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目的探讨Bax与Bcl-2在体外培养的皮层神经元的表达及非选择性一氧化氮合酶抑制剂NG-硝基-L-精氨酸甲酯(L-NAME)的干预作用。方法原代培养SD新生大鼠皮层神经元,并采用neuron specific enolase(NSE)免疫荧光方法进行细胞纯度鉴定。神经元随机分为正常组、N-甲基-D-天门冬氨酸(NMDA)组、NG-Nitro-L-arginine Methyl Ester(L-NAME)组,分别采用ELISA检测各组细胞中一氧化氮合酶(nitric oxide synthase,NOS)的表达,采用RT-PCR、Western blot分别检测各组细胞中Bax、Bcl-2mRNA及蛋白的表达。结果 NSE免疫荧光大多数细胞为NSE阳性细胞,神经元纯度高达90%;LNAME组中NOS及Bax的表达均高于正常组而低于NMDA组,Bcl-2的表达低于正常组而高于NMDA组。结论 L-NAME对NMDA诱导的神经细胞凋亡起明显的保护作用,其可能是通过上调Bcl-2的表达、下调Bax的表达发挥作用。
Objective To investigate the expression of Bax and Bcl-2 in cultured cortical neurons and the intervention of N-nitro-L-arginine methyl ester (L-NAME), a nonselective nitric oxide synthase inhibitor. Methods Primary cortical neurons of neonatal SD rats were cultured and neuronal specific enolase (NSE) immunofluorescence method was used to identify the purity of the cells. Neurons were randomly divided into normal group, N-methyl-D-aspartate (NMDA) group and NG-Nitro-L-arginine Methyl Ester (L-NAME) group. The expression of Bax and Bcl-2 mRNA and protein in each group were detected by RT-PCR and Western blot respectively, and the expression of nitric oxide synthase (NOS) was detected. Results The majority of NSE immunofluorescence cells were NSE positive cells, the purity of neurons was as high as 90%. The expression of NOS and Bax in LNAME group was higher than that in normal group and lower than that in NMDA group, while the expression of Bcl-2 was lower than that in normal group NMDA group. Conclusion L-NAME protects NMDA-induced neuronal apoptosis significantly, which may be through up-regulating the expression of Bcl-2 and down-regulating the expression of Bax.