【摘要】 目的探讨采用腺相关病毒血清型1型的衣壳蛋白构建的"假毒粒"型重组腺相关病毒（rAAV）1载体介导血管内皮生长因子（VEGF）促血管新生的可行性、有效性。方法以每个细胞10⁵vg rAAV1-绿色荧光蛋白（GFP）及 rAAV1-VEGF 载体量感染 C2C12细胞（小鼠成肌细胞）分化的肌管,荧光显微镜下观察 rAAV1-GFP 的转染效率。ELISA 法检测 rAAV1-VEGF 转染后细胞上清中VEGF 表达量。构建小鼠后肢缺血模型,术后10天每只小鼠股部缺血骨骼肌内注射3×10¹¹vg 的rAAV1-VEGF 载体,载体转染后1个月 ELISA 法检测小鼠缺血骨骼肌中 VEGF 蛋白表达量。载体转染后6周免疫组化检测小鼠缺血骨骼肌中毛细血管及小动脉新生情况。结果 rAAV1-GFP 感染后120 h 60%～80%的肌管可表达GFP。rAAV1-VEGF 载体感染后第3天细胞上清中分泌的 VEGF 表达量达到高峰,VEGF 浓度为（567.7±16.8）pg/ml。小鼠缺血骨骼肌中 rAAV1-LacZ 载体转染后1个月转染效率可达100%。rAAV1-VEGF 载体转染后1个月平均 VEGF 浓度为（205.4±36.1）pg/mg总蛋白,与对照组相比差异有统计学意义（P<0.01,n=5）。rAAV1-VEGF 载体转染有效促进了血管新生（P<0.001,n=6）,载体转染6周缺血骨骼肌中毛细血管计数与小动脉计数分别为（147.0±13.3）/mm²,（17.0±1.2）/mm²。结论新型"假毒粒"型 rAAV1-VEGF 载体可能是治疗缺血性心血管疾病更为优越的基因治疗载体。更多还原

【Abstract】 Objective To investigate the effects of vascular endothelial growth factor（VEGF）gene transfer with a new pseudotyped recombinant adeno-associated virus（rAAV）vector with AAV serotype 1 capsid protein（rAAV1）vector on neovascularization.Methods PBS,rAAV1-GFP and rAAV1-VEGF vectors were added in C2C12 derived myotubes[10⁵ vg（vector genomes）per cell].Transfer efficiency was determined by fluorescent microscope and VEGF protein concentration in the culture media measured by ELISA.Ten days following ischemia in a hindlimb ischemie mouse model,PBS,3×10¹¹vg rAAV1-LacZ vectors and rAAV1-VEGF165 vectors were injected in ischemic thigh muscles.VEGF protein at ischemic thigh muscle was measured by ELISA at 1 month after vector infection.Capillaries and arterioles were observed by immunohistochemical analysis at 6 weeks after vector infection.Results GFP expression was found in 60%-80% myotubes at 120 hours after rAAV1-GFP infection.VEGF protein peaked at the 3rd day post rAAV1-VEGF infection with an average concentration of（567.7±16.8）pg/ml.Transfer efficacy in ischemic thigh muscle was 100% one month post rAAV1-LacZ infection.The average concentration of VEGF protein in ischemic skeletal muscles is（205.4±36.1）pg/mg total protein in rAAV1-VEGF165 treated mice.Extensive angiogenesis[（147.0±13.3）/mm²]and arteriogenesis[（17.0±1.2）/mm²]were observed in ischemic skeletal muscles at 6 weeks post rAAV1-VEGF165 injection.Conclusion Gene transfer with the new psudotyped rAAV1-VEGF165 vector might be an effective therapeutic approach for ischemic cardiovascular diseases.更多还原