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目的建立人源5-羟色胺转运体(human serotonin transporter,hSERT)稳定表达细胞系,并对细胞系hSERT的结合活性和重摄取功能进行初步研究。方法采用脂质体转染法将hSERT转染于母细胞,即人胚胎肾(human embryo kidney,HEK)293细胞,应用抗生素G418(800μg/ml)压力筛选,并采用RT-PCR和Western印迹法在基因和蛋白水平对HEK293-hSERT细胞系进行鉴定和稳定性验证;采用放射性配体结合实验检测其与氚标西酞普兰([3H]-citalopram)的结合活性,并检测其对[3H]5-HT的重摄取功能。结果 RT-PCR和Western印迹结果表明,所建立的HEK293-hSERT细胞系在15代内(P1,P5,P10,P15)均稳定表达hSERT;放射配体结合实验表明,HEK293-hSERT细胞系与[3H]-西酞普兰具有明显的特异性结合;并且在100 nmol/L[3H]5-HT条件下,具有明显的5-HT重摄取功能,这一作用可被10μmol/L的氟西汀所阻断,而母细胞HEK293无此功能。结论本研究所建立的HEK293-hSERT细胞系可稳定表达SERT,并且具有内源性SERT的结合和重摄取功能,是一种稳定表达功能性hSERT的细胞系。
Objective To establish a human serotonin transporter (hSERT) stable expression cell line and to study the binding activity and reuptake function of hSERT in cell line. Methods Human embryo kidney (HEK) 293 cells were transfected with hSERT by lipofection method. The cells were screened by antibiotic G418 (800μg / ml) and analyzed by RT-PCR and Western blotting The HEK293-hSERT cell line was identified and stablized at gene and protein levels. The binding activity to [3H] -citalopram was tested by radioligand binding assay and its effect on [3H] 5-HT re-take function. Results RT-PCR and Western blotting results showed that HEK293-hSERT cell line stably expressed hSERT within 15 passages (P1, P5, P10, P15). Radioligand binding assay showed that HEK293- 3H] -citalopram with obvious specific binding; and under the condition of 100 nmol / L [3H] 5-HT, it has obvious 5-HT reuptake function, which can be induced by 10μmol / L fluoxetine Blocked, while the mother cell HEK293 no such function. Conclusion The HEK293-hSERT cell line established in this study can stably express SERT and has the function of binding and reabsorption of endogenous SERT. It is a cell line stably expressing functional hSERT.