缺氧诱导因子转染内皮祖细胞治疗大鼠缺血后肢

Treatment of transfection hypoxia inducible factor gene into endothelial progenitor cells for vascular regeneration in rat ischemic hind-limb

目的 使用缺氧诱导因子-1α（HIF-1α）转染内皮祖细胞（EPC）治疗大鼠后肢缺血,观察EPC、HIF-1α转染EPC对大鼠缺血后肢血管新生和肢体成活的影响.方法 制作SD大鼠后肢缺血模型,将动物随机分为3组,每组6只.将构建的HIF-1α基因真核表达载体转染入EPCs后通过尾静脉注射入大鼠体内,并与注射磷酸盐缓冲液（PBS）或EPC的大鼠进行比较,观察转染HIF-1α对新生血管形成的影响.结果 （1）EPC组、HIF组较PBS组肢体恢复率明显增加（P〈0.05）,EPC组肢体恢复率较HIF组差（P〈0.05）.（2）与PBS组比较,各时间点中EPC、HIF组微血管密度（MVD）均明显增多（P〈0.05）,HIF组较EPC组明显增高（P〈0.05）.（3）HIF组的HIF与血管内皮生长因子（VEGF）蛋白表达较PBS组、EPC组明显增多（P〈0.05）.PBS组Capase-3的表达较EPC组、HIF组明显增多（P〈0.05）.（4）术后7 d,各组的大鼠肢体灌注均明显降低,但EPC、HIF组细胞的血流灌注较PBS组多（P〈0.01）.术后14、21 d,与PBS对照组比较,HIF组的血流灌注恢复明显（P〈0.01）,EPC组血流灌注较HIF组少（P〈0.05）.结论 EPCs对大鼠缺血后肢的局部血管新生有明显促进作用,联合应用HIF-1α和EPCs有更优的治疗效果.

Objective To explore whether the overexpression of hypoxia inducible factor （HIF）transfection into endothelial progenitor cells （EPCs） is beneficial on eovascularization of rat ischemic hindlimb and limb survival.Methods SD rat ischemic hind-limb models were established,and randomly divided into three groups,six rats each group.Lipofectamine 2000 mediated Eukaryotic expression vector of HIF was transfected into EPCs from marrow,which then was injected into rats via vena caudalis.Observe how EPCs transfected with HIF gene gathered at the ischemic part and facilitated angiogenesis.Results （ 1 ） General limb disability： limb restoration rate of Group EPC,EPC ＋ HIF were obviously higher than that of Group PBS （P 〈0.05）.（2） Micro vessel density （MVD）： compared with Group PBS,there was a marked increase of MVD in Group EPC,HIF at each time point （P 〈 0.05 ）.14 and 21 days after operation,MVD in Group HIF was higher than that in Group EPC （P 〈0.05）.（3） HIF and Vascular endothelial growth factor （VEGF） Expression in ischemic limbs：compared with Group PBS,EPC,Expression of HIF and VEGF protein in Group HIF was significantly increased （P 〈 0.05）.There was obvious increase in expression of HIF and VEGF protein in Group EPC compared with Group PBS （ P 〈 0.05 ）.（4） Blood perfusion：seven days after operation,rat limb perfusion in all groups was reduced; 14,21 days after operation,compared with PBS controlled group,there was notable blood perfusion recovery in Group EPC and HIF （ P 〈 0.01 ）.Blood perfusion of Group EPC was less than that of Group HIF （ P 〈 0.05 ）.Conclusion EPCs transfected with HIF gene had a significant impact on angiogenesis in ischemic.Combined application of HIF gene enhanced the effect of EPCs treatment of augment naturally impaired neovascularization in animal model of experimentally induced limb ischemia.