原代缺氧缺糖损伤神经元模型Cdh1及其下游底物的表达

Expression of Cdh1 and its downstream substrates in primary neurons after oxygen-glucose deprivation

背景:课题组前期实验已证实Cdh1在大鼠海马、皮质均有大量表达,且体外实验发现神经元中Cdh1表达高于神经干细胞,可能与神经干细胞向神经元分化有关。但细胞周期末期促进复合物调节亚基Cdh1在缺血性神经元损伤中的作用,尚不明确。目的:体外构建原代缺氧缺糖损伤神经元模型,观察Cdh1及其下游底物表达变化。方法:取出生24 h内乳鼠大脑皮质,体外培养原代神经元并通过免疫荧光染色进行鉴定。使用无糖Earle’s液替代细胞培养液,利用三气培养箱充以氮气建立原代神经元缺氧缺糖模型,缺氧处理1 h后复氧。于缺氧前、缺氧缺糖损伤后6 h、1 d,3 d,7 d采用实时荧光定量PCR检测神经元Cdh1及其下游底物Skp2、Cyclin B1的表达。结果与结论:体外缺氧缺糖损伤后,原代神经元Cdh1及其下游底物Cyclin B1表达上调(P<0.05),Skp2表达均下调(P<0.05)。提示,体外缺氧缺糖损伤后神经元Cdh1表达升高,可能通过泛素化降解Skp2参与缺氧性神经元凋亡等病理过程。

BACKGROUND：Cdh1 has been shown to express in rat hippocampus and cortex in a large number. Moreover, in vitro test demonstrated that Cdh1 expression was higher in neurons than in neural stem cel s, which possibly associated with the differentiation of neural stem cel s into neurons. However, the effects of anaphase promoting complex Cdh1 on ischemic neuronal damage remain unclear. OBJECTIVE：To investigate the expression of Cdh1 and its downstream substrate in primary cultured neurons with oxygen-glucose deprivation. METHODS：Primary neurons from cortex of postnatal 24-hour rat pups were cultured in vitro, and identified by immunofluorescence staining. The oxygen-and glucose-deprived models were established by three gas incubator fil ed with nitrogen in sugar-free Earle’s solution. After 1 hour of hypoxia, reoxygenation was conducted. Real-time fluorescent quantitative PCR was used to detect the mRNA expression of Cdh1 and its downstream substrates Skp2, Cyclin B1 before hypoxia, 6 hours, 1, 3, 7 days after oxygen glucose deprivation. RESULTS AND CONCLUSION：After oxygen glucose deprivation, the expression of Cdh1 and Cyclin B1 in primary neurons was increased （P〈0.05）, while Skp2 expression was decreased （P〈0.05）. Above data indicated that Cdh1 expression in neurons increased after oxygen-glucose deprivation. It may degrade Skp2 and participate in hypoxic neuronal apoptosis by ubiquitination.