神经丝氨酸蛋白酶抑制剂对新生大鼠神经元损伤的保护作用

Protective Effect of Neuroserpin on Neuronal Damage in Newborn Rats

目的探讨神经丝氨酸蛋白酶抑制剂(NSP)在N-甲基-D-天冬氨酸(NMDA)诱导的神经元损伤模型中对神经元是否具有保护作用。方法将SD新生大鼠(出生24h内)的大脑皮质神经元在体外进行原代细胞培养,并进行神经元鉴定。培养7～10d后选取生长良好的神经元用于实验,吸净其培养液,并随机分为3组。1.正常对照组:加入正常神经元培养液;2.NMDA损伤组(NMDA组):在正常神经元培养液中加入NMDA,至终浓度为12.5μmol.L-1,构建NMDA神经元损伤模型;3.NSP干预组(NMDA+NSP组):在给予相同浓度NMDA的同时加入NSP,至培养液中NSP质量浓度为2.5mg.L-1。随后将3组细胞均放入培养箱中培养24h,收取细胞和培养液。通过免疫荧光化学染色法观察细胞形态学变化,通过LDH释放实验测定细胞毒性损伤,TUNEL试剂盒检测细胞凋亡率。结果与正常对照组比较,NMDA组神经元突触回缩,细胞网状结构被破坏,LDH释放量增加,TUNEL染色显示细胞凋亡率增高。与NMDA组相比,NMDA+NSP组细胞网状结构完整,LDH释放量及TUNEL染色阳性细胞率明显降低,差异均有统计学意义(P<0.05)。结论 NSP可以减轻NMDA对神经元的损伤,具有保护神经元的作用。

Objective To study the neuronal protective effect of neuroserpin（NSP） on the damage induced by N-methyl-D-aspartic acid（NMDA）.Methods The cerebral cortical neurons of newborn SD rats（within 24 h after born） were cultured and identified in vitro.The cultured medium of the neurons was absorbed after being cultured for 7-10 days,and then were divided into 3 groups randomly：1.normal control group with normal neuron cultured medium added;2.NMDA injured group（NMDA group） added NMDA into the normal neuron cultured medium,to a final concentration of 12.5 μmol.L-1;3.NSP treated group（NMDA＋NSP group） with the same concentration of NMDA and at the same time adding NSP to a concentration of 2.5 mg.L-1 in the medium.The 3 groups were subsequently placed in the incubator for 24 h,and then the cells and the cultured medium were harvested.Immunoflurescence was used to observe the morphological changes.The cytotoxicity and the apoptosis ratio of neurons were studied with LDH release assay and TUNEL assay,respectively.Results Compared with the normal control group,neurons of the NMDA injured group showed a synaptic retraction,and the reticular structure was destroyed,LDH releasing increased,and TUNEL staining had a higher apoptosis rate.Compared with the NMDA group,the NMDA＋NSP group showed a integrity of reticular structure,LDH releasing and TUNEL-positive cells were significantly reduced,the differences were statistically significant（Pa0.05）.Conclusion NSP can alleviate the neuronal injury induced by NMDA and protected the neurons.