HUVECs migration was evaluated.Briefly,in concentration-dependent experiments,HUVECs were treated with different concentration SDF-1α(1 ng/ml,10 ng/ml,100 ng/ml),respectively.Then in blocking experiments,HUVECs were preincubated with CXCR4 inhibitor 0.1mg/ml AMD3100,PI-3K inhibitor 50 M wortmannin or 10 μM LY294002,MAPK inhibitor 50 μM PD98059,PI-PLC inhibitor 10 μM U73122.Results Cultured HUVECs expressed SDF-1α receptor CXCR4,the efficiency of HUVECs migration into the filter increased gradually with the concentration increasing of SDF-1α,and HUVECs number in the filter was maximal when SDF-1α in 100ng/ml.HUVECs number in the filter decreased after HUVECs treatment with wortmannin,LY294002,PD98059,U73122,AMD3100,respectively.Importantly,the ability of HUVECs migration obviously decreased when HUVECs treated with U73122,AMD3100.Conlusion SDF-1α/CXCR4-mediated HUVECs migration is correlated with MAPK,PI-PLC and PKC signal pathways,and PKC signal pathway may be the central role for HUVEC migration.