病毒载体介导BDNF基因表达在大鼠神经元AD模型中的作用

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（Ca^2＋]）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Ca^2＋]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。

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

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.