Development and Characterization of a New Cell Line Derived from the Kidney of Large Yellow Croaker(Larimichthys crocea)
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摘要: 大黄鱼Larimichthyscrocea是福建省最重要的经济性鱼类之一。为扩充完善大黄鱼体细胞库，丰富其病毒学、细胞毒理学及细胞工程学研究手段，以大黄鱼肾脏组织为材料，进行组织细胞体外培养条件的探索，构建大黄鱼体细胞体外培养技术体系，并在此基础上建立大黄鱼肾组织细胞系（YCK）。原代培养使用组织块贴壁翻瓶法，标准传代培养是以0.25%胰蛋白酶消化细胞，使用添加10%胎牛血清（FBS）的M-199，在27℃下进行密闭培养，每72 h完全换液1次。该细胞系主要由上皮样细胞构成，在上述培养条件下生长旺盛，经过400多天的连续培养，已经成功传代超过70次，第65代YCK细胞的群体倍增时间为36.12 h。对常用的鱼类细胞培养基进行适应性筛选，确认M-199是最适合该细胞系的培养介质，也可适用于其他大黄鱼细胞的培养。对该细胞系的染色体分析表明：虽然出现了异倍化现象，但该系依然符合2n=48的二倍体细胞系。Abstract: Large yellow croaker (Larimichthys crocea) is an important economic species in Fujian. To develop virological, toxicological and cell engineering techniques, as well as resources for advanced researches on the fish, this study had developed a new cell line derived from the fish kidneys(YCK). The obtained YCK has been successfully conserved in the M-199 medium supplemented with 10% fetal bovine serum at 27℃ over 400 days with a medium-change every 72 h, and satisfactorily sub-cultured more than 70 times during the interim. A parallel test of commonly applied culture media was conducted to get the conclusion that M-199 being the most suitable medium for YCK. This newly established cell line consisted predominantly of epithelial-like cells, and was proven to be diploid with a modal chromosome number of 2n=48.
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Key words:
- large yellow croaker (Larimichthys crocea) /
- kidney /
- cell line
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图 1 大黄鱼肾组织细胞的原代和传代培养
注：A为大黄鱼肾脏原代培养，第12 d；B为第6代YCK细胞(标尺100 μm)；C为第27代YCK细胞；D为第70代YCK细胞。
Figure 1. Primary culture and subculture of cells from kidney of L. crocea
图 2 大黄鱼肾组织细胞系YCK在不同培养基的生长情况
注：A为L-15中培养的YCK细胞；B为RPMI-1640中培养的YCK细胞。
Figure 2. Comparison of the growth characteristics of YCK in different culture media
图 3 不同培养温度下大黄鱼肾组织细胞系YCK生长趋势
Figure 3. Growth kinetics of YCK under different temperatures
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