神经元细胞氧化应激模型中FOXO3基因及其下游凋亡基因BIM表达研究

Study on mRNA of FOXO3 gene and its downstream apoptosis gene BIM expression in neuronal oxidative stress model

为了研究在小鼠神经元细胞氧化应激模型中FOXO3基因及其下游凋亡基因BIM的表达情况,并进一步探讨FOXO3基因在神经退行性疾病中的临床意义,试验利用H_(2)O_(2)构建神经元细胞氧化应激模型,通过Western-blot分析和细胞活力检测验证该模型,采用实时荧光定量PCR方法检测FOXO3基因及其下游凋亡基因BIM的相对表达量,利用Si-RNA干扰FOXO3基因及其蛋白的表达,并再次利用实时荧光定量PCR和细胞活力检测方法分析FOXO3基因表达量与细胞死亡的关系。结果表明:与正常神经元细胞相比,经H_(2)O_(2)处理的神经元细胞中Cleaved-Caspase3表达量明显升高,细胞活力显著降低(P<0.05),FOXO3基因(P<0.05)及其凋亡基因BIM(P<0.01)相对表达量上升,即成功构建出神经元细胞氧化应激模型;用Si-RNA技术进行干扰后,FOXO3基因及其蛋白的相对表达量明显下降,与仅用H_(2)O_(2)处理的神经元细胞相比,凋亡基因BIM的相对表达量显著下降(P<0.05),神经元细胞活力显著增加(P<0.05)。说明FOXO3基因及其下游凋亡基因BIM的表达在神经元细胞氧化诱导死亡过程中有重要的促进作用。

To study the expression level of FOXO3 gene and its downstream apoptosis gene BIM in the oxidative stress model of mouse neurons, and to further explore the clinical significance of this gene in neurodegenerative diseases, In this study, H_(2)O_(2) was used to construct the oxidative stress model of neurons, which was verified by Western-blot analysis and cell viability detection. The relative expression levels of FOXO3 gene and its downstream apoptosis gene BIM were detected by real-time fluorescence quantitative PCR. Si-RNA interference was used to inhibit the expression of FOXO3 gene and its protein, and the relationship between FOXO3 gene expression and cell death was analyzed by real-time quantitative PCR and cell viability detection. The results showed that compared with the normal neurons, the expression of Cleaved-Caspase3 was significantly increased, the cell viability was decreased(P<0.05), and the expression of FOXO3 gene(P<0.05) and apoptosis gene BIM(P<0.01) were increased in the experimental group treated with H_(2)O_(2). The oxidative stress model of neurons was established successfully. The expression of FOXO3 gene and its protein decreased significantly after Si-RNA interference. Compared with normal neurons, the expression level of apoptosis gene BIM in neurons treated with H_(2)O_(2) was increased(P<0.05). The activity of neurons was significantly increased(P<0.05). These results indicated that the mRNA expression levels of FOXO3 gene and its downstream apoptotic gene BIM played an important role in promoting in the process of oxidative death of neurons.