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  

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

作　　者：鄢华[1] 吴小兵 郭艳红[3] 张鹏[4] 陈莉[4] 董小岩 王贵松[3] 高炜[3] 

机构地区：[1]武汉市亚洲心脏病医院 [2]本元正阳基因有限公司 [3]北京大学第三医院心内科,100083 [4]北京大学医学部心血管所

出　　处：《中华心血管病杂志》2007年第1期69-73,共5页Chinese Journal of Cardiology

基　　金：国家高技术研究发展计划(2001AA217111,2002AA2Z3324)

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^(11)vg 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)/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。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 × 10^11vg 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 rAAVl-VEGF165 vector might be an effective therapeutic approach for ischemic cardiovascular diseases.

关 键 词：新生血管化 生理性 血管 内皮生长因子 腺相关病毒 

分 类 号：R54[医药卫生—心血管疾病]