GLP-1R激动剂的细胞筛选模型的建立  

Establishment of cell screening model for GLP-1R agonists

作　　者：高华山 佟伟霜 范卫卫 张科 白现广 李彦娇 GAO Huashan;TONG Weishuang;FAN Weiwei;ZHANG Ke;BAI Xianguang;LI Yanjiao(Pingdingshan University,Pingdingshan 467000,China)

机构地区：[1]平顶山学院医学院,河南平顶山467000

出　　处：《沈阳药科大学学报》2019年第11期1011-1019,共9页Journal of Shenyang Pharmaceutical University

基　　金：河南省科技厅科技攻关项目(172102310211、182102110166、192102310087);高等学校科学技术研究重点项目(18A180026、20B350006);平顶山学院高层次人才启动基金(PXY-BSQD-2018011,PXY-BSQD-2018010);平顶山学院培育基金(PXY-PYJJ-2019007).

摘　　要：目的构建靶向GLP-1R的高通量筛选模型。方法构建过表达GLP-1R的重组质粒和响应GLP-1信号通路的报告载体,共转染到HEK293细胞中,筛选得到稳定转染的单克隆细胞株,并用阳性药进行模型验证。结果成功构建了表达GLP-1R的重组质粒pcDNA3.1(+)-HuGLP-1R和响应GLP-1信号通路的报告载体PGL4.22-CRE-vip-GFP,共转染到HEK293细胞,筛选得到稳定转染细胞株CG-HEK293,经GLP-1刺激后,确定GFP的最佳表达时间为8 h,经过不同浓度的GLP-1刺激后,能够剂量依赖性地激活GFP的表达。结论构建了靶向GLP-1R的高通量筛选模型,该模型能够直观、稳定、高通量的筛选靶向GLP-1R的小分子或者GLP-1类似物,为长效的GLP-1类似物的研究和开发奠定了基础。Objective To construct a high-throughput screening model for GLP-1 R.Methods The recombinant plasmid expressing GLP-1 R and the reporter vector in response to GLP-1 signaling pathway were constructed and transfected into HEK293 cells.The stably transfected monoclonal cell lines were screened and validated by positive drugs.Results The recombinant plasmid pcDNA3.1(+)-HuGLP-1 R expressing GLP-1 R and the reporter vector PGL4.22-CRE-vip-GFP in response to GLP-1 signaling pathway were successfully constructed and co-transfected into HEK293 cells.The transfected cell line CG-HEK293 was stimulated by GLP-1 to determine the optimal expression time of GFP for 8 h.After stimulation with different concentrations of GLP-1,the expression of GFP could be activated in a dose-dependent manner.Conclusion A high-throughput screening model targeting GLP-1 R is constructed.This screening model allows visualization,stabilization and high-throughput screening of small molecules against GLP-1 R or GLP-1 analogs,laying the foundation for the research and development of long-acting GLP-1 analogues.

关 键 词：GLP-1受体 cAMP响应元件 GFP报告基因 细胞模型 

分 类 号：R91[医药卫生—药学]