耳下腺腺房細胞の初代培養系への遺伝子導入

抄録

Acinar cells of salivary parotid gland produce saliva by two types of secretion; water fluid and exocytosis of proteins like amylase. Exocytosis of amylase is mainly regulated by intracellular cAMP, and VAMP2, one of the SNARE proteins, is essential for cAMP-dependent amylase secretion. However, it is difficult to investigate the molecular mechanism of parotid acinar cells since there is no stable cell line that has the ability to release amylase upon stimulation. To examine the functions of parotid acinar cells, we tried to establish a transfection system of exogenous genes to primary culture. Acinar cells were dispersed from rat parotid glands by enzyme digestion, and cultured in Waymouth's medium that contained rat serum. Cells that cultured in dishes coated with various extracellular matrix kept amylase activity, and have the ability to release amylase in response to isoproterenol at least for 48 h. The cells spread on the dish bases and formed filopodia although they have granules that contain amylase, suggesting they are derived from acinar cells. VAMP2 gene fused with enhanced green fluorescence protein (EGFP-VAMP2) was transfected to dispersed acinar cells and cultured for 48 h. EGFP-VAMP2 protein was observed to be correctly localized at granules that contained amylase. Therefore, the cells cultured by this method can be used to examine the effects of exogenous genes on functions of parotid acinar cells like regulated exocytosis and maturation of secretory granules. [Jpn J Physiol 54 Suppl:S78 (2004)]