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目的揭示星形胶质细胞对神经元内N-乙基顺丁烯二酰亚胺敏感性的融合蛋白(NSF)和AMPA受体的影响及其在癫痫发病中的作用。方法将马桑内酯激活的星形胶质细胞条件培养液(ACM)注入正常SD大鼠侧脑室,观察其行为变化;运用免疫组织化学方法观察其大脑皮质、海马内NSF免疫反应的变化;将培养的神经元随机分为对照组、ACM组及CNQX+ACM组,免疫细胞化学方法观察NSF表达的变化,Western blotting法检测NSF含量的变化。结果ACM组大鼠在注射后30min出现癫痫行为。免疫组织化学染色结果显示:1.ACM作用后2h及4h,大鼠大脑皮质及海马内NSF免疫反应阳性神经元数和平均吸光度明显降低(P<0.05);2.培养的神经元的免疫细胞化学染色结果显示,ACM组在作用4h及8h时,NSF免疫阳性反应产物与对照组比较明显减少(P<0.05)。Western blotting法检测NSF含量,在ACM作用4h及8h时较对照组及CNQX+ACM组明显减少(P<0.05)。结论ACM可下调神经元内NSF的表达,并导致动物痫性发作,而运用AMPA受体拮抗剂CNQX则能阻断其下调作用,提示ACM对NSF的下调作用可能与AMPA受体有关。
Objective To reveal the effects of astrocytes on N-ethyl maleimide-sensitive fusion protein (NSF) and AMPA receptors in neurons and their roles in epilepsy. Methods The astrocyte-conditioned medium (ACM) activated by macadamia was injected into the lateral ventricle of normal SD rats to observe its behavioral changes. The changes of NSF immunoreactivity in the cerebral cortex and hippocampus were observed by immunohistochemistry. The cultured neurons were randomly divided into control group, ACM group and CNQX + ACM group. The changes of NSF expression were observed by immunocytochemistry and the changes of NSF content by Western blotting. Results The rats in ACM group experienced epileptic behavior 30 min after injection. The results of immunohistochemical staining showed that: 1. The number of NSF immunoreactive neurons and the average absorbance of cerebral cortex and hippocampus were significantly decreased at 2h and 4h after ACM treatment (P <0.05); 2. The immune cells of cultured neurons The results of chemical staining showed that the NSF immunoreactive products in ACM group were significantly decreased compared with the control group at 4h and 8h (P <0.05). The level of NSF in Western blotting assay was significantly lower than that in control group and CNQX + ACM group at 4h and 8h (P <0.05). Conclusion ACM can down-regulate the expression of NSF in neurons and lead to epileptic seizures in animals. CNPX, an AMPA receptor antagonist, can down-regulate the expression of NSF, suggesting that the down-regulation of NSF by ACM may be related to AMPA receptor.