稳定表达CRFR1的HEK293细胞株建立与功能鉴定  

Development and evaluation of HEK293 cells stably expressing CRFR1

作　　者：葛治娟 于金梅[2] 马晓芸[2] 刘晓燕[2] 郑建全[2] 

机构地区：[1]中南大学药学院,湖南长沙410013 [2]军事医学科学院毒物药物研究所,北京100850

出　　处：《中国药理学通报》2014年第8期1113-1116,共4页Chinese Pharmacological Bulletin

基　　金：国家科技部"重大新药创制"科技重大专项(No2012ZX09301003-001);国家自然科学基金资助项目(No31350001)

摘　　要：目的建立稳定表达CRFR1的HEK293细胞株,并鉴定cAMP评价体系构建是否成功。方法采用Lipofectamine2000将CRFR1质粒转染至HEK293细胞中,经G418筛选单克隆阳性细胞,采用Western blot技术、RT-PCR法、免疫荧光法证实稳定表达CRFR1细胞株构建成功。并用CRF刺激稳定表达CRFR1的HEK293细胞株,绘制CRF刺激HEK293-CRFR1细胞释放cAMP的量效曲线。结果 Western blot、RT-PCR、免疫荧光结果表明,CRFR1受体在HEK293细胞系中成功表达。cAMP释放量效实验显示,CRF刺激HEK293-CRFR1细胞释放cAMP的EC50为(5.64±0.05)×10-10mol·L-1。结论成功建立了稳定表达CRFR1的HEK293细胞株,并且cAMP含量评价体系构建成功,为研究CRFR1的生物学功能及筛选CRFR1受体靶向药物奠定了基础。Aim To construct HEK293 cells stably expressing corticotropin releasing factor receptor 1 （CRFR1）, and evaluate its function by the cAMP assay. Methods Cultured HEK293 cells were transfected with CRFRl-expressing vector by Lipofectamine 2000 and were selected by using G418. CRFR1 expression was detected by Western blot, RT-PCR and immunofluorescence. Results Western blot, RT-PCR and immunofluorescence data revealed that the HEK293 cells expressed CRFR1 protein stably. Thedose-responsive relationship experiment revealed that CRF induced a CRFRl-mediated cAMP production in HEK293 cells with ECso = （5. 64 ±0.05） x 10^ -10 mol ·L-1. Conclusion HEK293 cell lines stably expressing CRFR1 were constructed successfully, which would provide a cellular model to facilitate the research on the biological function of CRFR1 and CRFRl-targeted drug screening.

关 键 词：促肾上腺皮质激素释放因子 CRFR1 HEK293细胞 转染 CAMP G蛋白偶联受体 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]