摘要
目的构建GLP-1受体激动剂高通量筛选细胞模型。方法构建pEGFP-GLP-1R-3C重组质粒,转染HEK293T细胞,用G418和流式细胞仪进行筛选。所构建的细胞系命名为HEK293T-GLP-1R-3C-eGFP细胞系。Western blot和激光共聚焦检测GLP-1R-3C-eGFP蛋白的表达。将环磷酸腺苷(cyclic adenosine monophosphate,cAMP)响应元件报告基因转染HEK293T-GLP-1R-3C-eGFP细胞,不同浓度的GLP-1刺激后,用一步法荧光素酶报告基因检测试剂盒检测发光值反映细胞cAMP水平,采用cAMP试剂盒(ELISA)进行验证。结果成功构建HEK293T-GLP-1R-3C-eGFP细胞系。不同浓度GLP-1刺激后,发光值变化趋势与细胞cAMP水平变化趋势相似。本实验中Z′因子的值为0.52。结论本研究建立了基于重组HEK293T细胞株,其可用于GLP-1受体激动剂高通量筛选。
Aim To establish a high-throughput screening cell model for GLP-1 receptor agonists.Methods A pEGFP-GLP-1R-3C recombinant plasmid was constructed and transfected into HEK293T cells.The cells were screened with G418 and flow cytometry.The established stable cell line was named HEK293T-GLP-1R-3C-eGFP cell line.The expression level of GLP-1R-3C-eGFP protein was confirmed by Western blotting and laser confocal microscopy.Then cyclic adenosine monophosphate(cAMP)response element reporter gene was transfected into the HEK293T-GLP-1R-3C-eGFP cells.The luminescence values were detected by One-Step Luciferase Reporter Gene Assay Kit after stimulation with different concentrations of GLP-1 peptide.The luminescence values reflected the cellular cAMP level,which was verified using the cAMP kit(ELISA).Results HEK293T-GLP-1R-3C-eGFP cell line was successfully constructed.The relative light unit change trend after stimulation with different concentrations of GLP-1 was similar to that of the cellular cAMP level change trend.The value of Z′in this experiment was 0.52.Conclusions A recombinant HEK293T cell line is established,which can be used for high-throughput screening of GLP-1 receptor agonists.
作者
王芮
杨紫鑫
柯志强
成细瑶
苏正定
WANG Rui;YANG Zi-xin;KE Zhi-qiang;CHENG Xi-yao;SU Zheng-ding(Cooperative Innovation Center of Industrial Fermentation(Ministry of Education&Hubei Province),Hubei University of Technology,Wuhan 430068,China;Hubei Key Laboratory of Diabetes and Angiopathy,Xianning Medical College,Hubei University of Science and Technology,Xianning Hubei 437100,China;School of Light Industry and Food Engineering,Guangxi University,Nanning 530004,China)
出处
《中国药理学通报》
CAS
CSCD
北大核心
2023年第9期1793-1798,共6页
Chinese Pharmacological Bulletin
基金
湖北省教育厅科学研究计划指导性项目(No B2021224)。
