内皮祖细胞移植和VEGF的定向表达在转基因Alzheimer小鼠神经功能修复中的应用

Alzheimer disease (AD) is a common neurodegenerative disease among the elderly, but no effective cure has been established so far. The occurrence and development of AD are often accompanied by cerebrovascular disease and brain hypoxia in consequence. In our previous studies, we have preliminarily confirmed that intravenous transplantation of endothelial progenitor cells (EPCs) may enhance the angiogenesis of the brain of patients after cerebral infarction. Circulating EPCs were not only able to pass through the blood-brain barrier and regulate neo-vascularization in stroke patients; it has also been reported that EPCs were involved in the neuron regeneration and the enhancement of the immune system in brain through the cytokine vascular endothelial growth factor (VEGF) pathway. The aim of this proposal is to find a new strategy for the treatment of AD. For this purpose, an adenovirus expression system, Ad.pET1-VEGF, will be built to co-express mouse VEGF and a green fluorescent protein (GFP), under the control of a fragment of the mouse endothelin-1 promoter (-384 - -1). The expression of VEGF and GFP can be then triggered by vascular damage-caused hypoxia within the transplanted EPCs, facilitating the establishment of VEGF concentration gradient around damaged tissues, recruitment and migration of EPCs toward this region, and also identification of EPCs and VEGF expression in the tissues via examining GFP expression under microscope. After transfection of Ad.pET1-VEGF into EPCs, the transfected EPCs will be intravenously transplanted into APPswe/PS1dE9 transgenic AD mice that manifest cerebral amyloid angiopathy syndrome. Its effects on the cerebrovascular system, the nervous system, the brain immune system, as well as the change of cognitive behavior of AD mice will be investigated. We hope that this study may help us to broaden and deepen our understanding of the diverse and critical functions of EPCs and VEGF in AD brains, the basis of AD pathology, and to explore a feasible cell therapy for the prevention and treatment of AD.

Alzheimer病（AD）是一种常见的老年神经变性疾病，常伴随着脑血管的损伤和因此造成的脑组织缺氧，但迄今为止一直缺乏有效的临床治疗手段。血液中的内皮祖细胞（EPCs）被证明可穿过血脑屏障参与脑血管的修复，而其诱导因子VEGF参与脑内神经细胞的再生和免疫功能的增强。为此，我们拟构建小鼠Endothelin-1启动子控制表达VEGF和绿色荧光蛋白GFP的腺病毒Ad.pET1-VEGF，使VEGF和GFP能在组织缺氧区域的移植EPCs中定向表达，并方便实验观察。将其转染入EPCs后通过静脉移植到APP/PS1转基因AD小鼠体内，观察其对AD小鼠脑内血管系统、神经系统和免疫系统，以及行为认知能力的影响。通过本课题的研究，可能了解EPCs和VEGF在AD小鼠受损神经功能的全面修复过程中起到的多重作用，探索可能的细胞基因疗法，为AD的临床防治提供新的思路。

Alzheimer病（AD）是一种常见的老年神经变性疾病，常伴随着脑血管的损伤和因此造成的脑组织缺氧，但迄今为止一直缺乏有效的临床治疗手段。血液中的内皮祖细胞（EPCs）被证明可穿过血脑屏障参与脑血管的修复，细胞因子VEGF参与脑内神经细胞的再生和脑内免疫系统的增强。为此，我们用密度梯度离心法从小鼠股骨和胫骨内提取单个核细胞，分离、培养并通过免疫荧光法、荧光色素吸收法和流式细胞术鉴定得到EPCs；将鉴定成功的EPCs用含有绿色荧光蛋白（GFP）基因的腺病毒进行转染。同时构建了小鼠Endothelin-1启动子控制表达VEGF和绿色荧光蛋白GFP的腺病毒Ad.pET1-VEGF，使VEGF和GFP能在组织缺氧区域定向表达，并方便实验观测。将其转染入EPCs后通过静脉移植EPCs到APP/PS1转基因AD小鼠体内，观测到了其对AD小鼠脑内血管系统、神经系统和免疫系统，以及对AD小鼠行为认知能力的影响。希望通过本课题的研究，能够更加深入地理解EPCs和VEGF在AD小鼠受损神经功能的全面修复过程中起到的多重而关键的作用，探索可能的细胞基因疗法，为AD的临床防治提供新的思路。