表达BDNF的脂肪干细胞对 5xFAD小鼠海马结构重建的实验研究

Pathologically, AD is characterized by the progressive accumulation of β-amyloid (Aβ) plaques and neurofibrillary tangles within the brain. Together, these pathologies lead to marked neuronal and synaptic loss and corresponding impairments in cognition. Current treatments, and recent clinical trials, have failed to modify the clinical course of AD; thus, the development of novel and innovative therapies is urgently needed. Recent studies have suggested that stem cell transplantation could serve as a potential therapeutic strategy to halt or ameliorate the inexorable disease progression. But it is unclear which types of stem cells and nerve factors work on synaptic structure. Acetylcholine transferase activity decreased in the hippocampus of 4-6 months age 5 × FAD mouse model of Alzheimer’s disease leading abnormal behavior. Due to BDNF is a key factor for the synaptic plasticity, improve ASCs differentiation into neurons, and also can through the blood brain barrier. In this project, We use ASCs designed to transient over-express BDNF, transplant into hippocampus of 4-6 months age 5xFAD mouse model of Alzheimer's disease to test animal model behavior; observe hippocampus structure, synaptic density, synapse formation, synapse plasticity, dendritic apine morphology by using TEM; evaluate the induction of LTP in CA1 area of hippocampus; observe the expression of synapsin, synaptophysin, GAP-43 , NEP and Choline acetyltransferase by immunofluorescence. The aim is to provide the molecular mechanism of BDNF and ASCs improving AD hippocampus for further study.

阿尔茨海默病（AD）是一种以记忆和认知损害为主要特征的神经系统退行性疾病，发病机理不明确，尚无有效治疗手段。目前尚不清楚各类干细胞及神经因子对于海马突触结构有何影响。4-6月龄5×FAD 小鼠出现海马依懒的行为异常，海马内乙酰胆碱转移酶活性下降。BDNF是神经突触塑形的关键因子并有助ASCs向神经元定向分化，且ASCs可通过血脑屏障。因此，本课题拟采用转染BDNF基因的ASCs 对4-6月龄5×FAD海马内移植，通过观察小鼠行为学、海马形态学及电生理学的改变，并通过免疫荧光检测synapsin、synaptophysin, GAP-43 、NEP及ChAT的表达，观察BDNF和ASCs对AD小鼠突触塑形影响，探讨其对海马神经元功能重建的形态学及生理学机制，为深入研究BDNF及ASCs改善AD病变的分子机制提供依据。

神经元丢失是阿尔茨海默病（AD）患者的常见病理改变。神经元的丢失可以恢复吗？为了解决这个重要问题，课题组将表达BDNF的脂肪组织干细胞（ASC）移植到7月龄5×FAD转基因小鼠的海马体中。移植六个月后，使用免疫组织化学（淀粉样蛋白为6E10抗体，海马中追踪ADSC的Flag-Tag抗体），实时荧光定量PCR和Western印迹（BDNF，NEP-2和MAP2抗体）。我们发现，移植后6个月，海马中存在活的BDNF-ASC。将这些细胞迁移至RSD区，RSGc区和CA1 oriens层海马中。同样在海马血管中发现了一些BDNF-ASCs。BDNF-ASCs组的海马区Aβ斑块减少。BDNF-ASCs移植显着增加5×FAD转基因小鼠海马中BDNF和NEP的表达。此外，与对照相比，海马神经元的树突更长且分布更密集。对5×FAD转基因小鼠行为学缺陷有一定的改善作用。课题组的研究发现可能为使用BDNF-ASCs移植治疗AD提供新的治疗策略。