ABSTRACT
Aim To establish a cell model to detect the activity of somatostatin (SST) by targeting somatostatin receptor 2 (SSTR2), and to provide a simple and stable evaluation method for the drug screening of SSTR2 agonists and somatostatin analogues (SSTA). Methods The target gene of SSTR2 was integrated into the pEGFP-N3 vector, and the recombinant plasmid was constructed and transfected into HEK293 cells. After G418 screening, positive clone was selected and the stable cell lines were obtained by expanding culture. The stable cell lines were identified by fluorescence cell imaging, Western blot and qPCR. A calcium flow detection system was established to optimize cell number, fluorescence dye concentration and incubation time. Finally, the screening model was used to detect the different batches of the marketed somatostatin preparation Stilamin. Results SSTR2 stable cell lines were successfully constructed, and the receptors were mainly distributed on the cell membrane. The optimal conditions for calcium flow detection were determined as follows; 30 000 cells/Well, Fluo-4/AM indicator concentration was 3 p,mol • L -1 ~5 u,mol • L-1 , incubation time was 45 min. Under this condition, EC50 value of Stilamin in different batches was stable. Conclusions SSTR2 overexpressed stable cell lines are successfully constructed and calcium flow detection method is optimized to provide a simple and stable model for the screening of somatostatin receptor agonists.