不同途径导入rAAV-VEGF165基因对大鼠脑缺血边缘区血管内皮细胞生长因子表达的影响

背景VEGF基因可促进脑缺血边缘区的血管生长,哪种导入途径的表达效果更理想值得探讨.目的探讨脑池内及静脉导入rAAV-VEGF165基因对大鼠脑缺血边缘区血管内皮细胞生长因子(vascularendothelial growthfactor,VEGF)表达的影响,为VEGF基因治疗脑缺血提供实验依据.设计析因设计.地点和对象实验地点深圳市人民医院动物实验中心;暨南大学医学院病理教研室.健康雄性SD大鼠48只,SPF级.干预措施用线栓加环扎法建立SD大鼠持续性大脑中动脉闭塞(middlecerebralartery occlusion,MCAO)模型.SD大鼠48只,随机分为脑脊液导入组、静脉导入组、梗死组和假手术组,治疗组于术后24h内将rAAV-VEGF165基因通过小脑延髓池或静脉内导入.各组分别取6只大鼠于术后7d和14 d断头取脑,免疫组织化学染色检测VEGF的表达.主要观察指标各组大鼠不同时间脑组织VEGF阳性细胞密度.结果最终进入统计分析的大鼠保持为4组,每组12只,无缺失值.各组脑缺血边缘区VEGF阳性细胞密度(个/高倍视野,×400)分别为7d组脑脊液导人组74.90±2.33、静脉导人组36.27±2.61、梗死组24.27±1.69和假手术组5.65±0.47(P＜0.05);14d组脑脊液导入组95.03±1.55、静脉导人组69.17±4.29、单纯梗死组29.95±1.05和假手术组7.30±0.76(P＜0.05).结论在rAAV为载体介导下,VEGF165基因可以通过脑池内及静脉内导人转染到大鼠缺血脑组织中并表达VEGF,促进新生血管的形成,保护神经细胞,治疗脑缺血.

Impact of rAAV-VEGF165gene introduced with different pathways on the expression of vascular endothelial growthfactor in cerebral ischemic penumbra in rats

BACKGROUND: VEGF gene promote the growth of blood vessel. Which introducing pathway achieve a more ideal expression remains further discussion.OBJECTIVE: To investigate the impact of rAAV- VEGF165 gene transferred through both intravenous route and cerebrospinal fluid(CSF) route on the expression of vascular endothelial growth factor(VEGF) in cerebral ischemic penumbra for providing a laboratorial gist in the VEGF gene therapy in cerebral ischemia.DESIGN: A factorial design.SETTINGS and MATERIALS: Study was conducted in the animal laboratorial center of Shenzhen People' s Hospital and the Department of Pathology of Medical College of Jinan University. Forty-eight male SD rats in a SPF grade were selected.INTERVENTION: A model of permanent middle cerebral artery occlusion (MACO) was established by nylon suture embolization and cerclage in SD rats. Forty-eight SD rats were randomly divided into CSF route group, intravenous route group, infarct group and sham-operation group. The rAAV-VEGF165 gene was transferred through CSF injection or intravenous injection within 24 hours in the therapy groups. On the 7th and 14th day, the brains of the six rats from each group were removed after the animals were executed for immunohistochemical analysis to detect the expression of VEGF gene in ischemic penumbra(IP).MAIN OUTCOME MEASURES: The density of the VEGF Immunoreactive(IR) -positive cell in the cerebral tissue over time in the rats from each group.RESULTS: The numbers of rats finally entered into statistical analysis were four groups with 12 rats each without any loss. The density of VEGF-IR-positive cell(under high power field, x 400) in cerebral IP of each group at the 7th day was 74. 90 ±2.33 in CSF group, 36. 27 ±2.61 in intravenous group, 24.27±1.69) in infarct group and 5.65±0.47 in sham-operation group respectively(P ＜ 0.05); and at the 14th day was 95.03 ± 1.55 in CSF group, 69.71 ±4. 29 in intravenous group, 29.95 ±1.05 in infarct group and 7.30 ± 0. 76 in sham-operation group respectively (P ＜ 0. 05).CONCLUSION: Under the mediation of rAAV, VEGF165 gene can be transfected into the cerebral ischemic tissue through both CSF route and intravenons route to express VEGF, which can promote the formation of new vessels and protect neurocytes to treat cerebral ischemia.