脑缺血再灌注后突触后膜NMDA受体重塑性机制的研究

The mechanism study onpostsynaptic NMDA receptorremoldability after cerebral ischemia reperfusion

目的探讨脑缺血再灌注后神经元突触后膜NMDA受体发生迁移重塑的机制。方法实验使用脑缺血再灌注Wistar大鼠模型,分为无缺血再灌注对照组(Ctrl),缺血再灌注0.5h组(I/R-0.5h),缺血再灌注4h组(I/R-4h),缺血再灌注24h组(I/R-24h)。再灌注时间达到后将脑组织取出,并制成脑组织匀浆液(HOM),利用差速离心法分离突触后膜组分(SYN)和突触外膜组分(EXTRA),通过Western Blot分析Src蛋白激酶的表达分布及NMDA受体NR2A和NR2B的表达分布。结果 Western Blot分析发现,脑缺血再灌注0.5h后,突触后膜组分Src蛋白激酶出现超激活状态(P<0.01),NR2A和NR2B在突触外膜组分中的表达显著降低(P<0.01),而NR2A和NR2B在突触后膜组分中的表达升高(P<0.05);在再灌注4h后,Src蛋白激酶激活程度更加明显(P<0.01),NR2A和NR2B在突触外膜组分中的表达仍显著下降(P<0.05),但NR2A和NR2B在突触后膜组分中却出现不同程度的下降。结论脑缺血再灌注后神经元突触后膜出现Src蛋白激酶超激活状态,并介导NMDA受体NR2A和NR2B的表达分布出现重塑性,NMDA受体从突触周膜向突触后膜转移聚集。

Objective To investigate the mechanism of neuronspostsynaptie NMDA receptorremoldability after cerebral ischemia reperfusion. Methods In this study, Wistar rats with global cerebral ischemia-reperfusion model divided into is the normal group （Ctrl）,ischemia-reperfusion 0.5 h group （I/R-0.5 h）,ischemia-reperfusion 4 h group （I/R-4 h） and ischemia-reperfusion 24 h group （I/R-24 h）. After arriving reperfusion, the brain tissue is removed and the hom- ogenization buffer （HOM） was prepared. Synaptic membrane fraction （SYN） and Extrasynaptic membrane fraction （EXTRA） are separated by differential centrifugation. Expression distribution of Src protein kinase and expression dis- tribution of NMDA receptor NR2A and NR2B are analyzed by Western Blot. Results Western Blot analysis results indicate that the Src protein kinase appeared hyperactive in the SYN after ischemia-reperfusion 0.5 h （P〈0.01） ,meanwhile the expression of NR2A and NR2B is decreased significantly in EXTRA （P〈0.01） and increased in SYN （P〈 0.05）. After ischemia-reperfusion 4 h, the Src protein kinase activation degree more obvious （P〈 0.01）, and the expression of NR2A and NR2B in EXTRA are still decreased significantly （P〈0.05） ,but NR2A and NR2B in SYN reduced at different degrees. Conclusion After cerebral ischemia reperfusion,Src protein kinase super activated state in post synaptic membrane of neurons mediating theremoldability of NMDA receptor NR2A and NR2B expression distribution,the NMDA receptor is gathered from Extrasynaptic membrane to Synaptic membrane.