CAJ | 학술논문

采用组织块培养法对泥鳅Misgurnus anguillicaudatus鳍组织细胞进行原代培养,并采用胰蛋白酶消化法传代,目前已传63代。原代培养和早期传代培养所用培养基为DMEM/F12,并添加体积分数为20%的胎牛血清( FBS)、10 ng/mL 人碱性成纤维细胞生长因子( bFGF )、20 ng/mLⅠ型胰岛素样生长因子(IGF-Ⅰ)和10μg/mL硫酸软骨素。30代以后所用培养基为20% FBS-DMEM/F12,在CO2培养箱(5%,25℃)中培养,培养的鳍细胞形态为成纤维细胞样,群体倍增时间为56.85 h,特征染色体数目为50条；细胞经液氮冷冻保存30 d,解冻复苏后,存活率为81.31%±1.46%。病毒敏感性试验结果表明,泥鳅鳍细胞系对鲤春病毒血症病毒( SVCV)敏感,病毒滴度为105.62 TCID50/mL,而对鱼类诺达病毒( YGNNV)不敏感。泥鳅鳍细胞系的建立丰富了鱼类细胞系的种类,并为泥鳅毒理学、病毒学的研究提供了科学±据。
채용조직괴배양법대니추Misgurnus anguillicaudatus기조직세포진행원대배양,병채용이단백매소화법전대,목전이전63대。원대배양화조기전대배양소용배양기위DMEM/F12,병첨가체적분수위20%적태우혈청( FBS)、10 ng/mL 인감성성섬유세포생장인자( bFGF )、20 ng/mLⅠ형이도소양생장인자(IGF-Ⅰ)화10μg/mL류산연골소。30대이후소용배양기위20% FBS-DMEM/F12,재CO2배양상(5%,25℃)중배양,배양적기세포형태위성섬유세포양,군체배증시간위56.85 h,특정염색체수목위50조；세포경액담냉동보존30 d,해동복소후,존활솔위81.31%±1.46%。병독민감성시험결과표명,니추기세포계대리춘병독혈증병독( SVCV)민감,병독적도위105.62 TCID50/mL,이대어류낙체병독( YGNNV)불민감。니추기세포계적건립봉부료어류세포계적충류,병위니추독이학、병독학적연구제공료과학±거。
Primary cell culture of fin from loach Misgurnus anguillicaudatus was carried out by tissue explant meth-od and the cells were subcultured for 63 generations until by the special and combined aseptic processing method in which the fin tissue was immerged in 10% povidone-iodine for 15 min, and then in penicillin and streptomycin mixture (500 IU/mL penicillin and 500 μg/mL streptomycin) for 30 min. The medium DMEM/F12 used in the primary culture and early subculture was supplemented with 20% fetal bovine serum( FBS) ,10 ng/mL basic fibro-blast growth factor(bFGF), 20 ng/mL insulin-like growth factor-I(IGF-Ⅰ)and 10 μg/mL chondroitin sulfate (pH 7. 2). Only 20% FBS-DMEM/F12 was used as medium after 30 generations, and the fin cell line was cul-tured under the conditions of 25 ℃ with 5% CO2 for 56. 85 h, with chromosome number of 50 in the samples col-lection from 100 metaphase plates. The cell livability of(81. 31%±1. 46%)was fund after thawing in 30 day frozen cells. The virus sensitivity test revealed that the cell line was susceptible to the infection of spring viremia of carp virus ( SVCV) , with viral titer of about 105. 62 TCID50/mL, and not to susceptible to fish nodavirus. Establishment of fin cell line from loach makes an increase in kinds of the fish cell lines, and provides the foundation for the re-search of toxicology and virology.