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。
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 ofhBDNF and Bcl-2 protein were determined by immunocytochemical staining. Laser scanning confocal microscopy （LSCM） was used to observe the changes of [Ca^2＋]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 [Ca^2＋]i was maintained in BDNF-treated cells with beta-amyloid exposure. Conclusion hBDNF expression can effectively protect cultured rat hippocampal cells fi-om beta-amyloid-induced apoptosis through inhibiting the intracellular calcium overload and increasing the expression of Bcl-2 protein.
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2006年第10期1388-1393,共6页
Journal of Southern Medical University
基金
陕西省自然科学基金(2003K10-G83-2)~~
关键词
阿尔茨海默病
脑源性神经营养因子
腺伴随病毒
钙超载
Alzheimer＇s disease
brain-derived neurotrophic factor
adeno associated virus
calcium overload
