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宽口裂腹鱼中肾组织细胞系建立的初步研究
代金彩,聂竹兰,刘洁雅,洪继彪
塔里木大学动物科学学院 新疆建设兵团塔里木畜牧科技重点实验室 塔里木珍稀鱼类研究中心 新疆 阿拉尔 843300
摘要:
鱼类细胞培养是进行种质保存、基因功能分析、细胞工程育种等研究的重要材料和模型。为了建立濒危的宽口裂腹鱼(Schizothorax eurystomus)中肾组织细胞系,本研究以其中肾组织为材料,以组织块移植法启动细胞原代培养,待细胞稳定后再进行传代培养,建立宽口裂腹鱼中肾组织细胞系,命名为EUM10。分析宽口裂腹鱼中肾细胞系冻存复苏能力和最适生长条件,且通过PCR技术检验污染状况,用线粒体基因片段分析鉴定细胞的来源。结果显示,宽口裂腹鱼中肾组织细胞的最佳培养基为DME/F-12,最适温度为25℃,最适血清浓度为20%;细胞呈悬浮生长,生长曲线为“S”型曲线,第10代中肾组织细胞系细胞的群体倍增时间为23.26 h;第6代细胞液氮冷冻保存6个月后,复苏存活率达91.91%;经污染鉴定无污染现象;EUM10第10代细胞线粒体16S rRNA序列分析结果与宽口裂腹鱼基因序列一致性为99.81%,表明细胞系来自宽口裂腹鱼。本研究成功建立了宽口裂腹鱼中肾组织细胞系,可将宽口裂腹鱼中肾细胞系作为体外体系运用于宽口裂腹鱼的研究,如细胞遗传学、基因功能分析等,可为宽口裂腹鱼的基础研究和育种工作提供基础资料。
关键词:  宽口裂腹鱼  中肾组织  细胞系  细胞培养
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Preliminary study on the establishment of kidney tissue cell lines in Schizothorax eurystomus
DAI Jincai1,2, NIE Zhulan1,2, LIU Jieya1,2, HONG Jibiao1,2
1.College of Animal Science, Tarim University;2.Key Laboratory of Tarim Animal Husbandry Science and Technology, Xinjiang Production and Construction, Alar, Xinjiang 843300, China
Abstract:
Fish cell culture is an important model for germplasm preservation, gene function analysis, and cell engineering breeding. Therefore, it is of great significance to establish cell lines for the endangered Schizothorax eurystomus. The aim of this study was to establish a kidney tissue cell line for S. eurystomus, for which mesonephric tissue was used. The primary cell culture was initiated by tissue block transplantation and the subculture was carried out after the cells were stabilized. A kidney cell line (EUM10) was established. To analyze the cryopreservation and resuscitation ability of renal cell lines and the optimal growth conditions for S. eurystomus, PCR was used to detect contamination and mitochondrial gene fragment analysis was used to identify the source of the cell. The results showed that the optimal medium for S. eurystomus kidney tissue cells was DME/F-12, the optimal temperature was 25℃, and the optimal serum concentration was 20%. The cells grew in suspension and the growth curve was S-shaped. The growth curve shows that the population doubling time of the kidney tissue cell line cells in the 10th generation was 23.26 h. After 6 months of cryopreservation with liquid nitrogen, the recovery/survival rate was 91.91% (6th generation). S. eurystomus shares 99.81% of the gene sequence identity with a split-bellied fish from Tarim, which proves that the cell line originates from a wide-mouthed split-bellied fish. In this study, a kidney cell line for S. eurystomus was successfully established. Kidney cell lines can be used as in vitro systems to study various aspects of fish, such as cytogenetics and gene function analysis. This study provides the basic data for the initial research into and breeding of S.eurystomus.
Key words:  Schizothorax eurystomus  Kidney tissue  Cell line  Cell culture