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目的:研究沙土鼠脑缺血再灌注后海马突触体N-甲基-D-天冬氨酸(NMDA)受体亚基2B(NR2B)酪氨酸磷酸化调节的机制。方法:沙土鼠双侧颈总动脉结扎形成前脑缺血模型;NR2B酪氨酸磷酸化通过免疫沉淀和免疫印渍分析。结果:脑缺血15分钟导致蛋白酪氨酸磷酸化水平明显下降;再灌注引起包括180kDa蛋白在内的多种蛋白酪氨酸磷酸化水平快速(再灌注15分钟)、持续(至少48小时)升高。免疫沉淀和免疫印渍证实,180 kDa条带为NR2B。缺血15分钟,再灌注6小时,NR2B酪氨酸磷酸化明显高于对照组,为对照组的1.8倍,而NR2B蛋白表达量则无变化。缺血前腹腔注射非竞争性NR拮抗剂氯胺酮或L-型电压门控钙通道(L-type VGCC)阻滞药硝苯地平,对NR2B酪氨酸磷酸化水平升高有明显的拮抗作用,而对NR2B蛋白表达量均无影响。在此条件下,非NMDA受体拮抗剂6,7-二硝基喹恶啉土卫四(DNQX)对NR2B酪氨酸磷酸化水平无影响。酪氨酸蛋白磷酸酶(PTP)抑制剂钒酸钠使脑缺血再灌注诱导的NR2B酪氨酸磷酸化进一步增加,而酪氨酸蛋白激酶(PTK)抑制剂金雀异黄素则使其减少。Src能与NR2B免疫共沉淀。结论:沙土鼠脑缺血再灌注NR2B的酪氨酸磷酸化的升高是通过NR和L-type VGCC介导的;PTK和PTP参与脑缺血再灌注NR2B酪氨酸磷酸化的调节,与NR2B以物理方式结合的Src可能在这种
AIM: To investigate the mechanism of tyrosine phosphorylation of N-methyl-D-aspartate (NMDA) receptor subunit 2B (NR2B) in hippocampal synaptosomes after gerbil cerebral ischemia / reperfusion. Methods: The bilateral common carotid arteries were ligated to form forebrain ischemia model. The tyrosine phosphorylation of NR2B was analyzed by immunoprecipitation and immunoblotting. RESULTS: Cerebral ischemia for 15 minutes resulted in a significant decrease in protein tyrosine phosphorylation; reperfusion resulted in rapid (multiple reperfusion for 15 minutes) multiple protein tyrosine phosphorylation including 180 kDa protein for at least 48 hours Rise. Immunoprecipitation and immunoblotting confirmed that the 180 kDa band was NR2B. At 15 minutes after ischemia and 6 hours after reperfusion, the tyrosine phosphorylation of NR2B was significantly higher than that of the control group, 1.8 times that of the control group, while the NR2B protein expression remained unchanged. Preischemic intraperitoneal injection of non-competitive NR antagonist ketamine or L-type voltage-gated calcium channel (L-type VGCC) blockers nifedipine on NR2B tyrosine phosphorylation increased significant antagonism, While NR2B protein expression had no effect. Under these conditions, DNQX, a non-NMDA receptor antagonist, had no effect on NR2B tyrosine phosphorylation. Sodium orthovanadate, a tyrosine protein phosphatase (PTP) inhibitor, further increases NR2B tyrosine phosphorylation induced by cerebral ischemia-reperfusion, while genistein, a tyrosine protein kinase (PTK) inhibitor, cut back. Src co-immunoprecipitates with NR2B. CONCLUSIONS: The increased tyrosine phosphorylation of NR2B in gerbils following cerebral ischemia-reperfusion is mediated by NR and L-type VGCC. PTK and PTP are involved in the regulation of tyrosine phosphorylation of NR2B in cerebral ischemia reperfusion rats, Src that NR2B physically binds may be in this