Development And Characterization Of Brain And Testis Cell Lines From Turbot (Scophthalmus Maximus)

Fish cells have provided important tools to study virology,environmental toxicology,fish genetic breeding and resources conservation,endocrinology,developmental biology,fish physiology and so on.In 1962,Wolf and Quimby established the first fish cell line RTG-2 in the world.To this day,the number of fish cell lines has increased to more than four hundred from freshwater,anadromous and marine Fish.These Cell lines were acquired from fin,skin,gill,liver,kidney,spleen,embryo,gonad and so on.Cultured fish cells have more advantages than live fish as the experimental material: not only the materials are cheap and easy to obtain but also the experimental condition could be controlled accurately and the experiment could be repeated.Therefore,the research on fish cell culture and the establishment of fish cell lines has the far-reaching significance.Several cell lines from the embryo,fin,liver,kidney and heart have been established from the turbot.The brain and testis cell lines have not been established from the turbot.S.maximus brain cell line SMB and S.maximus testis cell line SMT was established by tissue block method from turbot brain in this research.The two cell lines were maintained in Dulbecco's modified Eagle's medium–F12 medium(DMEM–F12,1:1)supplemented with Hepes,fetal bovine serum(FBS),antibiotics,basic fibroblast growth factor(b FGF),and 2-Mercaptoethanol(2-ME).The cultured SMB cells were composed of fibroblast-like cells and epithelial-like cells.So far it have been subcultured for more than 40 passages.The cultured SMT cells were mainly composed of fibroblast-like cells.So far it also have been subcultured for more than 40 passages.These cells grew well in the temperature 18~30 oC and grew fastest at 30 oC.Chromosome analyses indicated that the 30 th passage SMB and SMT cells exhibited chromosomal aneuploidy.43% of the SMB cells maintained a normal diploid chromosome number(2n=44t).28% of the SMT cells maintained a normal diploid chromosome number(2n=44t).The transfection efficiency was up to about 30%,when the SMB and SMT cells were transfected with pEGFP-N3 plasmid The SMB and SMT cells' Sequence of cytochrome oxidase subunit I(CO?)gene showed a 99% consistency with turbot(Gen Bank Accession No.: KJ205427).Then we can get the conclusion that the species of SMB and SMT were from turbot by mitochondrial DNA CO? gene.The Japanese flounder Paralichthys olivaceus is a widely cultivated marine fish species in China.However,intensive aquaculture has resulted in outbreaks of viral diseases with high mortalities.Lacking of disease-resistant varieties is also a prominent problem.After then,fish cell culture has become an essential research technology which has been used extensively,ranged from genetics,virology,immunology,genomics,fish genetic breeding.In the present study,the primary culture of spleen,Liver and kidney of Japanese flounder had been started by trysin disgestion and tissue block method.TO sum up,S.maximus brain cell line SMB was established by tissue block method from turbot brain and S.maximus testis cell line SMT was established by tissue block method from turbot testis in this research.Some research was explored in the application of cell lines and laid foundation of using cell lines to study theory and experiment.In addition,the research about the Japanese flounder Paralichthys olivaceus in vitro culture provided the basis for the cell lines establishment.