摘要
为研究红鳍东方鲀Takifugu rubripes鳍细胞系对该鱼病毒病诊断和治疗的影响,采用组织块培养法启动红鳍东方鲀鳍组织细胞的原代培养,用胰蛋白酶消化法进行传代培养,目前红鳍东方鲀鳍细胞传至40代,为成纤维细胞样细胞,原代培养和早期传代培养使用的培养基为添加了20%胎牛血清的DMEM/F12高糖培养基,添加的生长因子分别为终浓度为10 ng/m L的人碱性成纤维细胞生长因子(b FGF)、终浓度为20 ng/m L的Ⅰ型胰岛素样生长因子和终浓度为20μg/m L的硫酸软骨素,30代以后使用含20%胎牛血清的DMEM/F12培养基,将细胞于25℃、5%CO2恒温培养箱中培养。结果表明:生长曲线显示,细胞在0～2d内处于潜伏期,2～4 d时进入对数期,4～5 d时进入平台期,5 d后开始转入衰退期;红鳍东方鲀鳍细胞系的倍增时间为50. 24 h;细胞经超低温保存60 d,解冻复苏后测定其存活率为80%;经微卫星位点扩增,凝胶电泳后的条带证实细胞系来源于红鳍东方鲀。研究表明,红鳍东方鲀鳍细胞系的建立丰富了鱼类细胞系的种类,也为红鳍东方鲀病毒病的研究及疫苗制备等提供了数据资料。
A tissue block culture method is adopted to initiate primary culture of fin tissue cells of redfin puffer Takifugu rubripes,and the trypsin digestion method is used for subculture.At present,the fin cells of redfin puffer have passed to 40 generations and become the fibroblast-like cells.The medium used for primary culture and early subculture was DMEM/F12medium supplemented with 20% fetal bovine serum,with basic fibroblast growth factor(bFGF)final concentration of 10ng/mL,type I insulin-like growth factor of 20ng/mL(IGF-1),and the chondroitin sulfate final concentration of 20μg/mL.DMEM/F 12 medium containing20%fetal bovine serum was used,and cells were cultured in a constant temperature incubator at 25℃ and 5% CO2 in the 30th generation.The growth curve showed that the cells were in the incubation period from 0 to 2 days,in the logarithmic growth period from 2 to 4 days,in the plateau growth period from 4 to 5 days,and in the recession period5days afterwards.The culture doubling time was found to be 50.24h in redfin puffer fin cell line with survival rate of 80%,in 60 days cryopreservation,after thawing and resuscitation.Microsatellite site amplification and gel electrophoresis revealed that the cell line was originated from redfin puffer.The establishment of the redfin puffer fin cell line enriches the species of fish cell lines and lays a foundation for the research of viral diseases and vaccine preparation in redfin puffer.
作者
王缙云
李霞
秦艳杰
李状状
张晓雨
梁艳
姜志强
WANG Jin-yun;LI Xia;QIN Yan-jie;LI Zhuang-zhuang;ZHANG Xiao-yu;LIANG Yan;JIANG Zhi-qiang(Key Laboratory of Marine Bio-resource Restoration and Habitat Reparation in Liaoning Province,Dalian Ocean University,Dalian 116023,China;Key Laboratory of Mariculture and Stock Enhancement in North China s Sea,Ministry of Agriculture and Rural Affairs,Dalian Ocean University,Dalian 116023,China)
出处
《大连海洋大学学报》
CAS
CSCD
北大核心
2018年第6期703-708,共6页
Journal of Dalian Ocean University
基金
国家海洋公益性行业科研专项(201405003)
关键词
红鳍东方鲀
细胞系
细胞培养
生长特性
Takifugu rubripes
cell line
cell culture
characterization
