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〈0.05）;the cell viability decreased as the dosage of 3-MA increased.3.The expression of LC3 in 100 μg·L-1 BDNF group was up-regulated at 1 hour,3 hours and 5 hours after hypoxia compared with those of the control group（Pa〈0.05）. Conclusion BDNF may protect neurons from hypoxia injury,which may attribute to its activation of autophagy.
出处
《实用儿科临床杂志》
CAS
CSCD
北大核心
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)
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
brain-derived neurotrophic factor； oxygen deprivation； neuron； autophagy； microtubule-associated protein light chain 3；
