【作者】 刘科；

【导师】 唐文渊；

【作者基本信息】 重庆医科大学 ， 外科学， 2008， 博士

【摘要】 目的:血管内皮细胞生长因子( vascular endthelial growth factor ,VEGF)是一种存在于血管内皮细胞的特异性的促血管生长因子,对血管内皮细胞有强烈的促分裂、趋化作用和神经再生、修复作用。在脑缺血缺氧情况下,VEGF呈现表达上调。在创伤性脑损伤(traumatic brain injury ,TBI)的动物实验和临床研究中已发现VEGF的表达,认为TBI后VEGF的表达和上调与脑组织的自身保护有关[1-4]。本研究应用腺病毒(adenovirus, Ad)为载体的VEGF-165基因(Ad-VEGF-165)经损伤局部脑皮质处理创伤性脑损伤大鼠,观察脑挫裂伤周围组织在Ad-VEGF-165基因治疗后VEGF表达变化和CT灌注像及细胞凋亡之间的关系,研究外源性VEGF-165基因治疗TBI的可能性,探讨VEGF基因的脑保护作用,希望为颅脑创伤后的脑保护提供一些实验根据。方法:1.通过构建稳定表达的Ad- VEGF-165基因载体体系,并经损伤处脑皮质注入Ad- VEGF-165处理创伤性脑损伤大鼠,通过RT-PCR和Western blot检测脑伤后6 h、24 h、3 d、7 d、14 d VEGF-165在损伤局部的表达改变。2.用激光共聚焦显微镜系统(laser scanning confocal microscopy,LSCM)和荧光双标技术动态检测Ad- VEGF-165基因治疗TBI后VEGF-165表达和细胞凋亡的改变,了解外源性VEGF基因治疗TBI与细胞凋亡的关系。3.采用CT灌注像扫描(CT perfusion,CTP)动态检测Ad- VEGF-165基因治疗TBI前后损伤局部脑组织脑灌注的变化,观察外源性VEGF基因治疗与脑灌注的关系。结果:1.实验成功进行了携带重组腺病毒Ad-VEGF-165基因载体的构建、鉴定。外源性Ad-VEGF-165导入损伤局部脑组织后,VEGF在mRNA和蛋白质水平均有稳定的表达,并且其表达水平显著高于外伤组和质粒对照组。2.激光共聚焦显微镜观察到创伤性脑损伤后VEGF表达上调,TBI+VEGF基因组VEGF表达更为明显;TBI+VEGF基因组的细胞凋亡在伤后2 4h、3 d、7 d与TBI组比较有显著的减少,细胞凋亡与VEGF的关系密切。3.CT灌注像显示创伤后1 h损伤局部有明显的(cerebral blood flow, CBF)、脑血容量(cerebral blood volume ,CBV)降低。伤后24 h CBF、CBV开始增高,伤后3d时达到高峰。伤后14d CBV基本恢复正常。TBI+VEGF组伤后3d、7d和14d脑灌注明显高于TBI组。结论:VEGF与创伤性脑损伤关系密切。研究表明,不仅在损伤后随着时间的不同VEGF表达发生动态的改变,而且Ad-VEGF-165基因对于创伤性脑损伤后局部脑灌注有一定改善,并对减轻脑损伤组织细胞凋亡有一定的作用。从而证实了外源性的VEGF基因治疗对于创伤性脑损伤的有效性,为基因治疗脑外伤的临床应用的可能提供了一定的实验基础。更多还原

【Abstract】 Objective: Vascular endthelial growth factor (VEGF), produced by vascular endothelial cells, has been recognized as a one of the most important factor with angiogenesis, neuroprotection and nerve regeneration. Hypoxic in brain tissue due to traumatic brain injury (TBI) will induce the increased expression of VEGF. Researchers pointed out that the upgrade of VEGF expression on the experimental and clinical researches was evidenced the relationship between the VEGF and the neuroprotection. The present study was to investigate the expression level of VEGF in TBI on rat following a treatment of Ad-VEGF-165 gene applied to the injury sector using laser scanning confocal microscopy (LSCM), RT-PCR and Western blot, and the relationship between the expression level and the apoptosis, CT perfusion (CTP). This study provides both rationale and experiment evidences for neuroprotection after TBI using Ad-VEGF-165 gene.Materials and Methods:1. A recombinant adenoviral (rAd) vector carrying VEGF-165 which can be used for gene therapy in TBI was constructed, and its expression in vitro and in vivo was researched. There was an increased expression of VEGF in different time after a gene treatment by injected to the cortex of injury sector.The expression level of VEGF and apoptosis after TBI following a treatment of Ad-VEGF-165 gene was detected by immunofluorescence using LSCM.3. The dynamic CT perfusion imaging were obtained in different time on pre- and post-therapy of Ad-VEGF-165 gene to observe the changes of cerebral perfusion.Results:1. Ad-VEGF-165 gene was constructed successfully by recombinant techenic. RT-PCR and Western blot shows that the expression of VEGF mRNA and protein has been increased in injury sector after TBI with gene thearyp.2. A decreased apoptosis with increased VEGF expression observed by LSCM remarkably on 24h, 3d, 7d after TBI following an Ad-VEGF-165 gene therapy, and showed a close relationship between apoptosis and VEGF.3. The cerebral perfusion on CT perfusion imaging after TBI represents a lower CBF and CBV in early stage, and higher in 24h, 3d, 7d which indicates a hyperperfusion due to unsound hemodynamics after TBI. An improved cerebral perfusion was significant for TBI with gene therapy to build collateral circulation.Conclusions: VEGF expression in the TBI could be great associated with neuroprotection following brain harm due to hypoxia, and applying Ad-VEGF-165 to the cerebral cortex in injury sector would be an exercisable method to improve cerebral perfusion and come down apoptosis. This research provides both rationale and experiment evidences for designing high effectiveness way to the gene treatment of TBI. It lays foundation for the research on clinical application of angiogenic gene therapy of TBI.更多还原