【摘要】 目的探讨与膜-细胞骨架连接蛋白家族结合的磷酸化蛋白50(EBP50)对人脐静脉内皮细胞(HUVEC)内Akt1磷酸化水平、细胞分泌明胶酶的活性以及细胞骨架表达和分布的影响,阐明其影响人血管内皮细胞增殖、迁移及成管的分子机制。方法构建EBP50的真核重组表达载体,将重组质粒分别稳定转染HUVEC细胞系,经Western blot法验证后,采用MTT法检测细胞的增殖活性;应用划痕法检测细胞的迁移能力;应用Matrigel法检测细胞的成管能力;应用Western blot检测EBP50对Akt1磷酸化水平的影响;应用明胶酶谱法检测HUVEC细胞分泌的明胶酶活性;应用免疫荧光观察细胞骨架的分布。结果转染的外源性EBP50 cDNA片段已整合到基因组中;与对照组细胞比较,EBP50能显著抑制细胞的增殖、迁移及成管能力,并且EBP50明显下调Akt1的磷酸化水平(t=2.98,P<0.01);同时使HUVEC分泌的MMP2活性下降,并影响血管内皮细胞内细胞骨架的分布。结论EBP50可以抑制HUVEC细胞的增殖、迁移及成管,其机制可能与调整Akt1的磷酸化水平、调节细胞分泌明胶酶的活性及影响微丝细胞骨架分布有关。更多还原

【Abstract】 Objective To explore the effects of ezrin-radixin-moesin-binding phosphoprotein 50(EBP50) on the biological properties of human umbilical vein endothelial cells(HUVEC) and related mechanisms.Methods The biological effects of EBP50 on HUVEC were investigated.Western blotting was used to detect the expression of EBP50 in cells.The 3-4,5-Dimethyl-2-thiazolyl-2,5-diphenyl-2Htetrazolium bromide(MTT) assay and scarification test were used to measure the proliferation and migration activity of HUVEC.The effect of EBP50 on the phosphorylation levels of Akt1 and the changes of matrix metalloproteinase(MMP)-2 and MMP-9 levels were detected by western blotting and gelatin zymography analysis.Results Over expression of exogenous EBP50 in HUVEC significantly inhibited the proliferation,migration and tube formation of HUVEC.EBP50 also significantly down regulated Akt1 phosphorylation and the secretion of MMP-2 compared with control cells.Conclusions EBP50 might serve as a potential target for manipulating neovascularization related diseases.This discovery contributes to a better understanding of the bioactivity of EBP50 in angiogenesis.更多还原