云纹石斑鱼心脏细胞系的建立与鉴定

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摘要鱼类细胞系在鱼类病毒学及功能基因组学方面发挥着重要作用。云纹石斑鱼(Epinephelus moara) 是一种具有重要养殖价值的海水鱼类，尚未见细胞系建立的报道。本研究以云纹石斑鱼的心脏组织为材料，建立了云纹石斑鱼心脏细胞系(epinephelus moara heart cell line, EMH)。细胞系所用的培养基是在L15基本培养基中添加了HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)，抗生素，胎牛血清(fetal bovine serum, FBS)，β-巯基乙醇(2-Mercaptoethanol, 2-ME)和碱性成纤维生长因子(basic fibroblast growth factor, bFGF)的完全培养液。EMH细胞已传至20多代，由成纤维样细胞和上皮样细胞组成。本研究检测了温度及FBS浓度对EMH细胞生长的影响。EMH细胞在18~30 ℃能正常生长，其最适生长温度为30 ℃，在36 ℃时不能正常生长。FBS浓度在10%~20%细胞均能快速生长，最适FBS浓度为15%。染色体分析表明，具有正常二倍体染色体数目(2n=48)的细胞所占的比例为62%。用脂质体法将含增强绿色荧光蛋白(enhanced green fluorescent protein, EGFP)基因的质粒载体pEGFP-N3转入EMH细胞后，EGFP成功表达。EMH细胞系的建立将为病毒的分离、鉴定和功能基因的研究奠定基础。

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	刘肖峰
	陈松林
	沙珍霞
	吴娅红
	于洋
	王娜
	修旺珊

关键词 ：云纹石斑鱼, 细胞系, 核型, 增强绿色荧光蛋白(EGFP)

Abstract：Cell lines provide an important in vitro system for investigations in virology and functional genomics in fish. A new heart cell line, epinephelus moara heart cell line (EMH), was established from Kelp Bass (Epinephelus moara). The cell line was maintained in Leibovitz's L15 medium supplemented with HEPES, antibiotics, 2-Mercaptoethanol (2-ME), fetal bovine serum (FBS) and basic fibroblast growth factor (bFGF). The cultured EMH cells, fibroblastic and epithelial in morphology, could be subcultured once every 4 or 5 days and had been subcultured for more than 20 passages. The suitable temperature for the cell growth was 18~30 ℃ with the optimum growth at 30 ℃ and an unfavorable growth at 36 ℃. The suitable FBS concentration for the cell growth was 10%~20% with the optimum concentration at 15%. Cytogenetic analysis revealed that 62% of the cells maintained a normal diploid chromosome number (2n=48) in the EMH cells at Passage 20. The fluorescent signals were observed in EMH cells after the cells were transfected with enhanced green fluorescent protein (EGFP) reporter plasmid. The EMH established here can be used as a useful tool for transgenic and genetic manipulation studies and may be potentially used for fish virus isolation, propagation and vaccine development.

Key words： Epinephelus moara Cell line Karyotype Enhanced green fluorescent protein (EGFP)

收稿日期: 2015-03-20 出版日期: 2015-07-24

通讯作者: 陈松林 E-mail: chensl@ysfri.ac.cn

引用本文:

刘肖峰陈松林沙珍霞吴娅红于洋王娜修旺珊. 云纹石斑鱼心脏细胞系的建立与鉴定[J]. , 2015, 23(10): 1394-1400.

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http://journal05.magtech.org.cn/Jwk_ny/CN/ 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2015/V23/I10/1394

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