rAAV1介导血管内皮生长因子基因表达对大鼠脑缺血新生血管形成及神经功能恢复的影响

Recombinant AAV1 mediated vascular endothelial growth factor gene expression promotes angiogenesis and improves neural function：experiment with rats

目的探讨血管内皮生长因子（VEGF）基因治疗对大鼠脑缺血的治疗作用及其机制。方法成年雄性SD大鼠64只，分为对照组和治疗组，每组均32只，采用立体定向微量注射的方法将相同滴度的rAAV1-VEGF（治疗组）和rAAV1-LacZ（对照组）注人大鼠侧脑室，21d后制作大脑中动脉区缺血再灌注（MCAO）模型，于不同时间点对各组动物进行神经损伤程度评分（NSS），以免疫定量分析法检测各组大鼠脑组织中VEGF的表达量、以免疫组织化学染色检测脑内VEGF的表达部位和微血管密度（MVD），并行vWF和BrdU的免疫荧光双重标记，采用尾静脉注射FITC-葡聚糖法行脑微血管灌注成像，并对各组缺血半暗带区域的脑微血管灌注情况进行评估。结果（1）治疗组的NSS评分有明显改善；（2）VEGF可在脑内多个结构表达，其VEGF表达量是对照组的27倍；（3）治疗组MVD为157±13、对照组为89±9（P〈0．05），且治疗组半暗带区域可见大量BrdU阳性的内皮细胞，而对照组未见；（4）缺血半暗带区域微血管显影面积为（152617±13076）μm^2／mm^2，对照组为（91658±6577）μm^2／mm^2（P〈0．05）。结论rAAV1-VEGF可介导VEGF在大脑内表达，并对脑缺血大鼠神经功能的恢复具有促进作用，其治疗机制可能与VEGF促进新生血管形成并改善脑组织供血有关。

Objective To investigate the therapeutic effect of vascular endothelial growth factor （VEGF） gene expression mediated by recombinant AAV1 （rAAV1） vector in brain ischemia and the mechanism thereof. Methods Sixty-four SD rats were randomly divided into 2 equal groups and received intra-ventricular injection with rAAV1-VEGF or rAAVl-lacZ as controls. 21 days later the rats underwent transient middle cerebral artery occlusion （MCAO）. Neurological severity score （NSS） was recorded 1, 2, 3, 7, 14, and 21 days after MCAO. 48 rats were sacrificed 21 days after MCAO and brains were taken out from 48 rats. Immune quantitative analysis was used to identify the quantity of VEGF expression. Immunohistochemistry was used to identify the site of VEGF expression. Immunoflnoreseence double labeling of yon Willebrand factor （vWF） and 5-bromodeoxy-uridine （BrdU） was performed to detect the proliferation of endothelial cells. Fluoreseein isothiocyanate （ FITC ） -dextran was infused into the caudal vein of 8 rats from each group and then the rats were killed with their brains taken out to evaluate the cerebral microvessel perfusion and microvessel density. Results The NSSs of the VEGF group 7, 14, and 21 days after MCAO were all significantly lower than those of the control group （ all P 〈 0. 05 ） , and the VEGF165 protein expression quantity was 27 times as that of the control group （P 〈 0.05 ） . Immunohistochemistry demonstrated that VEGF expression was distributed mainly in the caudate putamen, corpus callosurn, choroid plexus, and hippocampus in the VEGF group, while no expression was detected in the control group. The microvessel density of the VEGF group was 157 ± 13, significantly higher than that of the control group [ （ 89 ± 9 ）, P 〈 0. 05 ]. BrdU ＋/vWF ＋ endothelial cells were detected in the area adjacent to the MCAO. The density of microvessel infused with FITC-dextran was （ 152 617 ± 13 076） μm^2/mm^2 in the VEGF group, significantly higher than that of the control group [ （ 91 658± 6577 ） μm^2/mm^2, P 〈 0. 05]Conclusion rAAV1 mediates the VEGF gene expression in multiple structures in the brain and attenuates the neurological deficit of MCAO. VEGF gene transfer may stimulate angiogenesis and improves blood supply in brain. Neovascnlarization may be a therapeutic strategy for brain ischemia.