Establishment Of Cell Lines, Inducement And Application From Several Important Marine Fishes.

The research of fish cell culture has developed rapidly since Wolf and Quimby established the first fish cell line- RTG-2 in 1960s for the first time. After then, fish cell culture has become an essential research technology which has been used extensively, ranged from virology, environmental toxicology, cytobiology, oncology, genomics, genetics and environmental protection. Cultured fish cells have more advantages than live fish as the experimental material: 1. the materials are cheap and easy to obtain; 2. the experimental condition could be controlled accurately and the experiment could be repeated.In the present study, two cell lines from the heart muscle of Cynoglossus semilaevis and the kidney of Verasper variegates have been established. The induced differentiation of two embryonic cell lines has also been studied. A gene of turbot (Scophthalmus maximus) GRIM19 was screened from a turbot spleen cDNA library. The expression of GRIM19 has been studied using two turbot cell lines.The two cell lines, HTHC and SHKC were cultured in minimum essential medium (MEM) supplemented with fetal bovine serum (FBS) and 10 ng ml-1 basic fibroblast growth factor (bFGF). HTHC cells were subcultured more than 30 times and SHKC cells 40 times. The effects of FBS, bFGF and temperature on the growth of HTHC and SHKC cells had been studied. FBS promotes cell growth at certain concentration and the optimum concentration of FBS was found to be 20% in the medium. bFGF enhanced cell living and propagation ability. The suitable temperature for growth was 20℃to 30℃with the optimum growth at 24℃and a reduced growth at <20℃. The double time of SHKC cells was determined to be 44.8 h. Chromosome analysis revealed that 52% cells maintained normal diploid chromosome number (n=46) in the SHKC cells and 42% in HTHC cells. The HTHC and SHKC cells have been successfully transfected with green fluorescent protein (EGFP) reporter plasmids and the expression of EGFP gene in the cells indicated the possible utility of the cells in gene expression studies. The HTHC and SHKC cells were infected by lymphosystis disease virus (LCDV) and found to be susceptible to the virus by cytopathic effect (CPE) observation. The infection was confirmed by PCR and electron microscopy experiments, which proved the existence of the viral particles in the cytoplasm of the virus-infected cells. In summary, the technology of establishing kidney and heart muscle cell lines is established. It is the first time that HTHC and SHKC were established in the world. Some researches are explored in the application of cell lines and lay foundation of using cell lines to the study in theory and application.Embryonic stem cells are undifferentiated permanent cell lines derived from inner cell mass or primordial germ cells of early developing embryos. In this study, two embryonic cell lines had been induced to study the multifunction of embryonic stem cells. HEC and FEC cells showed pluripotent and differentiation potential in vitro. Under the inducement of all-trans retinoic acid and low concentration of FBS, both of the ES-like cells differentiated into fibroblast-like cells; DMSO induced HEC cells into muscle cells. Under low cell concentration, both of HEC and FEC cells could also be induced into neuron-like cells, fibroblast-like cells, and muscle cells and so on. And FEC cells could also be induced into epidermic cells. Alkaline phosphate activity in HEC and FEC cells were both positive. All the results showed that both HEC and FEC cells were pluripotent embryonic cells. The HEC and FEC cells had been successfully transfected with green fluorescent protein (EGFP) reporter plasmids and the expression of EGFP gene in the cells indicated the possible utility of the cells in gene expression studies. The HEC and FEC cells were infected by lymphosystis disease virus (LCDV) and found to be susceptible to the virus by cytopathic effect (CPE) observation. The infection was confirmed electron microscopy experiments, which proved the existence of the viral particles in the cytoplasm of the virus-infected cells.A turbot (Scophthalmus maximus) gene associated with retinoic/ interferon induced mortality 19 protein-GRIM19 has been screened from a turbot spleen cDNA library. The expression in turbot different tissues, the different embryonic stages had been studied by RT-PCR. The complete cDNA of the turbot GRIM19 contained a 17 bp 5' UTR, a 435 bp open reading frame (ORF) encoding 144 amino acids and a 143 bp 3' UTR. Phylogenetic analysis showed that the turbot GRIM19 clustered with Hippoglossus stenolepis. RT-PCR demonstrated that turbot GRIM19 was expressed in spleen and head kidney from uninfected adult. GRIM19 expressed during the early stages of embryo development and gradually increased. The expression of GRIM19 was also dramatically increased after challenge in turbot liver, spleen and head kidney and the expression are most strong in head kidney. The expression reached highest after infection of 24 h in liver and in spleen, the expression was always enhanced. Furthermore, the turbot GRIM19 was induced after 48 h in turbot embryonic cells (TECs) after challenge with Vibrio anguillarum. These results indicated that the turbot GRIM19 played an important role in turbot immune response. Since GRIM19 is a mortality-correlative gene, in the turbot dying tissues infected by Vibrio anguillarum, the GRIM19 expresion was increased in liver, head kidney, heart and spleen. In a word, we report GRIM19 in turbot for the first time. The studies show GRIM19 is an important gene in turbot immunity response process and more research need to be processed for the more GRIM19 functions.