锦鲤组织细胞体外培养的初步研究

为建立来源于锦鲤的细胞系,本文采用组织块法,对来源于锦鲤(Cyprinus carpio)鳍条、吻端、肌肉、心脏、鳔、肠道、卵巢等组织的细胞进行原代培养和传代培养.在25℃下,添加20％胎牛血清、0.2μg/mL表皮生长因子(EGF)和25ng/mL成纤维生长因子(FGF)的L-15培养基中进行培养.结果显示,鳍条、心脏、鳔、吻端、肌肉、肠道、卵巢分别在原代培养第3、6、12、13、15、3、3天,有细胞从组织块迁出;对长至单层的鳍条、心脏、肌肉细胞进行传代培养,鳍条细胞已传至第39代,心脏和肌肉细胞分别传到第2代和第4代;鳍条、心脏和肌肉细胞分别呈现上皮细胞样、上皮细胞样和上皮细胞样与成纤维状混合型.第6代锦鲤鳍细胞的染色体计数,结果显示,细胞染色体数目分布范围为55～154条,2n=100.病毒敏感性实验,发现锦鲤鳍条细胞对草鱼出血病病毒(GCRV)和鲤春病毒血症病毒(SVCV)都敏感,且在24h内出现细胞病变(CPE);但对斑点叉尾鮰病毒(CCV)不敏感.锦鲤鳍条细胞系的建立为后期建立更多的锦鲤细胞系和鱼类病毒研究奠定了基础.

Study on the in vitro Primary Culture of Tissue Cells from Ornamental Carp, Cyprinus carpio

To establish cell lines from ornamental carp(Cyprinus carpio),primary cultures of fin,snout,muscle,heart,swim bladder,intestine and ovary of ornamental carp were initiated at 25 ℃ in L-15 medium supplemented with 20% fetal bovine serum,0.2 μg/mL epidermal growth factor(EGF) and 25 ng/mL fibroblast growth factor(FGF) by using the tissue explants technique.The cells of fin,heart,swim bladder,snout,muscle intestine and ovary tissue migrated out around the tissues at day 3,6,12,13,15,3 and 3 respectively.The confluent monolayers of cells were rinsed twice with 0.25% trypsin-EDTA solution.The second generation of heart cells,the third generation of muscle cells and the thirty-ninth generation of fin cells were obtained.The morphologies of heart cells and fin cells were both epithelioid,muscle cells were both epithelioid and fibroblast-like.Karyotype analysis showed the number of chromosome ranged from 55 to 154 in the fin cells at passage 6 with a distinct peak at 100 diploid chromosomes.The fin cells were sensitive to Grass Carp reovirus(GCRV) and Spring viremia of carp virus(SVCV),and visible cytopathic effect(CPE) appeared in 24 hours.But the fin cells were not sensitive to Channel Catfish virus(CCV).The fin cell line could lay a foundation for the establishment of more cell lines from ornamental carp and the research of fish virology.