三突变型低氧诱导因子-1α对人微血管内皮细胞增殖及VEGF蛋白表达的影响

摘要

Objective To investigate the effects of a recombinant adenovirus-mediated triple mutant hypoxia-inducible factor-la (HIF-lα) on the proliferation and vascular endothelial growth factor (VEGF) expression in human microvascular endothelial cells (hMVECs).Methods The adenovirus vector of the triple mutant HIF-lα (Ad-HIF-lα564/402/803),adenovirus vector of wild-type HIF-lα (Ad-HIF-lαnature),Ad-lacZ and Ad-Null were amplified in HEK293A cells,and the adenoviruses were purified and titrated.Dual luciferase reporter assay system was employed to detect the transcriptional activities of wild-type and triple mutant HIF-lα.After infection of the hMVECs with the adenoviruses,the cellular protein expressions of HIF-lα and VEGF were detected using Western blotting,and the cell proliferation was assessed by MTS assay.Results The transcriptional activity of the triple mutant HIF-lα was significantly higher than that of wildtype HIF-lα in the infected hMVECs (P<0.001).The protein levels of HIF-lα and VEGF in cells infected with Ad-HIF-lα564/402/800 were significantly higher than those in cells infected with other adenoviruses,and HIF-lα dose-dependently up-regulated VEGF protein expression.The absorbance was significantly higher in Ad-HIF-lα564/402/800 group than in the other groups (P<0.01) on the third and fifth days after infection.Conclusion The recombinant adenovirus-mediated triple mutant HIF-lα expression is stable under normoxic condition.The triple mutant HIF-lα can up-regulate the expression of VEGF protein in hMVECs to promote the cell proliferation.%目的 观察重组腺病毒三突变型低氧诱导因子-1cα(HIF-1α)对人微血管内皮细胞(hMVECs)增殖及VEGF蛋白表达的影响.方法 三突变型HIF-1α腺病毒载体(Ad-HIF-1α564/402/803)、野生型HIF-1α腺病毒载体(Ad-HIF-1αnature)、Ad-LacZ及空载腺病毒载体(Ad-Null)分别在HEK293A细胞大量扩增,氯化铯梯度离心纯化,终点稀释法测定病毒滴度；双荧光素酶报告系统检测三突变型HIF-1α与野生型HIF-1α的转录活性；各重组腺病毒载体以最佳转染复数转染体外培养的hMVECs,Westem blotting测定HIF-1α和VEGF蛋白表达；MTS检测Ad-HIF-1α564/402/803对细胞增殖的影响.结果 三突变型HIF-1α转录活性显著高于野生型HIF-1α(P＜0.001):Ad-HIF-1α564/402/803组HIF-1α和VEGF蛋白表达量高于Ad-HIF-1αnature及其他组；VEGF蛋白表达水平与HIF-1α呈剂量依赖关系.Ad-HIF-1α564/402/803组第3、5天吸光度值均显著高于Ad-HIF-1αnature及其他组(P＜0.01).结论 三突变型HIF-1α在体外常氧条件下稳定高效表达,可诱导hMVECs VEGF蛋白表达上调,促进hMVECs增殖.