Construction of Ndfip1 plasmid and its expression in neuroblastoma SH-SY5Y cells / 吉林大学学报(医学版)

ABSTRACT

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.