GIT1和ERK1/2在兔脊髓缺血再灌注损伤中作用的初步研究

The role of GIT1 and ERK1/2 in reperfusion of ischemic spinal cord

目的:研究G蛋白耦联受体激酶相互作用分子1(G-protein-coupled receptor kinase interacting protein 1,GIT1),细胞外信号调节激酶1/2(Extracellular signal-regulated kinases1 and 2,ERK1/2)在脊髓缺血再灌注损伤过程中的作用。方法:电镜观察缺血30min再灌注不同时间段[A组(对照组)再灌注0min、B组再灌注30min、C组再灌注2h、D组再灌注8h]脊髓组织标本的形态学改变,Westernblot检测各组脊髓标本中ERK1/2和GIT1的表达及其磷酸化,免疫共沉淀分析GIT1同活化的ERK1/2(phospho-ERK1/2,pERK1/2)相互作用的变化,免疫组织化学分析pERK1/2在神经细胞内的定位表达。结果:电镜结果显示D组脊髓神经细胞出现早期凋亡的征象;Western blot结果显示pERK1/2的表达、GIT1的磷酸化在C、D二组较对照组明显增加且均随再灌注时间延长呈明显增加的趋势;免疫共沉淀结果显示GIT1-pERK1/2结合力在C、D两组较对照组明显增加;免疫组织化学结果显示pERK1/2明显滞留于C、D两组的脊髓神经细胞胞浆区内。结论:缺血再灌注损伤过程中ERK1/2的活化以及在胞核外区域的滞留可能导致了脊髓神经细胞的凋亡,而GIT1作为细胞浆内局部黏附蛋白同pERK1/2的结合可能导致了其在胞浆区的滞留。

Objective：To investigate the role and mechanism of G-protein-coupled receptor kinase interacting protein 1 （GIT1） and Extracellular signal-regulated kinases 1 and 2 （ERK1/2） in reperfusion of ischemic spinal cord. Methods：The morphology of spinal cord was observed by electron microscope in different time courses after ischemia/reperfusion injury[group A（control group）,group B （ischemia 30 min/reperfusion 30 min）,group C（ischemia 30 min/reperfusion 2 h）,group D（ischemia 30 min/reperfusion 8 h）]. The phosphorylation of ERK1/2 and GIT1 was detected by Western blot. The interaction between GIT1 and pERK1/2 was detected by coimmunoprecipitation. The localization of pERK1/2 was analyzed by immunohistochemistry. Results：The apoptosis of neurocytes of spinal cord undergoing ischemia/reperfusion injury in group D was detected by electronmicroscope. The phosphorylation of ERK1/2 and GIT1 and the interaction between GIT1 and pERK1/2 significantly increased in group C and D. Immunohistochemistry results showed that the stagnation of pERK1/2 was observed in the cytoplasma of neurocytes in group C and D. Conclusion：The stagnation of pERK1/2 in the cy.toplasm might act as apoptosis signals contributing to the apoptosis of neurocytes of spinal cord after ischemia/ reperfusion injury,and the interaction between GIT1 and pERK1/2 might cause the stagnation of pERK1/2 in the cytoplasm of neurocytes.