脑源性神经生长因子经自噬对胚鼠神经元缺氧损伤的保护作用  

Brain-Derived Neurotrophic Factor Through Autophagy to Protect Oxygen Deprived Cerebral Cortical Neurons in Embryonic Rats

作　　者：陈艾[1] 周晖[1] 童煜[1] 毛萌[1] 

机构地区：[1]四川大学华西第二医院儿科,成都610041

出　　处：《实用儿科临床杂志》2012年第17期1351-1354,共4页Journal of Applied Clinical Pediatrics

基　　金：国家自然科学基金(30973215)

摘　　要：目的探讨脑源性神经生长因子(BDNF)对胚鼠缺氧神经元是否有保护作用及其机制是否与激活自噬有关。方法将SD大鼠胚鼠(孕17～19 d)的大脑皮质神经元在体外进行原代细胞培养,并进行神经元鉴定。培养7～10 d,选取生长良好的神经元用于前后两部分实验。1.第一部分随机分为3组,对照组:不加入药物,只作缺氧处理;3-甲基腺嘌呤组(3-MA组):提前加入不同浓度的3-MA后作缺氧处理,依次为5 mmol.L-13-MA组、10 mmol.L-13-MA组、20 mmol.L-13-MA组;BDNF组:提前加入不同浓度的BDNF后作缺氧处理,依次为50μg.L-1BDNF组、100μg.L-1BDNF组、200μg.L-1BDNF组。观察不同剂量3-MA、BDNF对缺氧神经元损伤的作用。之后以细胞计数盒-8(CCK-8)测定各组细胞活力,确定干预缺氧神经元的最佳药物浓度。2.第二部分分为3组,对照组:单纯缺氧,不加药物;100μg.L-1BDNF组:加入100μg.L-1BDNF;10 mmol.L-13-MA组:加入10 mmol.L-13-MA。免疫蛋白印迹法检测3组缺氧神经元在不同缺氧时间点细胞自噬微管相关蛋白轻链3(LC3)的表达情况,以LC3Ⅱ/ac-tin的相对表达量判断自噬发生的程度。结果 1.免疫荧光鉴定:与未缺氧神经元比较,缺氧神经元突触回缩,细胞网状结构被破坏。2.神经元细胞活力:50μg.L-1BDNF组缺氧神经元细胞活力最强,100μg.L-1BDNF组缺氧神经元细胞活力其次(Pa<0.05);随3-MA剂量加大,各剂量组神经元活力明显降低。3.免疫印迹:于缺氧1 h、3 h、5 h,100μg.L-1BDNF组缺氧神经元的LC3表达量,均较对照组相应时间点明显上调(Pa<0.05)。结论 BDNF通过自噬途径对缺氧损伤神经元起保护作用。Objective To investigate the protective effects of brain-derived neurotrophic factor（BDNF） on oxygen-deprived cerebral cortical neurons,and its relation to autophagy activation. Methods The cerebral cortical neuron cultures were prepared from SD rat E17-19 embryos and identified in vitro.After being cultured for 7-10 days,immunofluorescence was used to observe the morphological changes.1.The cultured medium of the neurons was assorted,and then divided into 3 groups randomly for the 1st phase test：normal neuron cultures as the control group;3-methyladenine（3-MA）group：deprived of oxygen 1 h beforehand,adding 3-MA into the normal neuron cultured medium to reach a final concentration of 5 mmol·L-1,10 mmol·L-1,and 20 mmol·L-1;BDNF group：deprived of oxygen 24 h beforehand,adding BDNF into the normal neuron cultured medium to reach a final concentration of 50 μg·L-1,100 μg·L-1 and 200 μg·L-1.The purpose of the 1st phase test was to determine the cell viability in each group of BDNF as wall as 3-MA of the oxygen-deprived neurons by cell coun-ting kit-8.2.Also the 2nd part test aimed to perceive the expression of microtubule-associated protein light chain 3（LC3） in 3 groups randomly by Western blot at 1 h,3 h,5 h after oxygen was deprived,and the intensity of autophagy was determined based on the levels of LC3 Ⅱ/actin expression.Seven-day mature neurons were divided into 3 groups randomly,control group：normal neuron cultured by only oxygen deprived;3-MA group：ahead of 1 h of oxygen deprived,adding 3-MA into the normal neuron cultured medium to the final concentration of 10 mmol·L-1;BDNF group：ahead of 24 h of oxygen deprived,adding BDNF into the normal neuron cultured medium to the final concentration of 100 μg·L-1. Results 1.Compared with the normal neurons,neurons of the oxygen-deprived groups showed synaptic retraction,and the reticular structure was destroyed.2.The cells of the 50 μg·L-1 BDNF group showed the strongest cell viability,followed by 100 μg·L-1 BDNF group（P〈0.0

关 键 词：脑源性神经生长因子 氧气剥夺 神经元 自噬 自噬微管相关蛋白轻链3 

分 类 号：R363[医药卫生—病理学]