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目的 探讨血小板活化因子(PAF)所致的神经元损伤是否涉及N 甲基 D 天(门)冬氨酸/突触 后密度蛋白93(NMDA/PSD93)信号通路。方法 细胞培养系统培养原代野生型和PSD93基因敲除型小鼠皮 质神经元;0.3μmol/LPAF处理神经元24h或5μmol/LPAF受体拮抗剂(BN52021),10μmol/L非竟争性 NMDA受体拮抗剂(MK 801)和60μmol/L神经性一氧化氮合成酶(nNOS)抑制剂(L NAMA)预处理,碘化 物/钙黄绿素染色检测细胞凋亡;免疫印迹检测野生型和基因敲除型小鼠皮质神经元中的多种蛋白表达;细 胞免疫组化和共聚焦显微镜观察在同一神经轴突上共同表达多种蛋白;放免法测定神经元细胞蛋白中环鸟 苷磷酸(cGMP)活性。结果 (1)PSD93基因敲除型神经元不表达PSD93外,与野生型一样表达PSD95、N 甲基 D 天(门)冬氨酸受体(NR2A)和nNOS。(2)神经轴突共同表达PSD93、NR2A和nNOS。(3)PSD93基因敲除型 小鼠皮质神经元减少PAF对神经的毒性作用,并降低其cGMP活性。结论 PAF通过NMDA/PSD93途径致神 经细胞损伤。
Objective To investigate whether neuronal damage induced by platelet activating factor (PAF) involves NMDA / PSD93 signaling pathway. Methods Primary culture of wild-type and PSD93 gene-knockout mouse cortical neurons was performed in cell culture system. Neurons of BN52021 and BN52021 cells treated with 0.3μmol / L LPA for 24h or 5μmol / L, (MK 801) and 60 μmol / L Nnos inhibitor (L NAMA) were used to pretreat the cells. Apoptosis was detected by iodide / calcein staining. Western blotting was used to detect wild type and gene knockdown Type mouse cortical neurons. Immunocytochemistry and confocal microscopy were used to co-express multiple proteins on the same axon. Radioimmunoassay was used to determine cyclic guanosine monophosphate (cGMP) activity in neuronal cellular proteins. Results (1) PSD93 gene knockout neurons expressed PSD95, N-methyl-D-aspartate receptor (NR2A) and nNOS as wild type without expressing PSD93. (2) axons co-express PSD93, NR2A and nNOS. (3) PSD93 knockout mice cortical neurons reduce the toxic effect of PAF on nerve and reduce its cGMP activity. Conclusion PAF induces neuronal damage through NMDA / PSD93 pathway.