马桑内酯激活的星形胶质细胞条件培养液及CNQX对正常大鼠脑内及培养的神经元内NSF的影响

THE EFFECT OF CORIARIA LECTON-ACTIVITED ASTROCYTE-CONDITIONED MEDIUM AND CNQX ON NSF OF THE CEREBRAL OF NORMAL RATS AND CULTURED NEURONS

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 explore the effects of astrocytes on the N-ethylmaleimide-sensitive fusion protin, （NSF） and AMPA receptor of the neurons as well as their function in epileptogenesis. Methods ACM was injected into lateral ventricle of SD rats and the behaviour changes were observed; Immunohistochemical method was used to assess the changes of the expression of NSF in the cerebral cortex and hippocampus; The cultured neurons were divided into control group, ACM group and CNQX ＋ ACM group at random, immunocytochemistry was used to assess the changes of the expression of NSF, Western blotting was used to assess the changes of the content of NSF of the cultured neurons. Results Seizure was observed in ACM group 30 min after injecting ACM. the immunoreaction of NSF in hippocampus and cerebral cortex of rats were depressed than those of the control group 2h, 4h after injecting ACM （ P 〈 0.05） ; The immunoreaction of NSF in cultured neurons of the ACM group was weaker than those of the control group 4h, 8h after ACM （ P 〈 0.05）. By mean of Western blotting, the concentrations of NSF of cultured neurons were depressed than those of the control group and the CNQX ＋ ACM group 4h, 8h after ACM （ P 〈 0.05）. Conclusion The results mentioned above indicated that the ACM can be reduced the expressions of NSF and result in epileptogenesis. CNQX can anatagonize this effect, showing that the down-regulation of ACM on NSF of rats and cultured neurons was partly mediated by AMPA receptor.