摘要
背景：有研究者发现激活细胞外信号调节激酶的上游激酶-细胞外信号调节激酶（Extracellular signal—regulated kinase，ERK）途径并没有促神经元存活的作用，还有一些研究者发现激活该信号通路不仅不能对细胞起到保护作用，反而可促进细胞死亡。目的：实验拟观察体外培养神经元急性缺氧损伤中ERK1／2通路在脑源性神经营养因子抗缺氧损伤中的作用。设计、时间及地点：随机对照分组设计，于2005—03／2006—04在华西医院移植工程及移植免疫实验室完成。材料：孕17～19d的SD雌鼠，清洁级，体质量350～400g：ERK1／2特异性阻滞剂U0126购白Calbiochem公司；脑源性神经营养因子购白R＆D公司。方法：体外培养孕17～19d的胚鼠大脑皮质神经元，7d后作缺氧培养，并随机分为5组，缺氧组：单纯神经元缺氧培养；脑源性神经营养因子组：缺氧培养前30min加入脑源性神经营养因子50μg／L；U0126组：缺氧培养前1h加入10μmol／L的U0126；U0126＋脑源性神经营养因子组：缺氧培养前1h加入10μmol／L的UO126，30min后加入50μg/L脑源性神经营养因子；基线对照组：不做缺氧培养也不加入任何物质。主要观察指标：采用四甲基偶氮唑盐法检测0-8h内各组之间细胞存活率的差别；蛋白免疫印迹检测0-6h内各组神经元ERK1／2和磷酸化ERK1／2、cAMP反应元件结合蛋白（cAMP responsive element—binding protein，CREB）和磷酸化CREB的表达变化。结果：①脑源性神经营养因子组神经元活性在缺氧后各时间点显著高于缺氧组（P〈0．01）；U0126＋脑源性神经营养因子组细胞活性在缺氧后各时间点显著低于脑源性神经营养因子组（P〈0．01）。②外源性脑源性神经营养因子对总ERK和总CREB的表达无影响，但可显著增加磷酸化ERK和磷酸化CREB的表达：U0126对总ERK表达无影响，但可显著抑制脑源性神经营养因子作用后磷酸化ERK的上调，同时U0126也可明显抑制脑源性神经营养因子介导的磷酸化CREB的表达。结论：阻滞ERK1／2传递后脑源性神经营养因子对缺氧神经元的保护作用迅速降低，提示ERK1/2是体外培养神经元缺氧损伤中陆源件神绎营养因子保护作用的重要涂径。
BACKGROUND： It is reported that to stimulate upstream kinase-extracellular signal-regulated kinase （ERK） pathway could not promote neuron living. Other studies also confirmed that to activate this signal pathway could not protect cells, but enhance cell death.
OBJECTIVE： To investigate the effect of ERK1/2 pathway on a protective effect of brain-derived neurotrophic factor （BDNF） on hypoxia-cultured neurons in vitro.
DESIGN, TIME AND SETTING： The randomized control grouping experiment was performed at the Laboratory of Transplant Engineering and Immunology of West China Hospital from March 2005 to April 2006.
MATERIALS： The pregnant 17-19 day female SD rats, weighing 350-400 g, were used in this study. U0126 and BDNF were respectively purchased from Calbiochem and R＆D.
METHODS： Embryonic rat cortical neurons were cultured in vitro. At 7 days, cultured neurons were treated with anaerobic cultivation and randomly divided into five different groups. Neurons in the hypoxic group received anaerobic cultivation. Neurons in the BDNF group were administered with 50 μ gFL BDNF 30 minutes before hypoxia. Neurons in the U0126 group were treated with 10 μ mol/L U0126 1 hour before hypoxia. Neurons in the U0126＋BDNF group were given 10 μ mol/L U0126 1 hour before hypoxia, and 30 minutes later, 50 μ g/L BDNF. Neurons in the baseline control group were intact.
MAIN OUTCOME MEASURES： MTT assay was used to detect the viability of neurons in each group at 0-8 hours. ERK1/2, phosphate ERK1/2 and cAMP responsive element-binding protein （CREB）, phosphate CREB expressions were detected by western blotting during 0-6 hours. RESULTS： The neuron viability in the BDNF group was significantly higher than in the hypoxic group at every time point after hypoxia （P 〈 0.01）. However, the neuron viability in the U0126＋BDNF group was significantly lower than in the BDNF group at every time point （P 〈 0.01）. No changes in total ERK and CREB levels were found when added BDNF, but phosphate ERK and phosphate CREB expressions were increased. U0126 had no influence on total ERK expression, but it can inhibit the up-regulation of phosphate ERK and phosphate CREB triggered by BDNF.
CONCLUSION： Protective effect of BDNF on hypoxic neurons rapidly decreases after blocking ERK1/2 transmission. This indicates that ERK1/2 pathway plays an important role in BDNF protective effect during in vitro cultured neurons against hypoxic injury.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2008年第20期3884-3888,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
国家自然科学基金资助项目(30371489
30400478)~~
