Establishment And Characterization Of Cell Lines From Spotted Knifejaw(Oplegnathus Punctatus)

As powerful tool,fish cell lines have been widely used in virology,toxicology,physiology,endocrinology and genetics.The research of fish cell culture began in1960 s.In 1962,the gonadal cell line RTG-2 of Oncorhynchus mykiss established by Wolf and Quimby that was the beginning of fish cell line.Since then,the research about fish cell line has developed rapidly.By the first half of 2010,275 fish cell lines had been established worldwide.The established fish cell lines which came from various materials of fish is contained embryos,brain,eyes,gills,bones,muscles,epidermis,fins,tumors and visceral tissues.Fish cell line has been applied to fish virology,pathology,physiology,genetically modified,tumorigenesis,cytoskeleton and DNA damage repair.Spotted knifejaw(Oplegnathus punctatus),commonly known as spotted snapper and black golden drum,which belongs to Perciformes,Oplegnathidae,Oplegnathus,and mainly distributes in the temperate and tropical waters of China,Korea and Japan.O.punctatus is an excellent marine fish species.While,in recent years,the outbreak of rainbow virus and other viral diseases have caused great economic losses of the industrial breeding of porpoise.The establishment of fish cell line as an in vitro culture system will become an important tool for the related research of fish species because of its low cost,good repeatability and precise control of conditions.In this study,O.punctatus liver cell line(OPL)and brain cell line(OPB)were established by tissue block method from O.punctatus liver and brain,respectively.The cell lines were maintained in L-15 which was supplemented with antibiotic(mycillin),fetal bovine serum(FBS),antibiotics,basic fibroblast growth factor(bFGF),and 2-mercaptoethanol(2-ME).The cultured OPL cells,which showed fibroblastic,had been subcultured to more than 30 passages under a good proliferating state.And,the cultured OPB cells,which showed fibroblastic and epithelioid,had been subcultured to more than 30 passages under a good proliferating state too.By comparing the effects of different medium of L-15,DMEM/F12,DMEM and MEM on cells growth,the result showed the optimum medium was L-15.The effects of temperature,FBS and bFGF on the growth of OPL and OPB cells were examined.The cells grew well in the temperature of 18~30 ?,and the optimum growth temperature was 24 ?.The results showed the significant growth rate of OPL and OPB cells were accompanied with the increase of serum concentration range of0 %~20 %.In addition,bFGF was benefit forgrowth of OPL and OPB cells.Chromosome analysis indicated that the passage 20 cells exhibited chromosomal aneuploidy in OPL and OPB cells.The normal diploid karyotype(2n=2sm+46t)accounted for 68 % and 64 %,separately.And the significant fluorescent signals were detected in OPL and OPB cells after transfection with pEGFP-N3 plasmid.The CO I gene was used to identified that OPL and OPB cell lines were originated from O.punctatus.Moreover,it suggested the OPL and OPB cell lines could be potentially used for study the mechanisms of infective viruses and gene function analysis.The isolation and culture of tissue cells from spleen,kidney and heart tissues of O.punctatus were also studied by tissue culture method.The culture medium used for cell culture is L-15 complete medium.The primary cultured cells of various tissues have various morphologies.Kidney cell line was cultured to the seventh generation,spleen and heart cell lines were cultured to the tenth generation.In vitro culture of spleen,kidney and heart tissues of O.punctatus laid a foundation for cell function research,and provided a basis for the establishment of cell lines of spleen,kidney and heart in the future.In summary,the liver and brain cell lines of O.punctatus were established by the primary culture method of tissue culture,and the application of cell lines was studied from the characteristics of cell growth and the transfection of exogenous genes,which laid a foundation for the related research on cell lines in the future.At the same time,the preliminary study on the in vitro culture of spleen,kidney and heart cells of O.punctatus provides a basis for the establishment of cell lines of spleen,kidney and heart.