Establishment of a continuous embryonic cell line from Japanese flounder Paralichthys olivaceus for virus isolation
Establishment of a continuous embryonic cell line from Japanese flounder Paralichthys olivaceus for virus isolation
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DOI：
10.3354/dao060241
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发表时间：
2004-09-08
影响因子：
1.4
通讯作者：
Shi, CY
中科院分区：
文献类型：
--
作者：
Chen, SL;Ren, GC;Shi, CY
以海水养殖鱼类牙鲆（Paralichthys olivaceus）原肠期胚胎为材料，建立了比目鱼胚胎细胞系（FEC）。FEC细胞在补充有抗生素、胎牛血清（FBS）、鲈鱼血清（SPS）和碱性成纤维细胞生长因子（bFGF）的Dulbecco改良Eagle培养基（DMEM）中培养。细胞小而圆，在培养中生长活跃、稳定。检测了温度、FBS浓度和bFGF对FEC细胞生长的影响。细胞在24至30 ℃之间生长良好，但在18 ℃以下生长速率降低。FEC细胞在含15%FBS的培养基中的生长速度高于含7.5%FBS的培养基。培养基中添加碱性成纤维细胞生长因子也显着增加了生长速率。染色体分析显示FEC细胞具有正常的二倍体核型，2n = 48。细胞培养物经冷冻保存后获得高存活率。检测了该细胞系对鱼病毒的易感性。测试的两种病毒显示在FEC细胞上诱导CPE（细胞病变效应）。通过电镜进一步阐明了感染鱼虹彩病毒的FEC细胞培养物。在病毒感染的FEC细胞的细胞质中发现了许多病毒颗粒。这些结果表明，FEC细胞系可用于鱼类病毒的分离和研究。
A continuous cell line, the flounder embryonic cell line (FEC), was established from gastrula-stage embryos of a marine cultured fish, the Japanese flounder Paralichthys olivaceus and cultured for more than 200 d with more than 60 passages. FEC cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with antibiotics, fetal bovine serum (FBS), sea perch serum (SPS), and basic fibroblast growth factor (bFGF). The cells were small and round, and grew actively and stably in culture. The effect of temperature, FBS concentration and bFGF on FEC cell growth was examined. Cells grew well between 24 and 30 degreesC, but had a reduced growth rate below 18 degreesC. The growth rate of FEC cells in medium containing 15 % FBS was higher than that in medium containing 7.5% FBS. Addition of bFGF to the medium also significantly increased the growth rate. Chromosome analysis revealed that FEC cells have a normal diploid karyotype with 2n = 48. High survival rate was obtained after cryopreservation of cell cultures. The susceptibility of the cell line to piscine viruses was examined. Two viruses tested were shown to induce CPE (cytopathic effect) on FEC cells. FEC cell culture infected with fish iridovirus was further elucidated by electron microscopy. Many virus particles were found in the cytoplasm of the virus-infected FEC cells. These results indicated that the FEC cell line could be potentially used to isolate and study fish viruses.