RESUMO
This chapter describes the propagation and characterization of transplantable insulinoma cells as model of insulin-producing pancreatic islet cells in the rat. Here, the cells are propagated by transplantation into rats followed by harvesting after growth for approximately 1 month. The cells are then purified by Percoll density gradient centrifugation and characterized by pulse-chase radiolabelling and immunoprecipitation of the insulin-related peptides. The results show that the transplantable insulinoma cells produce insulin in a manner similar to that found in normal pancreatic islets.
Assuntos
Técnicas de Cultura de Células/métodos , Imunoprecipitação/métodos , Insulinoma/patologia , Neoplasias Pancreáticas/genética , Animais , Proliferação de Células/genética , Humanos , Insulina/genética , Secreção de Insulina/genética , Insulinoma/genética , Ilhotas Pancreáticas/crescimento & desenvolvimento , Ilhotas Pancreáticas/patologia , Neoplasias Pancreáticas/patologia , RatosRESUMO
Pulse radiolabelling of cells with radioactive amino acids such is a common method for investigating the biosynthetic rates of proteins. In this way, the abundance of newly synthesized proteins can be determined by several proteomic techniques including 2D gel electrophoresis (2DE). This chapter describes a protocol for labelling pancreatic islets with 35S-methionine in the presence of low and high concentrations of glucose, followed by subcellular fractionation enrichment of secretory granule proteins and analysis of the granule protein contents by 2DE. This demonstrated that the biosynthetic rates of most of the granule proteins are co-ordinately regulated in the presence of stimulatory glucose concentrations.