重组腺病毒三突变型低氧诱导因子-1α对血管新生的影响

Effects of recombinant adenovirus-mediated triple mutant of hypoxia inducible factor-1α in modulating angiogenesis

目的研究重组腺病毒三突变型低氧诱导因子-1α(Ad-HIF-1α-Ala564-Ala402-Ala803,简称Ad-HIF-1α-564/402/803)对体外血管新生的影响。方法将前期构建的Ad-HIF-1α-564/402/803在HEK293A细胞中进行扩增,用氯化铯浓度梯度离心法进行腺病毒纯化,终点稀释法测定病毒滴度,提取病毒DNA,进行PCR及PCR产物测序鉴定三突变型HIF-1α基因;X-Gal染色法测定重组腺病毒转染效率;Ad-HIF-1α-564/402/803、Ad-HIF-1αnature、Ad-Null分别转染hMVECs后,观察hMVECs在Matrigel上毛细血管管腔样结构的形成情况;Ad-HIF-1α-564/402/803、Ad-HIF-1αnature、Ad-Null和PBS分别转染鸡胚绒毛尿囊膜(CAM)模型后,利用Image Pro Plus6.0软件收集数据并计算各组CAM血管面积比。结果经PCR及基因测序鉴定,扩增纯化后的腺病毒所携带的目的基因信息无丢失或变异,病毒滴度达1011～1012PFU/ml;X-gal染色显示MOI为100pfu/cell时,转染效率趋于稳定;Ad-HIF-1α-564/402/803转染hMVECs后Matrigel上的管腔数目显著多于Ad-HIF-1α-nature组、Ad-Null及对照组;基因转染CAM72h后,Ad-HIF-1α-564/402/803组CAM上微小血管数目较Ad-HIF-1α-nature组、Ad-Null组、PBS组明显增多;Ad-HIF-1α-564/402/803组血管面积比与Ad-HIF-1α-nature组、Ad-Null组、PBS组相比,差异有统计学意义(P值分别为0.01、0.000、0.000)。结论Ad-HIF-1α-564/402/803能明显促进hMVECs毛细血管管腔样结构的形成,同时可以促进CAM模型上微小血管新生,证实了Ad-HIF-1α-564/402/803在常氧情况下对血管新生有一定的促进作用。

Objective To investigate the effect of recombinant adenovirus-mediated triple mutant of hypoxia inducible factor-1α （Ad-HIF-1α-564/402/803） in modulating angiogenesis in vitro.Methods The recombinant adenoviruses Ad-lacZ,Ad-Null,Ad-HIF-1α-nature,and Ad-HIF-1α-564/402/803 were amplified in HEK293A cells and purified by ultracentrifugation in CsCl step gradient solutions,and the adenoviral titer was determined by end-point dilution assay.The recombinant adenovirus was confirmed by PCR and DNA sequence analysis,and the infection efficiency was observed by X-gal staining.Human microvascular endothelial cells （HMVECs） were infected with Ad- HIF-1α-564/402/803,Ad- HIF-1α-nature,or Ad-Null to compare the number of capillary-like tube structures in vitro.The effect of Ad- HIF-1α-564/402/803,Ad-HIF-1α-nature,and Ad-Null on angiogenesis was evaluated using a chick embryo chorioallantoic membrane （CAM） model.Results PCR and gene sequencing suggested the correct construction of the recombinant adenovirus HIF-1α,and the adenoviral titer reached 1011-1012 PFU/ml.Infection of the hMVECs with Ad-HIF-1α-564/402/803 at the optimal multiplicity of infection of 100 pfu/cell resulted in a significantly greater number of capillary-like tube strucures than infection by Ad-HIF-1α-nature and Ad-Null （P=0.000）.Ad-HIF-1α-564/402/803 group showed significantly higher microvessel density than Ad-HIF-1α-nature,Ad-Null,and PBS groups,with also higher angiogenesis area to CAM area ratio （P=0.01,0.000,and 0.000,respectively）.Conclusion The triple mutant Ad-HIF-1α-564/402/803 can obviously promote the formation of capillary-like tube structures in vitro and modulate angiogenesis in the CAM model,suggesting the capacity of Ad-HIF-1α-564/402/803 in promoting angiogenesis under normoxic condition.