rAAV1载体介导外源性VEGF-165和Angiopoietin-1基因治疗大鼠脑缺血的疗效评价及其机制研究

rAAV1-mediated Gene Over-expression of VEGF-165 and Angiopoietin-1 in Rat Cerebral Ischemia Model: An Investigation on Its Efficacy and Possible Mechanisms

为研究经脑室途径联合应用血管内皮生长因子(VEGF)和血管生成素(Angiopoietin-1,Ang-1),治疗大鼠急性脑梗塞的效果,并探讨治疗的机制,采用脑立体定向输注的方法,将rAAV1-VEGF治疗载体或者rAAV1-VEGF和rAAV1-Ang-1的混合治疗载体,通过侧脑室转染途径对大鼠大脑中动脉阻塞缺血再灌注模型进行基因治疗。观测VEGF和Ang-1蛋白表达、血脑屏障通透性、脑微血管密度等指标,并对大鼠进行神经功能行为评分等。结果显示,联合应用VEGF和Ang-1治疗急性脑梗塞可降低血脑屏障通透性,减轻脑水肿,增加缺血灶周围脑区的微血管密度,改善大鼠的神经功能。由此得出结论:联合应用VEGF和Ang-1基因治疗大鼠急性脑缺血,可保护脑细胞,促进新生血管生成,减轻脑水肿,改善大鼠的神经功能。

To examine the effects of intra-ventricular pre-treatment with a combination of recombinant Adeno-Associated Virus vectors encoding VEGF （rAAV1-VEGF） and Ang-1 （rAAV1-Ang-I） on early stroke in a rat model of transient Middle Cerebral Artery Occlusion （tMCAO）, rAAV1-VEGF/rAAV1-Ang-1 or rAAV1- VEGF/rAAVI-null vector were delivered into the lateral ventricle of each rats. After eight weeks later, the rats were subjected to tMCAO for two hours. During the early stages of ischemic reperfusion, VEGF and Ang-1 expression levels, Blood Brain Barrier （BBB） permeability and cerebral microvessel density were determined and compared statistically between groups. Cerebral infarct volume and modified Neurological Severity Scores （NSS） were also determined to evaluate the therapeutic efficacy of rAAV1-VEGF/rAAV1-Ang-1. The results show that the intra-ventricular application of rAAV1-VEGF/ rAAV1-Ang-1, eight weeks before tMCAO, results in VEGF and Angl overexpression, and significantly reduces Evans blue permeability following ischemia （P〈0.05）. The microvessel density in the peri-infaret zone is significantly increased in the rAAV1-VEGF/ rAAV1-Ang-1 group as compared with the rAAVI-VEGF/rAAV1- null group（P〈0.05）. Cerebral infarct volume and NSS in the rAAV1- VEGF/rAAV1-A ng-1 group are significantly decreased as compared with the rAAV1-VEGF/rAAVI-null group （P〈0.05）. It is concluded that, in cerebral ischemia, the combination of Ang-1 and VEGF could be used early to promote the formation of mature neovessels and protect the injured cells, without inducing the side effects on BBB permeability. Early intra-ventricular injection of mixed rAAV1-VEGF and rAAV-Ang-1 may he a favorable therapeutic strategy in gene therapy for stroke.