浅析低氧促进体外培养的星形胶质细胞内β-catenin积聚的机制.docVIP

浅析低氧促进体外培养的星形胶质细胞内β-catenin积聚的机制 【摘要】 目的探讨低氧对星形胶质细胞内β-catenin积聚的机制。方法体外分离昆明小鼠海马星形胶质细胞，分别置于常氧和低氧条件下培养12h和24h后，利用RT-PCR和Western blot法检测星形胶质细胞内Wnt通路相关蛋白Wnt3、Wnt3a及磷脂酰肌醇-3激酶/Akt(PI3K/Akt)通路中磷酸化Akt(p-Akt)和磷酸化糖原合成酶激酶-3β(p-GSK-3β)蛋白的表达情况。结果常氧培养的海马星形胶质细胞内存在Wnt3的表达，低氧对海马星形胶质细胞内Wnt3的表达无影响(P0.05);常氧和低氧培养的海马星形胶质细胞内均不存在Wnt3a表达;低氧增加海马星形胶质细胞的p-Akt和p-GSK-3β蛋白水平(P0.05)。结论低氧通过增加海马星形胶质细胞的p-Akt蛋白和p-GSK-3β蛋白水平促进β-catenin积聚。 【关键词】 低氧;星形胶质细胞;Wnt;β-catenin;Akt;GSK-3β 　 ABSTRACT: ObjectiveTo explore the mechanism of hypoxia-promoted accumulation of β-catenin in astrocytes.MethodsAstrocytes were isolated, cultured and identified from Kunming mouse hippocampus in vitro, then cultured in hypoxia and traditional oxygen for 24 hours and 12 hours, respectively. The expressions of Wnt3 and Wnt3a in hippocampus astrocytes were detected by RT-PCR. The effect of hypoxia on the expressions of Wnt3 and Wnt3a in hippocampus astrocytes was analyzed by RT-PCR. The influence of hypoxia on the expressions of phosphorylated Akt and glycogen synthase kinase-3β(GKS-3β) protEin was analyzed by Western blot.ResultsWnt3 was expressed in hippocampus astrocytes and hypoxia did not affect its expression (P0.05). Wnt3a was not detected in hippocampus astrocytes in traditional culture or hypoxic culture condition. Hypoxia promoted the level of phosphorylated Akt and GKS-3β protEIn (P0.05).ConclusionHypoxia increases the expression of β-catenin in astrocytes through promoting the level of phosphorylated Akt and GKS-3β protein in vitro. 　　KEY WORDS: hypoxia; astrocyte; Wnt; β-catenin; Akt;glycogen synthase kinase-3β(GSK-3β) 　　低氧预适应是指轻度低氧和短暂缺血的重复预处理触发和动员机体内在的防护能力，从而对随后的严重低氧或缺血损伤产生强大的防御和保护作用[1]。在整体动物脑缺血预适应中，星形胶质细胞可能释放多种因子，作用于神经元[2-3]，但其具体的作用机制尚不清楚。研究表明，在低氧预处理后离体及在体动物均发生反应性星形胶质细胞增生，其作用可能与β-catenin的过度激活有关[4-5]。我们在前期工作中亦发现，β-catenin可促进(30±20)mL/L低氧条件下体外培养的神经前体细胞和胶质前体细胞增殖，并可在低氧培养的星形胶质细胞中积聚[6]，但其作用机制尚未明确。本实验选用海马星形胶质细胞，探讨低氧培养条件下，星形胶质细胞内β-catenin积聚的可能机制。 　　1材料与方法 　　1.1材料DMEM/F12、胎牛血清(FBS)购自Hyclone公司，兔抗胶质原纤维酸性蛋白(glial fibrillar