| Blunt snout bream (Megalobrama amblycephala), commonly known as Wuchang fish, affiliated to Cypriniformes, Cyprinidae, Abramidinae, Megalobrama. M. amblycephala is native to China, distributed originally to affiliated lakes of middle and lower reaches of the Yangtze River in China. In1964it was domesticated successfully, endowed with excellent biological characteristics for high survival rate, fast growth, herbivorous, high nutritive value, less disease and easy fishing, etc., now it has become one of the main farmed freshwater species in China. In recent years, the germplasm resources of M. amblycephala are under threat of recession and mixture due to overfishing, pollution of waters, inbreeding, and cultured population of M. amblycephala is gradually exhibiting early such as smaller sexual maturity individuals, lose of growth predominance and increase disease. Now, many studies have been conducted to understand its biology, immunology, nutrition, diseases and breeding, but there is limited information available on cultured cells derived from M. amblycephala. Therefore, this study established and characterized three new fish cell lines from the fin, muscle and heart of M. amblycephala, and used these three cell lines as in vitro systems for studying virology, environmental toxicology, genetics and so on. We hope these three cell lines as in vitro systems could be applied in all researches of M. amblycephala, provid basic datas for farming and breeding of M. amblycephala, and identify the more sensitive cell lines as bio-indicators to detect environmental toxic chemicals. The main results were as follows:(1) Development and characterization of three new cell lines from M. amblycephalaThree new fish cell lines (MAF、MAM and MAH) were developed from the fin, muscle and heart of M. amblycephala by using the tissue explants technique, and have been subcultured more than95,86and71times in more than a year. After3-6months of storage in liquid nitrogen, the viability of the MAF, MAM and MAH cells was92.92±5.38%,90.04±5.49%and91.54±6.55%, respectively. All the cell lines were optimally maintained at28-32℃in medium199supplemented with10%fetal bovine serum. Most MAF, MAM and MAH cells have chromosome number of48at passage10, which accounted for62%,59%and65%, respectively, it demonstrated that the three cells are normal diploid cells. DNA was isolated from passage30MAF, MAM and MAH cells, an expected PCR product size was obtained by amplification of12s rRNA from extracted DNA, subsequent comparative analysis of the identified sequences demonstrated a99-100%match with the known mitochondrial DNA sequence of M. amblycephala12S rRNA, the results demonstrated that MAF, MAM and MAH cells indeed originated from M. amblycephala. MAF, MAM and MAH cells were strongly positive for pancytokeratin, desmin, fibronectin and the proliferative marker Ki67, the results demonstrated the three cells to be fibroblastic, epithelial and muscular in nature.(2) The preliminary application of MAF cellsThe susceptibility of MAF cells to infection by four viruses, snakehead rhabdovirus (SHRV), spring viremia carp virus (SVCV), channel catfish virus (CCV) and grass carp reovirus (GCRV), was evaluated by cytopathic effects (CPE). The significant CPE of SHRV, SVCV and CCV infection was observed in MAF cells at1d,2d and2d post-inoculation, respectively. However, no CPE was found in GCRV-infected MAF cells. Furthermore, PCR assays detected prominent bands from SVCV, SHRV and CCV infected MAF cells, it demonstrated MAF cells infected by SVCV, SHRV and CCV. The viral titers of SVCV, SHRV and CCV in MAF cells reached1068,1082and1O5.9TCID50ml-1, respectively. Bacterial cytotoxicity studies showed that extracellular products from Aeromonas hydrophila were toxic to MAF cells, the morphological changes were shrinking, detachment and finally destruction of the monolayer. Cu2+was also cytotoxic to MAF cells and the24h-IC50value was144.48±3.25μmol/L. When MAF cells were transfected with pEGFP-N1plasmid, bright fluorescent signals were observed, and the transfection efficiency reached up to5%. These results suggest that the MAF cell line may provide a valuable tool for studying virus pathogenesis, as well as cytotoxicity testing and genetic manipulation studies.(3) The toxicity studies of perfluorooctanoic acid on MAM cellsCytotoxicity of perfluorooctanoic acid (PFOA) on MAM cells was measured by four endpoint systems:methyl thiazolyl tetrazolium (MTT) assay, cell protein (CP) assay, neutral red (NR) uptake assay and cell counting kit-8(CCK-8) assay. The result showed that PFOA was cytotoxic to MAM cells at all tested concentrations, and toxicity increased as the concentration of PFOA was progressively increased. The24h-IC50values of PFOA were240.81±10.66mg/L,243.96±11.65mg/L,248.63±13.95mg/L and223.62±13.97mg/L for MTT assay, CP assay, NR uptake and CCK-8assay, respectively. The48h-IC50values were214.01±10.48mg/L,219.86±10.13mg/L,235.57±8.43mg/L and210.40±8.32mg/L, respectively. For the four methods,24h-IC50and48h-IC50values were NR>CP>MTT>CCK-8, and there was no significant difference for24h-IC50and48h-IC50values measured by these foure methods (P>0.05). Moreover, the morphological changes of MAM cells were also studied at the concentration of50mg/L and100mg/L for48h. MAM cells were not full, tended to shrinking, and100mg/L group was more obvious. Under a scanning electron microscopy, the nucleus of MAM cells became small, mitochondrial swelled and an increased number of lipid particles after50mg/L PFOA exposure. The number of lipid droplets in MAM cells was significantly increased, and mitochondrial swelled with obvious ridge could be seen in MAM cells at the group of100mg/L. These results indicated PFOA could damage mitochondrial and affect lipid metabolism of MAM cells. This would suggest that the MAM cell line is a suitable bioindicator for the screening of the acute toxicity of PFOA. |
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