摘要
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。
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- 1αt 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. 1 mg/ml AMD3100, PI -3K inhibitor 50 M wortmannin or 10μM 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. Gonlusion 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.
出处
《郧阳医学院学报》
2007年第3期129-132,157,F0002,共6页
Journal of Yunyang Medical College
基金
湖北省自然基金资助项目(2005ABA079)
郧阳医学院科研启动基金资助项目(2005QDJ01)
湖北省卫生厅(JX3B29)
湖北省教育厅(Q200524003
B200624006)
