人源5-羟色胺转运体稳定表达细胞系的建立及其功能探究  

Establishment of a stable recombinant human serotonin transporter cell line and validation of its function

作　　者：安磊[1,2] 李静[1,2] 金增亮[1] 李云峰[1] 李锦[1] 张有志[1] 

机构地区：[1]军事医学科学院毒物药物研究所全军军事认知与心理卫生研究中心,北京100850 [2]大连医科大学药学院,辽宁大连116044

出　　处：《军事医学》2011年第9期681-684,共4页Military Medical Sciences

基　　金：国家自然科学基金资助项目(30973516;30901975;81173036;81072624;81001653);"重大新药创制"科技重大专项资助项目(2009ZX09103-025);国家重大基础研究发展计划(973计划)资助项目(2007CB512307)

摘　　要：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 stably-expressed human serotonin transporter（hSERT）cell strain in the human embryo kidney（HEK） 293 cell line and validate its binding and uptake function.Methods Lipofectamine transfection was applied to transfect hSERT into the parental cell HEK293.The transfected cells were stably selected by 800 ng/μl G418 sulfate,and the positive clones and their stability were identified by RT-PCR and Western blotting.The binding（-citalopram） and uptake（ 5-HT） function of hSERT was identified by radioligand binding assay.Results RT-PCR and Western blotting showed that the HEK293-hSERT cell line stably expressed hSERT RNA and protein in P1,P5,P10 and P15.Additionally,the hSERT expressed in HEK293-hSERT cells demonstrated significantly specific binding activity to serotonin selective reuptake inhibitor -citalopram.At the concentration of 100 nmol/L 5-HT,the hSERT showed significant 5-HT uptake function that could be inhibited by 10 μmol/L fluoxetine.The parental cell HEK293 did not possess these functions.Conclusion The established HEK293-hSERT cell line which possesses the function of endogenous hSERT is a stable functional hSERT transfected cell line.

关 键 词：5-羟色胺转运体(SERT) HEK293 抗抑郁药 5-HT 细胞转染 

分 类 号：R96[医药卫生—药理学]