【摘要】 目的在大鼠后肢缺血模型中,探讨联合转染促血管生成素1和血管内皮生长因子基因治疗对新生血管数量和侧支循环形成的影响,并观察不同基因剂量配比对新生血管形态和功能的影响。方法制备大鼠后肢血管闭塞病变模型,采用电脉冲法介导转基因,按不同剂量配比进行肌肉组织转染携带促血管生成素1基因的质粒和/或携带血管内皮生长因子基因的质粒。采用逆转录聚合酶链反应检测外源基因的表达、免疫组织化学染色检测缺血局部毛细血管和小动脉的数量和分布、通过后肢血管造影检测侧支循环建立的情况。结果单独转促血管生成素1基因或血管内皮生长因子基因治疗组血管生成数量略有增加,联合基因治疗组中,血管内皮生长因子质粒剂量为100μg时,随着促血管生成素1质粒剂量的增加,小动脉计数较同期空载质粒对照组分别增加0.90倍、1.40倍和1.45倍;当促血管生成素1质粒剂量为100μg时,随着血管内皮生长因子质粒剂量的增加,小动脉计数较同期空载质粒对照组分别增加1.90倍、1.07倍和1.39倍。血管通透性检测表明,随着血管内皮生长因子质粒剂量的增加,血管通透性渐增加,促血管生成素1质粒能降低新生血管的通透性;其中促血管生成素1质粒200μg联合血管内皮生长因子质粒100μg组在有效促进小动脉形成的同时血管通透性与正常对照组最为接近。结论联合血管生成素1和血管内皮细胞生长因子基因治疗能更有效促进小动脉形成,其中血管内皮生长因子质粒与促血管生成素1质粒比例为1:2时血管通透性最接近生理状态,是比较理想的联合基因治疗剂量配比。更多还原

【Abstract】 Aim To investigate the effect of combined gene transfer of angiopoietin-1(Ang-1) gene and vascular endothelial growth factor(VEGF) gene,with different proportions,on angiogenesis,arteriogenesis and neovascular structure and function in a rat model of hindlimb ischemia.Methods Expression plasmid,pcD2(control plasmid),pcD2/Ang-1,pcD2/VEGF,or pcD2/Ang-1+pcD2/VEGF at different proportions,was injected intramuscularly into ischemic hindlimb rat model,followed by electroporated.VEGF and Ang-1 expressions were evaluated by RT-PCR.Angiogenesis and arteriogenesis were assessed by capillary density,arteriole density and angiography.Vascular permeability was evaluated by Evans blue uptake assays.Results Collateral vessel development was assessed at 14 days after treatment.Vessel numbers increased slightly in the rat transfected with pcD2/Ang-1 or pcD2/VEGF compared with that in the rats transfected with pcD2 alone(P<0.05).In the rats transfected with pcD2/Ang-1+pcD2/VEGF,with the increase of pcD2/Ang-1 during the pcD2/VEGF at 100 μg,the number of artery increased and was 1.9,2.4 and 2.45 times higher than that of control rats after gene delivery.With the increase of pcD2/VEGF during the pcD2/Ang-1 at 100 μg,the number of artery increased and was 2.9,2.07 and 2.39 times higher than that of control.Evans blue amount in skeletal muscle increased companied with the dosages of pcD2/VEGF.Combination with pcD2/Ang-1 decreased the vessel leakage.Evans blue uptake in group of pcD2/VEGF 100 μg+pcD2/Ang-1 200 μg was the closest to that of the normal group(P>0.05).Conclusions The current study demonstrated that co-expression of Ang-1 and VEGF genes in the ischemic muscle effectively develop leakage-resistant vessels in rat model.At a ratio of pcD2/VEGF to pcD2/Ang-1 by 1:2 is the optimal dosage.Therefore,this approach may provide a more appropriate therapeutic strategy in ischemic vascular diseases.更多还原