自噬对早期氧糖夺模型下神经凋亡的影响.doc

自噬对早期氧糖剥夺模型下神经凋亡的影响 周迎，段满林* 周迎，221002，徐州，徐州医学院麻醉学院，E-mail：jesson1020@163.com *通讯作者：段满林，210002，南京，南京军区南京总医院麻醉科，E-mail： HYPERLINK mailto:dml9001@163.com dml9001@163.com Autophagy effects apoptosis of neurons under early oxygen-glucose deprivation ZHOU Ying，Duan Man-lin ZHOU Ying, Xuzhou 221002, Department of Anesthesiology, Xuzhou Medical College, E-mail：jesson1020@163.com Corresponding author: Duan Man-lin, Nanjing 210002, Department of Anesthesiology, General Hospital of Nanjing Military Region, E-mail： HYPERLINK mailto:dml9001@163.com dml9001@163.com 自噬对早期氧糖剥夺模型下神经凋亡的影响 【摘要】 目的 研究在氧糖剥夺的情况下体外培养的皮层神经元中自噬的表达，并探讨自噬在脑缺血缺氧损伤中的作用及相关分子机制。方法 原代神经元培养8-10天，用OGD (oxygen glucose deprivation)法建立小鼠皮层神经元体外缺血模型，用CCK-8法和Western Blot法分别检测各OGD时间点神经元的存活率和凋亡相关蛋白Caspase3的表达，Western Blot法检测自噬相关蛋白LC3B的表达，给予自噬诱导剂雷帕霉素和自噬抑制剂3-甲基腺嘌呤，运用Western blot技术检测二者对Caspase3表达的影响。结果 利用OGD法成功建立了小鼠皮层神经元体外缺血模型；CCK-8法结果证实了氧糖剥夺(OGD)处理对于神经元的损伤呈现一定的时间相关性；Western Blot显示，LC3B在OGD处理后立即上升，在1 h达到峰值(P0.05)，2 h又开始下降；用雷帕霉素处理后，抑制了Caspase 3的活化，减少神经元的凋亡(1 h)，而用3-MA处理后活化了caspase3，从而增加了神经元凋亡。结论 缺血缺氧早期自噬抑制神经元凋亡。 关键字：自噬；脑缺血缺氧；OGD；凋亡 Autophagy effects apoptosis of neurons under early oxygen-glucose deprivation 【Abstract】 Objective To study the expression of autophagy in primary cultured cortical neurons deprived of oxygen and glucose, and explore its possible function and molecular mechanism after cerebral ischemia in rats. Methods After primary cortical neurons was cultured 8-10 days, the model of oxygen-glucose deprivation (OGD) injury was established. Neuron viability and apoptosis rate ( Caspase3 ) were evaluated by CCK-8 assay and Western blot in different time periods after OGD treatment. The expression of specific protein of autophagy LC3B in OGD groups were detected by Western blot. Primary neurons pretreated with the inhibitor of autophagy 3-Methyladenine(3-MA) and the inducer of autophagy Rapamycin, Western blot assay was used to evaluate the expression of Caspase3. Results We have successfully established the