大黄鱼鳍细胞系的建立及久效磷对其毒性作用的研究

Establishment and Identification of a Continuous Fin Cell Line from Large Yellow Croaker,Pseudosciaena crocea and Cytotoxic Effects of Monocrotophos to the Fin Cells

为了建立大黄鱼（Pseudosciaena crocea）鳍细胞系,为其细胞毒理学、病毒学与细胞工程等研究奠定基础,本文采用酶消化法使用含有20%胎牛血清（FBS）的DMEM/F12、Leibovitz L-15和DMEM培养液（pH7.2）在22~28℃分别启动了大黄鱼鳍组织的体外培养,通过添加促细胞贴壁和分裂的物质在最适培养条件下对大黄鱼鳍细胞进行了原代培养和继代培养,并研究了久效磷对大黄鱼细胞系鳍细胞的毒性作用。体外培养结果显示,大黄鱼鳍细胞的最适培养液为20%FBS-DMEM/F12,最适培养温度为25℃,体外培养鳍细胞的形态主要为成纤维细胞样,22 d后便可形成汇合细胞单层,经过连续继代培养成功建立了大黄鱼的连续性鳍细胞系,目前已传至第112代;对第60代大黄鱼鳍细胞系细胞的鉴定结果显示,其群体倍增时间为50.96 h,分裂状态十分旺盛,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48条,并具有正常的6m＋6sm＋36t二倍体核型,证明所建立的细胞系确为大黄鱼鳍细胞系。不同浓度久效磷处理的检测结果显示,大黄鱼鳍细胞对久效磷敏感,20~160μg/mL久效磷可引起鳍细胞乙酰胆碱酯酶活性的持续性显著降低,引起超氧化物歧化酶和谷胱甘肽-S-转移酶活性的显著升高并随着浓度的升高而逐渐降低,久效磷对该细胞系细胞的48 h半抑制浓度（IC50）为43.05μg/mL,证实久效磷对大黄鱼鳍细胞具有显著的毒性作用。

For laying a solid foundation for cellular toxicology,virology and cell engineering studies,this article is about establishment and identification of large yellow croaker fin cell line and study of cytotoxic effects of monocrotophos to the fin cells.The fin tissues,digested with hyaluronidase and collagenase II,were cultured in three kinds of media（pH7.2） at different temperatures supplemented with 20% fetal bovine serum,carboxymethyl-chitooligosaccharide,chondroitinsulfate,basic fibroblast growth factor and insulin-like growth factor-I to initiate the primary culture and subculture.The culture in vitro showed that the optimum medium and temperature were Dulbecco＇s modified Eagle medium/F12 medium（DMEM/F12） and 25 ℃ respectively,and the fibroblastic fin cells grew steadily and formed monolayer 22 days later.The large yellow croaker fin cell line has been sucessfully established and subcultured to passage 112 now.The fin cells at passage 60 had a population doubling time of 50.96 h,indicating that the cells still proliferated actively.Karyotype analysis showed that the cells exhibited chromosomal aneuploidy but had a modal diploid chromosome number of 48 with 3 pairs of metacentrics,3 pairs of submetacentrics and 18 pairs of telocentrics.In the monocrotophos treated cells from 20 to 160 μg/mL,results showed that the activities of acetylcholinesterase（AChE） reduced sustainingly and significantly,the activities of superoxide dismutase（SOD） and glutathione S-transferase（GST） increased significantly but later reduced along with increased concentration of monocrotophos and the 48 h-IC50 values of monocrotophos to yellow croaker fin cells were 43.05 μg/mL.Results showed that monocrotophos have significantly cytotoxic effect to large yellow croaker fin cells.The established continuous large yellow croaker fin cell line has theoretical significance and practical value for laying a foundation to detect the organophosphorus pesticide and seting up a rapid effective bioassay system,and studing of cellular toxicology,virology and cell engineering as an ideal in vitro research system.