摘要
目的探讨NMDA受体激活引起的突触活动诱导Wnt非经典通路的活化。方法构建C57BL/6J胎鼠大脑皮层神经元原代培养体系,用NMDA处理神经元细胞,并结合Western blotting、双免疫荧光染色等技术,检测神经元细胞内Wnt非经典通路的相关蛋白的变化。结果免疫荧光染色显示成功建立了C57BL/6J胎鼠大脑皮层神经元体外培养体系,原代神经元细胞在体外培养10d生长良好,且纯度达90%;体外培养的神经元细胞内存在Wnt5a神经递质,经NMDA的刺激,发现Wnt非经典通路的两个标志性蛋白CaMKII和JNK的磷酸化水平显著增加,且Wnt非经典通路的一种受体Frizzled-5的蛋白表达水平也显著增加。进一步的研究显示,用NMDA竞争性抑制剂DAP5能够阻断NMDA引起的CaMKII和JNK蛋白的磷酸化水平的提高。结论 NMDA受体的激活会诱导Wnt非经典通路的活化。
Objective To investigate whether synaptic NMDA receptor activation induces Wnt non-canonical signaling pathway activation.Methods Various methods such as primary cortical neuron culture,immunofluorescence staining,double immunofluorescence staining,and Western blotting combined with NMDA activation were used to assess the increase of some proteins in Wnt non-canonical pathway.Results The methods of primary cortical neuron culture were established and the purity of neurons was enough for experiment.Wnt5a was expressed in the cells of the primary cortical cultures,and p-CaMKII and p-JNK expressions were markedly increased after NMDA stimulation.Moreover,NMDA competitive inhibitor DAP5 could inhibit the increase of NMDA-induced p-CaMKII and p-JNK phosphorylation.NMDA stimulation also led to Wnt non-canonical receptor Frizzled-5 rapidly increased.Conclusion Synaptic NMDA receptor activation can induce Wnt non-canonical signaling pathway activation.
出处
《中国实验动物学报》
CAS
CSCD
2011年第3期181-187,275,共8页
Acta Laboratorium Animalis Scientia Sinica
基金
国家自然科学基金(30873457)
广东省科技计划项目(2008A060202010
2010B050700019)资助项目