Abstract: 【Objective】 To investigate the inhibition of siRNA on the expression of Cytoglobin in hippocampus cell of rats in vitro. 【Methods】 Primary hippocampal neurons of newborn SD rats were cultured, and then identified them by Neu immunofluorescence antibody. siRNAs, which were designed against CYGB gene, were transfected into hippocampus cell with lipofectamine. The non-transfected cells and transfected with negtive-siRNA were taken as controls. The efficacy of transfection was detected by transfecting fluorescence-siRNA into cells, and the inhibitory effect of siRNA on mRNA level was detected by RT-PCR. 【Results】 The efficacy of transfection was about 70%. RT-PCR showed that the expression of CYGB was significantly decreased after transfecting siRNA-78 for 24 h(P＜0.01), 48 h(P＜0.001)and 60 h(P＜0.001), but it had no difference after 72 h. There was no difference in each time (24 h, 48 h, 60 h, 72 h, respectively) between siRNA-79/siRNA-80 and control group(P＞0.05). 【Conclusion】 The data of this study suggest that siRNA-78 designed against CYGB gene could significantly inhibit the expression of CYGB gene in hippocampus cell of rats in vitro after transfection for 24 h to 60 h.

Key words: siRNA, CYGB, gene, hippocampus, immunofluorescence

摘要： 【目的】 研究siRNA(small interfering RNA)对细胞红蛋白(Cytoglobin, CYGB)基因表达的抑制作用。 【方法】 取新生大鼠海马做神经元原代培养,NeuN免疫荧光抗体鉴定神经元纯度后,通过阳离子脂质体转染试剂将体外化学合成的针对CYGB基因的siRNA转入细胞中,以未转入siRNA细胞及转入阴性siRNA细胞为对照,通过转染荧光对照siRNA检测转染效率,用RT-PCR方法检测siRNA对CYGB表达的抑制作用。 【结果】 成功体外培养海马神经元,转染荧光对照siRNA结果示转染效率在70%左右,RT-PCR结果提示与阴性对照及空白对照相比,转染siRNA-78 后24 h(P＜0.01),48 h(P＜0.001)及60 h(P＜0.001)CYGB mRNA的表达明显减弱,72 h后表达无明显减少。而转染siRNA-79,siRNA-80在各时间点与对照相比没有明显差别(P＞0.05)。 【结论】 特异性针对CYGB的siRNA-78转染体外培养的大鼠海马神经元24~60 h能显著下调CYGB基因的表达。

关键词: siRNA, CYGB, 基因, 海马, 细胞免疫荧光