新型血清型1型重组腺相关病毒载体携带血管内皮生长因子基因促血管生成的实验研究

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10^5vg 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3×10^11vg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120h60％～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）ps／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）／mm^2,（17．0±1．2）／mm^2。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。

The in vivo and in vitro effects on neovascularization of VEGF gene transfer with a new pseudotyped rAAV1 vector

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 [10(5) 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 ischemic mouse model, PBS, 3 x 10(11)vg 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)/mm(2)] and arteriogenesis [(17.0 +/- 1.2)/mm(2)] 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.