条斑星鲽连续性鳍细胞系的建立与鉴定

Establishment and identification of a continuous fin cell line from barfin flounder Verasper moser

为了建立条斑星鲽鳍细胞系,为其细胞工程及病毒学研究奠定基础,对经Ⅱ型胶原酶和透明质酸酶联合消化法所得鳍组织碎块分别用DMEM/F12、L-15和M199培养液(pH7.2)在18～26℃进行了鳍组织的体外培养,并在所筛选出的最适培养液和培养温度下通过添加羧甲基壳寡糖、碱性成纤维样生长因子(bFGF)和I型胰岛素样生长因子(IGF-I)启动了鳍细胞的原代培养。体外培养结果显示,条斑星鲽鳍细胞的最适培养液为DMEM/F12培养液,最适培养温度为22℃,原代培养鳍细胞的生长分裂状态旺盛,细胞形态主要为成纤维样,20d后便可形成汇合细胞单层,经过连续的继代培养已建立了条斑星鲽的连续性鳍细胞系,目前已传至第135代;生长特性检测结果显示,第60代鳍细胞系细胞的群体倍增时间为56.9h,其生长分裂状态依然十分旺盛;染色体分析结果显示,第60代鳍细胞系细胞虽然出现了染色体的非整倍性,但其特征性染色体数目仍为46条,并具有2sm+44t的正常二倍体核型,证明所建立的细胞系确为条斑星鲽连续性鳍细胞系。该细胞系为条斑星鲽的细胞工程育种和病毒-细胞相互作用提供了一个理想的体外研究体系,具有重要的理论意义和应用价值。

To establish a barfin flounder fin cell line and lay a solid foundation for viral and cytotechnological studies,the fin tissues,digested with hyaluronidase and collagenase II,were cultured in 20% fetal bovine serum（FBS）-containing DMEM/F12 （1：1）,M199 and Leibovitz L-15 medium （pH7.2）,supplemented with 20% fetal bovine serum,carboxymethyl-chitosan,basic fibroblast growth factor （bFGF） and insulin-like growth factor-I （IGF-I）,at 18～26℃,respectively.The results of in vitro culture showed that the optimum medium of the fin cells was DMEM/F12 medium （pH7.2） with the above supplements,and the optimum temperature of them was about 22℃.By utilizing 20% FBS-DMEM/F12 medium with the above supplements,the primary culture of the fin cells was initiated at 22℃.And the fin cells,in fibroblastic morphology,grew rapidly,and formed a confluent monolayer 20 d after culture initiation.By successive subculture,a barfin flounder fin cell line was sucessfully established,and sub-cultured to passage 135 till now.Growth property analysis showed that the fin cells of the cell line at passage 60 had a population doubling time of 56.9h,which indicated that the cells had active proliferating abilities.Chromosome analysis showed that the cells exhibited chromosomal aneuploidy but still had a modal chromosome number of 46 which had a normal diploid karyotype of 1 pair of submetacentric and 22 pairs of telocentric chromosomes.All these indicate that a novel continuous barfin flounder fin cell line was sucessfully established in this study,which as an ideal in vitro researching system,will be of great theoretical and practical significance for cytotechnological breeding and cell-virus interaction studies of barfin flounder.