青鱼鳍条组织细胞系的建立及其生物学特性

Establishment and characterization of a cell line derived from fin of Mylopharyngodon piceus

采用组织块移植培养技术,对来源于青鱼(Mylopharyngodon piceus)的鳍条组织细胞进行原代培养,建立了青鱼鳍条组织细胞系,定名为BC-Fin。青鱼鳍条组织细胞为成纤维样细胞,已稳定传代培养50多代,其最适培养温度为25℃,最佳培养基为L-15,最适血清浓度为15%,在最适培养条件下,青鱼鳍条组织细胞的群体倍增时间为60.6 h。青鱼鳍条组织细胞液氮冷冻保藏6个月后,经台盼蓝染色,约(90.09±4.65)%的细胞具有细胞活性,复苏后的细胞生长旺盛。细胞染色体分析显示,第16代青鱼鳍条组织细胞的染色体数目为正常二倍体(2n=48),第41代细胞染色体众数为46。通过对离体培养细胞的线粒体中的16S rRNA基因进行特异性扩增,获得长度为320 bp的核酸片段,核酸序列比对分析结果表明,其与青鱼16S rRNA基因序列的一致性达98%,表明该细胞来源于青鱼。病毒敏感性试验结果显示,在感染草鱼呼肠孤病毒(GCRV)后BC-Fin细胞系可产生典型细胞病变效应,病毒滴度为105.33±0.21TCID50/m L,且PCR检测可检测出细胞培养的草鱼呼肠孤病毒,表明BCFin细胞系对草鱼呼肠孤病毒较敏感。

In order to rich the fish cell resourse and provide the important basic experiment materials for research of herring hemorrhagic disease etiology and pathogenesis,diagnostic technology,vaccine and immune prevention technology,a cell line,designated BC-Fin,has been established and characterized from the fin of Mylopharyngodon piceus by using tissue explant techniques. BC-Fin cell line has been continuously subcultured over 50 times and fully characterized in the aspects of morphological observation,cryopreservation,optimal growth kinetics,population doubling time and karyotyping,etc.BC-Fin cell monolayer consists of fibroblast-like cells and its optimal growth condition is： L-15 medium,15% FBS and25 ℃. Under such optimized condition the doubling time of BC-Fin cell numbers is about 60. 6 h. After 6 months cryopreservation in liquid nitrogen,BC-Fin cells exhibit a viability of about（ 90. 09 ± 4. 65） % with trypan blue staining. Chromosome analysis of BC-Fin cell line revealed that the chromosome number was diploid（ 2n = 48） at the 16 thpassage,and the chromosome modal number is 46 at the 41 thpassage. 16 S rRNA amplification,sequencing and analysis showed that the cloned fragment sequence has 98% identity with the mitochondrial 16 S rRNA of black carp. The results of virus sensitivity test indicated that BC-Fin cell line is susceptible to the infection of Grass carp reovirus（ GCRV）,a characteristic viral-induced cytopathic effect（ CPE） is observed and the viral titer（ TCID50/ m L） reaches about 105. 33 ± 0. 21. Moreover,PCR detection demonstrats that the targeted gene can be amplified by using specific primers.