利用in vitro 細胞培養和組織培養方式探討高滲透壓環境對廣鹽性硬骨魚鰓上細胞之影響

Abstract

Epithelial cells of fish gills interact directly with external environments. Hence changes of the environments (e.g., salinity, temperature, and heavy metal concentrations) were found to affect the viability of gill epithelial cells. NaCl induced hyperosmotic environment is a kind of environmental stress on gill epithelial cells of euryhaline teleosts. Experiments using in vitro cell and organ culture offered the advantage by eliminating the variable internal factors and reducing the sacrifices. In order to investigate the effects of increased ionic stress on gill epithelial cells, the in vitro cell culture and organ culture system were used in this study. In the first part, rainbow trout (Oncorhynchus mykiss) gill epithelial cell line (RTg-W1) was cultured at 19°C in the culture media as the control group (375 mosm•kg-1) or experimental group (1000 mosm•kg-1 made of culture media and NaCl). The apoptosis was evaluated by flow cytometry. The living, apoptotic, and necrotic cells were 80%, 15%, and 5%, respectively, among cultured cells in the control group. In experiment group of the cultured cells, however, the apoptotic cells were 40% and living and necrotic cells were 52% and 8% cells, respectively. The results indicated that the survival rate of gill epithelial cells (RTg-W1) decreased due to more apoptosis but not necrosis when the ionic stress elevated dramatically. In the second part, freshwater tilapia gill filaments were cultured in the control group (375 mosm•kg-1), NaCl group (600 mosm•kg-1 made of culture media and NaCl), and mannitol group (600 mosm•kg-1 made of culture media and mannitol) at 28°C for 24 hours. The scanning electron microscopic observation revealed that the structure of cultured gill filaments was destroyed after 24 hours of incubation in the control media. The Cl- channel, cystic fibrosis transmembrane conductance regulator (CFTR) was expressed in Na+/K+-ATPase (NKA) immunoreactive cells of the control, NaCl, and mannitol groups after cultured for 12 hours. Meanwhile, apoptosis of mitochondria-rich cells was found in the control, NaCl, and mannitol groups after 6 hours of incubation. The immunoblotting showed that the protein abundance of proliferating cell nuclear antigen (PCNA) and heat shock protein 70 (HSP 70) decreased in both NaCl and mannitol group at 6 hours after the incubation. In addition, significant increased of NKA protein abundance since 6 hours of incubation in all three groups. The NKA activity also showed significant increase at 6 hours after the incubation in all three groups. However, after 12 hours incubation, the NKA activity of NaCl groups was significantly higher than the other groups. Although the protein abundance of the NKA, PCNA, and HSP 70 was not significantly different between ion-induced and non-ion-induced hyperosmotic environments, increased NKA activity indicated that ion-induced hyperosmotic environment might activate the NKA-drived ionoregulatory pathway to increase the survivability of euryhaline teleosts.