Establishment Of Screening Of Agonist For GLP-1 Receptor With GFP Labeled GLP-1 Receptor Cell Line

GLP-1(glucagon-like peptide-1)is a polypeptide secreted by endocrine gut L-type cells,having the regulation of insulin secretion,stimulation of pancreatic β cell regeneration.It is an important target for the development of drug for diabetes.Currently the analogues for type Ⅱ diabetes have been listed.They are expensive,only subcutaneous injection,long-term medication and not convenient.Therefore,screening of hydrone GLP-1 analogs is one of the main directions of future development.However,the current cell screening technology of GLP-1 receptor agonists is mostly GLP-1 receptor activated by GLP-1 and intracellular cAMP levels detected after activation.Because cAMP levels in cells will be caused by more factors.The system can not be efficiently performed on a large number of effective screening of natural products.Therefore,it’s important to develope an efficient GLP-1 R cell screening model.The study will make full use of rich natural resources in Yunnan province.The method of directly fluorescent labeled with GLP-1 R cell line has been established.The technology not only realizes screening of the complex natural products with high specificity,but also significantly reduces the false.In this paper,GLP-1R/pCMV6 is used in the construction of human glucagon like peptide-1 receptor(GLP-1 R)and green fluorescent protein(GFP)supplyed by vector pCMV6-AC-GFP.Transfected U2OS cells have been obtained through resistance selection and flow cytometry technology.Microscopic imaging has been showed GLP-1 R was identified in the recombinant cells.The cells stay stable after department for more than 10 generations.No significant differences in the expression of different generations through western-bloting.Finally GLP-1R/U2OS cell line can express fluorescence labeled GLP-1 R stably.Then the biological function of this cell line was also analyzed to verify by Exendin-4.Detection of the cell strains with Exendin-4 can konw reaction time and dose effect relationship.The results showed that the cells produced fluorescent spots in different time.20min was set up when the cells appeared maximum number of pits and did not produce endocytosis.The optimum detection time is 20min.Different concentrations of Exendin-4 activated GLP-1R/U2OS cells.The results can get dose effect relationship curve and show a good linear relationship between 1×10-5mol/L and 1.6×10-10mol/L.EC50 is 46nM.When GLP-1R/U2OS cell line with the same concentration of Exendin-4 in 60min,pit average intensity of cell strain pretreated with Exendin9-39 was obviously inhibited.This study has been successfully constructed the vector GLP-1R/pCMV6,established the stable transfected U2OS cell line,and verified its function.The whole detection method are easy to be standardized,having good repeatability,having high receptor specificity and cheap.Screening of peptide and non peptide GLP-1 analogs by the high throughput has a good prospect.