半滑舌鳎脾脏、卵巢组织细胞离体培养的初步研究

The preliminary researches on half-smooth tongue sole spleen and ovary cells in vitro culture

实验采用胰酶消化法,对半滑舌鳎脾脏、卵巢组织细胞进行离体培养,结果显示,脾脏、卵巢细胞能够在添加20%FBS,50 mmol/L 2-Me,100 U/mL青霉素,100μg/mL链霉素,1mmol/L丙酮酸钠,10 ng/mLbFGF的MEM完全培养基中生长,两种细胞的形态均为成纤维样,脾脏细胞可传至第9代,卵巢细胞至今已传至第11代。脾脏、卵巢细胞染色体中期分裂相均为正常的二倍体核型(2n=42),且具有异型W染色体。20%的血清浓度更适用于卵巢细胞传代培养。利用脂质体可将含有RFP荧光报告基因的质粒成功转入脾脏细胞中。

The cell lines from different organs of half-smooth tongue sole were the ideal material for researches on fish virology, genetic screening and functional analysis, etc. In this study, the researches of the spleen and ovary cells from female individual in vitro culture were developed by trypsin digestion method. The results showed that the spleen and ovary cells were cultured in minimum essential medium （MEM） supplemented with 20% fetal bovine serum （ FBS ）, 50 mmol/L 2-Mercaptoethanol （ 2-Me）, 100 U/mL penicillin,100 μg/mL streptomycin, 1 mmol/L sodium pyruvate and 10 ng/mL basic fibroblast growth factor（bFGF）, and microscopically, the cells were composed of fibroblast-like cells. The spleen cells were subcultured for 9 passages. Up to now, the ovary cells had been subcultured for 11 passages. Chromosome analysis revealed that the spleen and ovary cells maintained normal diploid number（2n = 42）, containing the heterogametic W chromosome. In subculture,the Suitable concentration of FBS for the ovary cells was 20%. The spleen cells had been successfully transformed with red fluorescent protein（RFP）reporter plasmid.