中文摘要：

目的构建人源去甲肾上腺素转运蛋白（h NET）稳定表达细胞系,并对细胞系h NET的结合和重摄取功能进行研究。方法采用脂质体转染法将h NET转染于母细胞-人胚肾（HEK）293细胞（HEK293-h NET）;采用RT-PCR和Western蛋白质印迹法在基因和蛋白水平对HEK293-h NET细胞系进行鉴定和稳定性验证;采用放射性配基结合实验检测HEK293-h NET的结合和重摄取功能。并采用三环类抗抑郁药地昔帕明（DIM）和5-羟色胺/去甲肾上腺素双重重摄取抑制剂度洛西汀（DLX）,在0~1×10-4mol·L-1浓度梯度范围绘制药物与h NET的结合与重摄取抑制曲线,进一步验证HEK293-h NET细胞系的结合和重摄取功能。结果 RT-PCR和Western蛋白质印迹实验结果表明,所建立的HEK293-h NET细胞系在15代内均稳定表达h NET;放射性配基结合实验表明,HEK293-h NET细胞具有内源性h NET的结合和重摄取功能。并且,DIM和DLX与HEK293-h NET细胞中h NET具有明显的特异性结合（Ki值分别为2.45和3.40 nmol·L-1）,并可显著抑制h NET对去甲肾上腺素的重摄取作用（IC50分别为5.78和10.23 nmol·L-1）。结论成功建立的可稳定表达h NET的HEK293-h NET细胞系具有内源性h NET的结合和重摄取功能,可用于h NET靶标药物研究。

英文摘要：

OBJECTIVE To establish a stable recombinant human norepinephrine transporter（ h NET） in human embryo kidney（ HEK） 293 cell line and validate its binding and reuptake function.METHODS Lipofectamine transfection method was used to establish stably-expressed h NET in HEK293 cells. Then,RT-PCR and Western blotting were used to determine the expression of h NET in RNA and protein level,and ratioligand binding assays to validate its binding and reuptake function. Finally,a tricyclic antidepressant,desipamine（ DIM）,and a serotonin / norepinephrine（ NE） reuptake inhibitor,duloxetine（ DLX）,were used to validate the binding activity and reuptake function of HEK293-h NET cells. RESULTS The recombinant HEK293-h NET cells stably expressed h NET within 15 passages,and the h NET in HEK293-h NET cells possessed native binding and reuptake activities. Additionally,it was demonstrated that DIM and DLX had high affinity for h NET in the recombinant cells（ Ki= 2. 45 and3. 40 nmol·L- 1,respectively）,and also potently inhibited the reuptake of NE into the recombinant cells（ IC50= 5. 78 and 10. 23 nmol·L- 1,respectively）. CONCLUSION The stable recombinant HEK293-h NET cell line established in the present study possesses endogenous function of h NET,which may contribute to research of drugs targeting at h NET.