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目的:探讨E1A激活基因阻遏子(Cellular repressor of E1A-stimulated gene,CREG)对血管内皮细胞血管新生的影响及其机制。方法:将重组腺病毒CREG载体(Ad-GFP-CREG)和腺病毒GFP载体(Ad-GFP)转染人血管内皮细胞株(vascularendothelialcell,VE),转染后细胞分别命名为ADCREG和ADGFP。内皮细胞基质胶血管形成实验观察其比较3组细胞血管发生情况。应用Western blotting测定细胞中CREG、整合素连接激酶(integrin-linked kinase,ILK)和血管内皮生长因子(vascular endothelial growthfactor,VEGF)的表达。应用ILK激酶活性突变的质粒转染和不同浓度的VEGF中和抗体转染VE后进行细胞血管形成能力检测。结果:体内基质胶血管形成实验显示,腺病毒介导的CREG过表达显著促进VE的血管形成能力,血管密度、数量和交叉点均较对照组ADGFP和VE显著增多(P<0.01)。同时,Westernblot分析证实CREG过表达显著增加了ILK与VEGF的表达;转染ILK激酶失活质粒导致ADCREG成血管能力显著减弱(P<0.001)。应用VEGF中和抗体干预后,ADCREG细胞的成血管能力也呈现剂量依赖性下降。并且ILK激酶活性受到抑制的ADCREG细胞中VEGF表达降低。结论:CREG基因过表达通过活化ILK/VEGF信号通路促进了内皮细胞血管新生。
Objective: To investigate the effects of E1A-stimulated gene repressor (CREG) on angiogenesis of vascular endothelial cells and its mechanism. Methods: The recombinant adenovirus CREG vector (Ad-GFP-CREG) and adenovirus GFP vector (Ad-GFP) were transfected into human vascular endothelial cell line (VE). The transfected cells were named as ADCREG and ADGFP respectively. Vascular endothelialization was observed in matrigel in three groups. Western blotting was used to detect the expression of CREG, integrin-linked kinase (ILK) and vascular endothelial growth factor (VEGF) in the cells. The transfection of plasmid with ILK kinase activity mutation and VEGF neutralizing antibody at different concentrations were used to detect the vascularization ability of VE. Results: The results of in vivo matrigel angiogenesis showed that adenovirus-mediated over-expression of CREG significantly enhanced the vascularization ability of VE. The vascular density, number and intersection were significantly increased (P <0.01) compared with ADGFP and VE in control group. Meanwhile, Western blot analysis confirmed that overexpression of CREG significantly increased the expression of ILK and VEGF; transfection of ILK kinase-inactivated plasmids led to a significant decrease in ADCREG-forming ability (P <0.001). After intervention with VEGF neutralizing antibody, the angiogenic capacity of ADCREG cells also decreased in a dose-dependent manner. And VEGF expression was decreased in ADCREG cells in which ILK kinase activity was inhibited. Conclusion: Overexpression of CREG promotes endothelial cell angiogenesis by activating ILK / VEGF signaling pathway.