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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 effect of the recombinant adenovirus containing the triple-point mutants (HIF-1α-trip) gene on the vascular endothelial growth factor factor, VEGF expression, angiogenesis and perfusion. Methods Forty-eight male Wistar rats (body weight 0.200-0.250 kg) were randomly divided into 4 groups (n = 12): saline group (Saline group), gland Ad-Null group, Ad-HIF-1α group and Ad-HIF-1α-trip group. Immediately after modeling, normal saline, Ad-Null, Ad-HIF-1α, Ad-HIF-1α-trip0.5 ml and virus titer of 4 × 109 PFU were intramuscularly injected respectively. At 7 days after operation, Real-time PCR was used to detect the expression of HIF-1α and VEGF in ischemic tissue. The hind limbs of rats were examined by contrast enhanced ultrasound (CEU) immediately before and at 7, 14 and 28 days after the gene transfection. The animals were sacrificed at 28 days after transfection. The pathological sections of CD31 immunohistochemical microvessels density, MVD) count. Results The mRNA and protein levels of HIF-1α and VEGF were the highest in hindlimb hindlimb muscle of Ad-HIF-1α-trip group at 7 d after transfection (HIF-1α: 2.576 ± 0.019, VEGF: 2.498 ± 0.031), P <0.05]. CEU results showed that the improvement of blood perfusion of ischemic hindlimb in Ad-HIF-1α-trip group was significantly better than that in other groups {gene transfection 28 d A × β The normalized values of Saline vs Ad-Null versus 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) 0.05]. CD31 immunohistochemistry showed that the MVD count of ischemic hindlimb muscle in Ad-HIF-1α-trip group was significantly higher than that in other groups on the 28th day after transfection [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 can be effectively expressed in the ischemic limb muscle tissue of rats and promote angiogenesis and perfusion of ischemic limbs.