Separation and characterization of connective tissue cells expressing 43-kDa zinc-binding protein from common carp

Abstract

To separate the connective tissue cells expressing the 43-kDa zinc-binding protein from the common carp, mucosa of the digestive tract of the fish was removed by scraping with a glass slide, and the de-mucosa tissue was digested with a collagenase type IV solution. The cells collected from the collagenase-treated suspension were rather homogeneous, and more than 90% of the cells were round with a diameter of approximately 6 mm. Significant quantities of the 43 kDa zinc-binding protein were shown to be present on the cell surface of the approximately 6-mm cells. The mean zinc concentration in the cells was found to be 2.21 mg zinc/10 6 cells, which is approximately 20-30 times higher than that found in the other three fish species (grass carp, silver carp, tilapia) studied. The present work may provide a basis for the culture of these cells.

Role of high zinc levels in the stress defense of common carp

“…An indirect immunohistochemical staining experiment using antibodies against the 43-kDa zinc-binding protein indicated that in the common carp digestive tract, the protein is mainly located in the connective tissue of the lamina propria and submucosal layer [16]. Connective tissue cells, likely fibroblasts, contain this protein, which loosely binds zinc on its outward-facing side [17]. In fibroblasts, the 43-kDa protein had a single type of binding site specific for zinc.…”

“…Negative controls were performed by incubating cells with normal rabbit serum instead of primary antibody. Blood cells were observed using fluorescent microscopy [17].…”