大泷六线鱼三种组织细胞的有限传代及其生物学特性分析

Limited subculture and biological characteristic analysis of three cell lines from fat greenling(Hexagrammos otakii)

采用组织块培养法启动大泷六线鱼鳍、吻端和肾脏3种组织细胞的原代培养,并稳定传代培养30代、31代和35代。结果发现,用透明质酸酶和Ⅱ型胶原酶联合消化鳍和吻端组织后细胞分散效果更好。培养于添加5 ng/mL人碱性成纤维细胞生长因子(bFGF)、20μg/mL硫酸软骨素、40 ng/mLⅠ型胰岛素样生长因子(IGF-I)及20%胎牛血清的DMEM/F12培养基(pH 7.2)中的3种组织细胞生长分裂旺盛,均为成纤维样细胞。在此条件下第20代的鳍、吻端、肾脏组织细胞群体倍增时间分别为58.7,50.4和32.9 h。第25代大泷六线鱼3种组织细胞的特征性染色体数目均为48条。细胞经液氮冷冻保存60 d后,解冻复苏并经台盼蓝染色,3种细胞成活率分别达84.59%±1.07%、85.75%±1.03%和87.39%±1.05%。3种细胞现已保存在中国典型培养物保藏中心。3种组织细胞体外培养方法的建立为鱼类疾病防治和病理机制研究奠定了基础。

Fat greenling（Hexagrammos otakii）is one of the important commercial fishes along the northern coast of China, and its artificial breeding and culture have been developed recently. Primary culture of fin, lip and kidney from H. otakii was studied using tissue explant method. Until now cells of three tissues have been subcultured at passage 30 for fin,passage 31 for lip, passage 35 for kidney respectively. The results showed that the fin and lip tissues could be dispersed quickly after being digested with 0.5% hyaluronidase and 0.2% collagenase 1I. The optimal growth conditions for fin,lip and kidney tissues were 20% fetal bovine serum （ FBS）, 5 ng/mL basic fibroblast growth factor （ bFGF）, 20 Ixg/mL chondroitin sulfate and 40 ng/mL insulin-like growth factor-I to medium DMEM/F12 （ pH 7.2 ） at 25 ~C. The doubling time of fin, lip and kindey cell numbers was about 58.7 h,50.4 h and 32.9 h at the 20th passage. Karyotype analysis of 100 metaphase plates revealed that the feature diploid chromosome number was 2n = 48 in fin, lip and kidney cell at 25th passage. The cell livability of these three kinds of cell were （ 84.59±1.07 ） % , （ 85.75 ± 1.03 ） % and（87.39 ± 1.05 ） % , respectively, when recovered after being stored in liquid nitrogen for 60 d at the 20th. Now, the three kinds of cell have been preserved in China Center for Type Culture Collection （CCTCC）. The methods of cell culture in vitro could be used to prevent and cure fish diseases and the pathomechanism study.