BDNF转染神经干细胞可降低β-淀粉样蛋白对神经元的毒性  

Brain-derived neurotrophic factor transgenic neural stem cells can alleviate toxicity of β-amyloid to cultured neurons

作　　者：陈宇新[1] 杨璐军[1] 张凤兰[2] 钟慧霖 孙海涛[1] 肖志成[2,4] 姜晓丹[1] 

机构地区：[1]南方医科大学珠江医院神经外科,广东神经外科研究所,广东省脑功能修复与再生重点实验室,广州510282 [2]昆明医科大学分子临床研究院,云南省干细胞和再生医学重点实验室,昆明650500 [3]中国科学院广州生物医药与健康研究院华南干细胞与再生医学重点实验室,广州510530 [4]澳大利亚墨尔本大学免疫与干细胞研究中心,墨尔本VIC3010

出　　处：《中华神经医学杂志》2015年第1期2-8,共7页Chinese Journal of Neuromedicine

基　　金：国家自然科学基金（81171179、81272439）;广东省自然科学基金重点项目[（2013）S2013020012754];广东省教育厅重点实验室项目（3013CXZDA008）;广州市医药健康协同创新重大创新专项（201400000003-2）

摘　　要：目的探讨神经干细胞（NSCs）转染脑源性神经生长因子（BDNF）后对β-淀粉样蛋白（Aβ）毒性作用的抵抗能力及机制。方法用脂质体介导法将BDNF基因转染到C57小鼠NSCs内．经荧光显微镜及ELISA检测转基因NSCs及其分化细胞基因表达情况。体外培养小鼠神经元，将其分成3组：a组为神经元组，b组为神经元与NSCs共培养组，c组为神经元与BDNF转基因NSCs共培养组；每组设2平行组。培养3d后，向其中一平行组加入AB。蛋白，作为d、e、f组。通过ELISA和Western blotting检验记录24h和48h后神经元生存率以及各组Aβ、总tau蛋白、磷酸化tau蛋白水平。结果BDNF转基因干细胞及其子代细胞均有BDNF表达并持续1．5周左右。以a组细胞存活率为参照．24h后d-f组细胞平均存活率分别是51．86％±0．03％、73．68％±0．04％、85．34％±0．01％：48h后d-f组细胞平均存活率分别是38．76％±0．01％、59．65％±0．04％、75．6l％±0．07％；其中d、e组细胞存活率明显低于其他各组，差异有统计意义（P〈0．05）。a-c组Aβ蛋白未能检测到，d-f组Aβ蛋白水平较高，组间差异不具有统计学意义（p〉0．05）。各组细胞总tau蛋白水平差异不具有统计学意义（P〉0．05）。各组磷酸化tau蛋白水平差异有统计学意义（P〈0．05），其中d、e组磷酸化tau蛋白水平显著高于其他各组，差异有统计学意义（P〈0．05）。结论BDNF转基因NSCs可以持续表达BDNF基因产物，降低Aβ对神经元的毒性作用，同时通过减少细胞内磷酸化tau蛋白的水平提高细胞存活率。Objective To explore the resistance of neural stem cells （NSCs） in β-amyloid （Aβ） toxicity after being transfected with brain-derived neurotrophic factor （BDNF） and its mechenism. Methods NSCs in the C57 mice were transfected with BDNF gene by using lipofectamine technique; the BDNF gene expression in transfected NSCs and their differentiated cells were detected by immune-ftuorescent staining and ELISA. Rat neurons were cultured in vitro and divided into 3 groups： neurons group, neurons co-cultured with NSCs group, neurons co-cultured with BDNF transfected NSCs group; two-parallel group design was adopted. Three d after the group culturing, cells fi＇om each parallel group were added Aβ40 protein. Neuron survival of each group was recorded 24 and 48 h after addingAβ40 by ELISA. The Aβ, tau, and phosphorylating tau （ptau） levels were detected by Western blotting. Results BDNF-transfected NSCs could continuously express BDNF gene products during the first 1.5 weeks. Both transfected NSCs and their progenies expressed BDNF gene products. Using neurons group as reference, the mean survival rates of neurons plus Aβ40 group, neurons co-cultured with NSCs plus Aβ40 group, neurons co-cultured with BDNF transfected NSCs plus Aβ40 group were 51.86%±0.03%, 73.68%±0.04% and 85.34%±0.01% at 24 h after adding Aβ40, and 38.76 %±0.01%, 59.65 %±0.04%, 75.61%± 0.07% at 48 h after adding Aβ40; the survival rate in the neurons plus Aβ40 group and neurons co-cultured with NSCs plus Aβ40 group was significantly lower than the other groups （P〈0.05）. High level of Aβ protein was noted in the neurons plus Aβ40 group, neurons co-cultured with NSCs plus Aβ40 group and neurons co-cultured with BDNF transfected NSCs plus Aβ40 group, without significant differences between each two groups （P〉0.05）. The tau protein level showed no significant differences between each two groups （P〉0.05）, while the p-tan protein level was significantly different （that in the neurons co-cultured with N

关 键 词：Β-淀粉样蛋白 TAU蛋白 脑源性神经生长因子 神经干细胞 

分 类 号：R742.5[医药卫生—神经病学与精神病学]