CAJ | 학술논문

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Lipofectamine 2000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 West-ern 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)×10-10 mol · 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。
목적：건립은정표체CRFR1적HEK293세포주,병감정cAMP평개체계구건시부성공。방법채용Lipofectamine 2000장CRFR1질립전염지HEK293세포중,경G418사선단극륭양성세포,채용Western blot기술、RT-PCR법、면역형광법증실은정표체CRFR1세포주구건성공。병용CRF자격은정표체 CRFR1적 HEK293세포주,회제 CRF 자격HEK293-CRFR1세포석방cAMP적량효곡선。결과 West-ern blot、RT-PCR、면역형광결과표명, CRFR1수체재HEK293세포계중성공표체。 cAMP석방량효실험현시,CRF자격 HEK293-CRFR1세포석방 cAMP 적 EC50위(5.64±0.05)×10-10 mol · L-1。결론성공건립료은정표체CRFR1적HEK293세포주,병차cAMP함량평개체계구건성공,위연구CRFR1적생물학공능급사선CRFR1수체파향약물전정료기출。
Aim ToconstructHEK293cellsstablyex-pressing corticotropin releasing factor receptor 1 ( CRFR1 ) , and evaluate its function by the cAMP as-say.Methods CulturedHEK293cellsweretransfect-ed with CRFR1-expressing vector by Lipofectamine 2000 and were selected by using G418 . CRFR1 ex-pression was detected by Western blot, RT-PCR and immunofluorescence.Results Westernblot,RT-PCR and immunofluorescence data revealed that the HEK293 cells expressed CRFR1 protein stably. The dose-responsive relationship experiment revealed that CRF induced a CRFR1-mediated cAMP production in HEK293 cells with EC50 =(5. 64 ± 0. 05) × 10 -10 mol ·L-1.Conclusion HEK293celllinesstablyex-pressing CRFR1 were constructed successfully, which would provide a cellular model to facilitate the research on the biological function of CRFR1 and CRFR1-targe-ted drug screening.