【摘要】 建立大菱鲆(Scophthalus maximus)鳍细胞系,文中采用胰蛋白酶、透明质酸酶和Ⅱ型胶原酶消化法启动大菱鲆鳍组织的原代培养,并通过培养液配方和培养条件的优化成功进行大菱鲆鳍细胞的继代培养。研究结果显示,在pH=7.0~7.4、温度20~24℃的培养条件下,培养于含有表皮细胞生长因子、碱性成纤维细胞生长因子、羧甲基壳多糖、N-乙酰葡萄糖盐酸盐的Leibovitz-15培养液(20%胎牛血清)中的大菱鲆鳍细胞,其生长分裂状况最好,细胞形态为成纤维细胞样。经继代培养后,细胞生长分裂依然十分旺盛,第60代大菱鲆鳍细胞系的群体倍增时间为62.4 h,虽然出现了染色体的非整倍性,但其特征性染色体仍为44条。该细胞系细胞经液氮冻存后仍保持有原有形态和较高存活率。现已成功建立了连续性大菱鲆鳍细胞系,目前已传至第65代。该细胞系的建立对于查清病毒对大菱鲆细胞的感染途径与感染机理等具有重要的理论意义,对于病毒疫苗研制具有重要应用价值。更多还原

【Abstract】 To establish a cell line of turbot Scophthalus maximus,primary culture of turbot fin cells was initiated by trypsin,hyaluronidase and type II callagenase digestion methods,and the cells were successfully subcultured after optimization of the culture medium and culture conditions.The results showed that the optimal culture medium for turbot fin cells was Leibovitz-15 medium supplemented with 20% fetal bovine serum,basic fibroblast growth factor(bFGF),epidermal growth factor(EGF),N-Acetyl-D-glucosamine,and carboxymethyl-chitosan.And the optimal culture conditions for turbot fin cells were 20-24 ℃ and pH=7.0-7.4.The turbot fin cells were in fibroblast-like shape and grew actively under the optimal medium and culture conditions.The population-doubling time of the cell line established is 62.4 hours at passage 60.And chromosome analysis showed that the cells exhibited chromosomal aneuploidy with a modal chromosome number of 44.The cells maintained their original shape and high viability after cryopreserved and resuscitated.A continuous turbot fin cell line has been established which has been subcultured to passage 65.The established turbot fin cell line will provide an ideal research system for studying the pathways and mechanisms of infective viruses,and will lay solid foundations for viral vaccine development.更多还原