The novel analyzing assay for neuroprotection of the inhalation anesthetics by immuno-precipitation and the protein kinases related cerebral ischemia.

通过免疫沉淀和与脑缺血相关的蛋白激酶对吸入麻醉剂进行神经保护的新型分析方法。

基本信息

批准号：
17591652
负责人：
MATSUMOTO Shohei
金额：
$ 2.37万
依托单位：
Tokyo Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

项目摘要

Male C57/Black mice were decapitated after inhalation of isoflurane or sevoflurane, and N-methyl-D-aspartate (NMDA) receptor subunits NR2A, NR2B were measured. The interaction between these subunits and calcium-calmodulin dependent protein kinase II (CaMKII) was also measured. Immuno-precipitation and western blot analysis were used for these studies.Method : Mice ischemia model was made by decapitation. After decapitation, the skull was incubated in the 37℃ saline 3 or 5 min, then stored-80℃. Previous to the decapitation, 1MAC or 2MAC isoflurane or sevoflurane were inhalated. The cerebrum was dissected from the skull in the cold globe box (-28℃), then homogenized. Using these samples immuno-precipitation was performed targeting to phosphor-tyrosine. Then the samples were assayed by SDS-PAGE, following western blot analysis was performed with anti-NR2A antibody and anti-NR2B antibody. Likewise, the interaction between phosphor-CaMKII and NR2A, B subunits were also examined.Results : NR2A phosphorylation was decreased when isoflurane and sevoflurane inhalation. The change of NR2B was not obvious. There is a tendency that the change of NR2A phosphorylation is dozing dependent for inhalation anesthetics. The interaction between phosphor-CaMKII and NR2A. B subunits has not been evident so far.Conclusion : Inhalation anesthetics prevent CaMKII translocation to synaptic membrane, subsequently the interaction between phosphor-CaMKII and NR2A is decreased, resulting down regulation of NR2A phosphorylation which reeds to neuroprotecition.

吸入异氟烷或七氟硫烷后，将雄性C57/黑色小鼠斩首，并测量N-甲基-D-天冬氨酸（NMDA）受体亚基NR2A，NR2B。还测量了这些亚基与钙 - 钙统治蛋白激酶II（CAMKII）之间的相互作用。这些研究使用了免疫沉淀和蛋白质印迹分析。方法：小鼠缺血模型是通过斩首做出的。斩首后，将头骨在37盐水3或5分钟内孵育，然后储存80℃。在斩首之前，吸入了1MAC或2MAC异氟烷或七氟烷。从冷地球盒（-28℃）的头骨中解剖大脑，然后均匀化。使用这些样品对磷酸酪氨酸进行了免疫沉淀的靶向。然后通过抗NR2A抗体和抗NR2B抗体进行蛋白质印迹分析后通过SDS-PAGE分配样品。同样，还检查了磷酸-CAMKII和NR2A，B亚基之间的相互作用。分子：当异氟烷和七氟硫烷吸入时，改善了NR2A磷酸化。 NR2B的变化并不明显。有一种趋势，即NR2A磷酸化的变化是在吸入麻醉剂中进行打z的。磷酸-CAMKII和NR2A之间的相互作用。到目前为止，尚未证明B亚基：吸入麻醉药可阻止CaMKII转运至突触膜，随后磷酸 - camkii和NR2A之间的相互作用减少，导致NR2A磷酸化的调节，从而减少了对神经前测的NR2A磷酸化。