Ad-HIF-1α-Trip肌肉注射对大鼠缺血肢体血管新生和血液灌注的影响

Effects of Ad-HIF-1α-Trip transfer on angiogenesis and blood perfusion in a rat model of hind limb ischemia

目的研究重组腺病毒低氧诱导因子-1α三突变体(the recombinant adenovirus containing the triple-pointmutants HIF1-αgene,Ad-HIF-1α-trip)基因对大鼠缺血后肢血管内皮生长因子(vascular endothelial growth factor,VEGF)表达、血管新生及血流灌注的影响。方法 48只2月龄雄性Wistar大鼠(体质量0.200～0.250 kg)建立急性左后肢缺血模型,应用随机数字表法随机分成4组(n=12):生理盐水组(Saline组),腺病毒空载体组(Ad-Null组)、重组腺病毒低氧诱导因子-1α组(Ad-HIF-1α)、Ad-HIF-1α-trip组。分别在建模后即刻肌注生理盐水、Ad-Null、Ad-HIF-1α、Ad-HIF-1α-trip0.5 ml,病毒滴度4×109PFU。术后7 d,行Real-time PCR检测缺血组织HIF-1α及VEGF的表达情况。术前及基因转染后即刻、7、14、28 d对大鼠后肢进行对比超声(contrast enhanced ultrasound,CEU)检查,基因转染后28 d处死动物行病理切片CD31免疫组化微血管密度(microvascular density,MVD)计数。结果基因转染后7 d,在mRNA水平HIF-1α及VEGF在Ad-HIF-1α-trip组大鼠缺血后肢肌肉组织中表达最强[HIF-1α:(2.576±0.019);VEGF:(2.498±0.031),P<0.05];CEU结果显示:Ad-HIF-1α-trip组基因转染后大鼠缺血后肢血流灌注改善明显优于其他各组{基因转染28 d A×β标化值:Saline vs Ad-Null vs Ad-HIF-1αvs Ad-HIF-1α-trip:[(0.529±0.045)vs(0.535±0.062)vs(0.642±0.056)vs(0.895±0.049),P<0.05];CD31免疫组化示:转染后28 d,Ad-HIF-1α-trip组缺血后肢肌肉组织MVD计数明显优于其他各组[Saline vs Ad-Null vs Ad-HIF-1αvs Ad-HIF-1α-trip:(82.316±19.258)vs(89.662±21.374)vs(175.248±31.736)vs(213.426±48.073),P<0.05]}。结论 Ad-HIF-1α-Trip在大鼠缺血肢体肌肉组织能有效表达,促进缺血肢体的血管新生及血流灌注。

Objective To investigate the effects of the recombinant adenovirus containing the triple-point mutants HIF1-α gene（Ad-HIF-1α-trip） on the expression of VEGF,the angiogenesis and the blood perfusion in the rat model of ischemic hind limb.Methods After ligation of left femoral artery,totally 48 male Wistar rats were randomly divided into 3 control groups（n=12 in each group） and model group（n=12）.The control groups were subjected with normal saline（0.5 ml）,Ad-Null（4×109 PFU）or Ad-HIF-1α（4×109PFU） respectively by intramuscular injection.The model group was subjected with Ad-HIF-1α-trip（4×109 PFU）.The expression of HIF-1α and VEGF at mRNA level in the ischemic skeletal muscle were detected by real-time PCR in 7 d after gene transfer.The changes of blood perfusion of the ischemic hind limb were detected by contrast enhanced ultrasonography（CEU） before operation,immediately and on the 7th,14th and 28th day after gene transfer.The microvascular density（MVD） of the ischemic hind limb were observed through immunohistochemistry of CD31.Results On the 7th day after gene transfer,the expression of HIF-1α and VEGF at mRNA level was more significant in the Ad-HIF-1α-trip group than other groups（HIF-1α： 2.576±0.019;VEGF： 2.498±0.031,P〈0.05）.CEU revealed better blood perfusion in Ad-HIF-1α-Trip group than in other groups（the standardized value of A×β28 days after gene transfer： Saline vs Ad-Null vs Ad-HIF-1α vs Ad-HIF-1α-trip： 0.529±0.045 vs 0.535±0.062 vs 0.642±0.056 vs 0.895±0.049,P〈0.05）.Immunohistochemical assay for CD31 showed the MVD of Ad-HIF-1α-trip was more denser than other groups in 28 d after gene transfer（Saline vs Ad-Null vs Ad-HIF-1α vs Ad-HIF-1α-trip： 82.316±19.258 vs 89.662±21.374 vs 175.248±31.736 vs 213.426±48.073,P〈0.05）.Conclusion Ad-HIF-1α-Trip is efficiently expressed in the ischemic skeletal muscle,and improves the angiogenesis and blood perfusion of the ischemic limb effectively.