原代缺氧缺糖损伤神经元模型Cdh1及其下游底物的表达

摘要

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.%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1h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。