Establishment Of Three Novel Cell Lines From Large Yellow Croaker, Pseudosciaena Crocea, And Their Preliminary Applications Studies

Recently, the degradation of marine environment and outbreak of fish diseases have severely affected many marine fishes and caused great economic losses in aquaculture. Large yellow croaker, Pseudosciaena crocea, is one of the most important commercial fish also in face of these problems. Fish cell lines have been found to be rapid and effective bioassay systems to screen the cytotoxicity of environmental pollutants. On the other hand, fish cell lines can be used to study the function analysis of immune gene and immune mechanism. Fish cells also can be transfected with foreign genes, and will be a useful systerm in vitro for cell enginerring.By now, only several cell lines have been established from commercial marine fishes. Unfortunately, no cell line has been established from P. crocea. This study was conducted to establish three novel cell lines from P. crocea and to characterize their application in the studies of fish immunology and environmental pollutants monitoring and cell lines transfection.To establish fin, snout and spleen cell lines from P. crocea, fin tissues and snout tissues, which were digested with enzyme; and spleen tissues, which were digested without any enzyme, were used respectively to initiate the primary culture. The fin, snout and spleen cells in primary-culture were all in fibroblastic morphology and proliferated to confluence within 20 days. They grew at a steady rate during subsequent subcultures. Growth property studies and karyotype analysis ndicated was done. To date, fin cells have been subcultured up to passage 100, snout cells have been subcultured up to passage 80 and spleen cells have been subcultured up to passage 70. They are still in a good proliferating status. Three continuous cell lines from large yellow croaker fin, snout and spleen have been successfully established respectively.To establish a rapid and effective bioassay system to detect the organophosphorus insecticide and study their cytotoxic effects and possible toxic mechanisms, large yellow croaker fin cell line were used to determine the acute cytotoxic effects of monocrotophos by MTT assay and cell protein assay. The result showed that monocrotophos was toxic to yellow croaker fin cells at all tested concentrations in concentration-dependent manner. The 48h-IC50 values of monocrotophos to yellow croaker fin cells were 43.05 and 46.24μg/mL respectively. In the monocrotophos-treated cells, the activities of acetylcholinesterase (AChE), superoxide dismutase (SOD) and glutathione S-transferase (GST) changed significantly (P<0.05). Results suggested that monocrotophos could affect these enzyme activities with significant changes. And the AChE, SOD and GST activities of cells could be suitable biomarker for the detection of monocrotophos. All these indicated that large yellow croaker fin cell line could be used as a rapid and effective bioassay system.Clone the TLR2 cDNA fragment and analysis the immune function of TLR2 in the new establishment large yellow croaker snout cell line. Total RNA are extracted from large yellow croaker snout cells lines. A cDNA conserve fragment was obtained by RT-PCR from large yellow croaker snout cells. And 3'end of cDNA was obtained by 3'RACE (Rapid amplification of cDNA ends). Real-time quantitative PCR revealed that the expresstion of TLR2 mRNA increased in snout cells after stimulated with inactived Bacillus subtilis, Vibrio. harveyi and marine yeast. Results indicated that toll-like receptor2 not only can recognize the contents of Bacillus subtilis (Gram-positive bacteria) but also recognise Vibrio harveyi (Gram-negative bacteria) and marine yeast (fungus) in large yellow croaker snout cells.Large yellow croaker spleen cell line was transfected with pcDNA3.1-GFP by Lipofectamine2000TM. The optimal conditions of transfection were studied and the maximal transfection efficiency was 10%. Results indicated that Large yellow croaker spleen cell line is a useful study systerm for transfecting of foreign genes.The applications of three new established continuous cell lines of Large yellow croaker are studed respectively, but theirs values are much more and need to be studied further.