南方医科大学学报

南方医科大学学报 ›› 2006, Vol. 26 ›› Issue (10): 1388-1393.

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病毒载体介导BDNF基因表达在大鼠神经元AD模型中的作用

刘朝晖； 马东亮； 靳辉； 马延兵； 胡海涛；   

  1. 西安交通大学医学院解剖与组织胚胎学系环境与基因相关疾病教育部重点实验室； 西安交通大学医学院解剖与组织胚胎学系环境与基因相关疾病教育部重点实验室 陕西西安710061； 陕西西安710061；
  • 出版日期:2006-10-20 发布日期:2006-10-20
  • 基金资助:
    陕西省自然科学基金(2003K10-G83-2)~~

Protective effect of adeno-associated viral vector-mediated expression of human brain-derived neurotrophic factor in rat neurons against beta-amyloid-induced Alzheimer’s disease in vitro

LIU Zhao-hui, MA Dong-liang, JIN Hui, MA Yan-bing, HU Hai-tao Department of Anatomy and Histology-Embryology, Key Laboratory of Environment and Genes Related of Disease, Ministry of Education, School of Medicine, Xi’an Jiaotong University, Xi’an 710061, China   

  1. 西安交通大学医学院解剖与组织胚胎学系环境与基因相关疾病教育部重点实验室； 西安交通大学医学院解剖与组织胚胎学系环境与基因相关疾病教育部重点实验室 陕西西安710061； 陕西西安710061；
  • Online:2006-10-20 Published:2006-10-20

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摘要： 目的探讨重组腺伴随病毒(rAAV)载体介导表达人脑源性神经营养因子(hBDNF)基因以及表达的hBDNF对β淀粉样蛋白(Aβ)诱导的AD模型神经元保护效应的机制。方法使用分子克隆技术克隆了hBDNF基因,并且构建了携带hBDNF基因的rAAV病毒载体(AAV-hBDNF),使用病毒载体转染Aβ诱导损伤的海马神经元。使用MTT检测和流式细胞仪分析观察细胞凋亡变化,同时使用免疫细胞化学技术检测了BDNF蛋白以及Bcl-2抗凋亡蛋白的表达,使用激光共聚焦显微镜观察细胞内游离钙离子浓度([Ca2+]i)的变化。结果结果显示重组病毒对培养的海马神经元进行了有效的转染,BDNF蛋白表达水平明显增高,表达的BDNF对Aβ诱导的神经元损伤有显著的保护效应,在BDNF治疗组表现出抗凋亡蛋白Bcl-2的表达增高和有效地维持了[Ca2+]i平衡。结论表达的BDNF通过抑制Aβ依赖的细胞内钙超载和增加抗凋亡蛋白Bcl-2的表达,有效地保护神经元抵抗Aβ神经毒性引起的凋亡。 

Abstract: Objective To achieve expression of human brain-derived neurotrophic factor (hBDNF) mediated by recombinant adeno-associated virus (rAAV) and explore the mechanism of its neuroprotective effects in rat neurons against beta-amyloid-induced Alzheimer’s disease. Methods Using molecular cloning technique, rAAV vector containing hBDNF gene (AAV-hBDNF) was constructed to transfect SD rat hippocampal neurons exposed to beta-amyloid treatment. The changes in cell apoptosis were observed by MTT assay and flow cytometry, and the expression of hBDNF and Bcl-2 protein were determined by immunocytochemical staining. Laser scanning confocal microscopy (LSCM) was used to observe the changes of [Ca2+]i. Results The cultured rat hippocampal neurons were effectively transfected with AAV-hBDNF and expression of BDNF protein was obviously increased. hBNDF expression showed significant protective effects against beta-amyloid-induced neuronal damage, and the expression of Bcl-2 protein was increased significantly and the balance of [Ca2+]i was maintained in BDNF-treated cells with beta-amyloid exposure. Conclusion hBDNF expression can effectively protect cultured rat hippocampal cells from beta-amyloid-induced apoptosis through inhibiting the intracellular calcium overload and increasing the expression of Bcl-2 protein. 

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