Ndfip1质粒的构建及其在神经母细胞瘤SH-SY5Y细胞中的表达

Construction of Ndfip1 plasmid and its expression in neuroblastoma SH-SY5Y cells

目的:观察Nedd4家族反应蛋白1(Ndfip1)过表达条件下神经细胞中二价金属离子转运蛋白1(DMT1)的表达,探讨Ndfip1对DMT1的调控作用。方法:构建Ndfip1真核表达质粒,应用Ndfip1质粒转染人神经母细胞瘤SH-SY5Y细胞,检测转染效率。Ndfip1质粒转染的SH-SY5Y细胞为实验组,空质粒转染的SH-SY5Y细胞为对照组,应用免疫荧光法观察2组细胞中DMT1蛋白表达强度,采用蛋白印迹法定量检测2组细胞DMT1蛋白表达水平。结果: Ndfip1质粒经扩增、电泳分离和测序比对,证明质粒构建成功。Ndfip1质粒转染48 h后,与转染前比较,SH-SY5Y细胞中Ndfip1蛋白表达水平明显增加(P<0.01),证明转染成功。免疫荧光法检测,与对照组比较,实验组细胞中DMT1的荧光强度明显减弱。蛋白印迹法检测,与对照组比较,实验组细胞中DMT1蛋白表达水平明显降低(P<0.05)。结论: Ndfip1过表达可下调神经细胞中DMT1蛋白的表达,Ndfip1对DMT1具有负性调控作用,通过减少神经细胞中铁离子的蓄积对神经细胞起到保护作用。

Objective::To observe the expression of divalent metal transporter 1 (DMT1) in neurons under the condition of Ndfip1 overexpression,and to explore the regulation effect of Ndfip1 on DMT1.Methods:The Ndfip1 eukaryotic expression plasmid was constructed,and the human neuroblastoma SH-SY5Y cells were transfected with Ndfip1 plasmid;the transfection efficiency was detected.The SH-SY5Y cells transfected with Ndfip1 plasmid were used as experimental group,and the SH-SY5Y cells transfected with empty plasmid were used as control group.The expression intensities of DMT1 protein in the SH-SY5Y cells in two groups were observed by immunofluorescence method,and the expression levels of DMT1 protein in the SH-SY5Y cells in two groups were detected by Western blotting method.Results:The plasmid was amplified,electrophoretically separated and sequenced,and the results proved that the plasmid was successfully constructed.After transfection of SH-SY5Y cells with Ndfip1 plasmid for 48 h,the expression level of Ndfip1 was significantly increased compared with before transfection (P <0.01).The immunofluorescence results showed that the fluorescence intensity of DMT1 in the cells in experimental group was significantly decreased compared with control group.The Western blotting results showed that the expression level of DMT1 in the cells in experimental group was decreased (P <0.05) compared with control group.Conclusion:The overexpression of Ndfip1 can down-regulate the expression of DMT1 protein in the nerve cells,and Ndfip1 nerve has a negatively regulatory effect.