Establishment and characterization of an intestinal epithelial cell line from Japanese flounder (Paralichthys olivaceus)

Abstract

The Japanese flounder Paralichthys olivaceus is a high-value mariculture species. However, the aquaculture industry worldwide faces significant challenges due to flounder ascites, a serious disease caused by gram-negative bacterium Edwardsiella infection of fish cultures. Studies have confirmed that Edwardsiella tarda infection of flounder causes intestinal inflammatory disease. The recognition of pathogenic microorganisms by intestinal epithelial cells is the primary cause of inflammatory responses. In this study, a new cell line derived from the distal intestine (mainly the mid and posterior intestine) of Japanese flounder was established and characterized. This cell line, designated as the PoIEC line, was maintained in DMEM with 15% fetal bovine serum at 24 °C for >1 year, and subcultured >55 times. The PoIECs were epithelial-like cells with cobblestone-like shapes. Microvilli were observed on the surface of the PoIECs through transmission electron microscopy and the existence of epithelial markers was confirmed. PoIECs transfected with a pEGFP-N3 plasmid emitted an intense green fluorescence signal. Transfection efficiency of these cells was calculated to be approximately 37%, making this cell line a potential tool for future exogenous gene function research. Furthermore, PoIECs were stimulated by LPS and live E. tarda, and the expression profiles of immune-related genes (MHC Iα, MHC IIα, TNFα, IL-8, and IL-1β) confirmed that these cells recognized antigens and activated immune responses. In conclusion, a new cell line derived from the intestine of Japanese flounder was established, and this cell line was an ideal cellular platform for the study of mucosal immunity, probiotic flora and nutrient metabolism in the fish gut.