摘要
目的观察重组腺病毒三突变型低氧诱导因子-1α(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,Western 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。
Objective To investigate the effects of a recombinant adenovirus-mediated triple mutant hypoxia-inducible factor-1α （HIF-1α） 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-1α （Ad-HIF-1α 564
/402/803）, adenovirus vector of wild-type HIF-1α （Ad-HIF-1αnature）, 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-1α. After infection of the hMVECs with the adenoviruses, the cellular protein expressions of HIF-la 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-1α was significantly higher than that of wildtype HIF-1α in the infected hMVECs （P〈0.001）. The protein levels of HIF-1α and VEGF in ceils infected with Ad-HIF-1α 564
/402/803 were significantly higher than those in cells infected with other adenoviruses, and HIF-1α dose-dependently up-regulated VEGF protein expression. The absorbance was significantly higher in Ad-HIF-1α 564
/402/803 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-1α expression is stable under normoxic condition. The triple mutant HIF-1α can up-regulate the expression of VEGF protein in hMVECs to promote the cell proliferation.
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2012年第6期812-816,共5页
Journal of Southern Medical University
基金
国家自然科学基金(30370587)
国家自然科学基金青年基金(30800462)~~
关键词
低氧诱导因子-1Α
腺病毒载体
微血管内皮细胞
血管内皮生长因子
hypoxia-inducible factor-1α
adenovirus vector
vascular endothelial growth factor
microvascular endothelial cells
