水产学报2017，Vol.41Issue(3)：358-365,8.DOI:10.11964/jfc.20161010567
中华鳑鲏皮肤细胞的分离及其细胞系的建立
The establishment of skin cell separation method and its cell lineage of Rhodeus sinensis
摘要
Abstract
Skin was the first barrier to protect fish body from the out-environment,and was the main carrier of the body color.For lack of fish skin cell lines,investigation on skin function and color regulatory mechanism reached a plateau.In this paper,Rhodeus sinensis was used to investigate the fish skin cell separation method,with modified enzyme digestion ways.Rhodeus were kept in bacteriostasis and fungistasis solution for about 8-24 hours.After digesting in the 0.25％ Trypsin-EDTA for 3-5 min,Rhodeus were killed and stroked off the scales.Peeled fish skin,and removed muscle or other tissue which jointed to the skin in stereo microscope.Then skin was digested by collagenase Ⅳ and trypsin jointed digestion method.Later,Rhodeus Skin Derived Cells,RSDCs for short,were collected and cultured at 28 ℃,5％ CO2.The growth curve was drawn by hemacytometer counting method.The expression of epithelial markers,Keratin 18&Vinculin A,and endothelial label gene,collagen I,were tested by RT-PCR method at F0,F5 and F10 generation.RSDCs lines were obtained and cultured well with doubling time of 30 h and "S"-growth-curve,which was familiar with the other adherent cells.The RT-PCR results revealed that the expression of Keratin 18 and Vinculin A declined along with cell passage cultivation,while the expression of Collagen I was on the rise.Our researches indicated that modified enzyme digestion method should be ideal method to obtain fish skin cell.The obtained RSDCs growth curve was typical adherent cells "S" type.And the obtained RSDCs were derived from epithelium (ectoderm) and endothelium (mesenchyme).Meanwhile,along with passage cultivation,the proportion of epithelium declined.关键词
中华鳑鲏/皮肤细胞/细胞培养/酶消化法Key words
Rhodeus sinensis/skin cell/cell culture/enzyme digestion method分类
生物科学引用本文复制引用
李伟,王建国,郑蒙蒙,陈小江,熊良伟,袁圣,王权..中华鳑鲏皮肤细胞的分离及其细胞系的建立[J].水产学报,2017,41(3):358-365,8.基金项目
Researches on Conservation and Breeding Technology of Rhodeus Resources of Lixiahe Basin (Y2015-25)里下河流域鳑鲏鱼种质资源保护与品种选育技术研究(Y2015-25) （Y2015-25）
