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为丰富海水鱼类细胞系的数量,提供更多的基因功能研究及病毒分离、鉴定工具,本研究建立了一种新的细胞系,大菱鲆心脏细胞系(Scophthalmus maximus heart cell line,SMH)。该细胞系使用组织块法(tissue block method)启动原代培养,培养液是添加了胎牛血清(fetal bovine serum,FBS)、人类碱性成纤维生长因子(basic fibroblast growth factor,b FGF)、抗生素、β-巯基乙醇(2-mercaptoethanol,2-ME)、hepes(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)的DMEM/F12。结果显示,SMH形态呈典型的成纤维细胞样,在培养液中生长分裂旺盛,经230 d传代培养,成功传至58代。用带有绿色荧光蛋白(green fluorescent protein,GFP)的p EGFP-N3质粒转染SMH,发现GFP在细胞中成功表达,转染效率为40%左右。使用遗传霉素(geneticin,G418)对重组质粒细胞进行筛选,得到了一簇荧光表达效率高的细胞。染色体分析表明,具有正常二倍体核型(2n=44)的细胞占64%。线粒体细胞色素C氧化酶Ⅰ(cytochrome oxidase subunitⅠ,COⅠ)基因鉴定出该细胞系来源于大菱鲆。该细胞系的建立为大菱鲆功能基因及其他基于细胞系的研究奠定了基础,对大菱鲆的病毒感染途径和分子机制研究具有重要的理论意义。
In order to enrich the number of marine fish cell lines and provide more genetic function and virus isolation and identification tools, this study established a new cell line, Scrophthalmus maximus heart cell line (SMH) . This cell line was used to initiate primary culture using a tissue block method, supplemented with fetal bovine serum (FBS), basic fibroblast growth factor (b FGF) Antibiotics, 2-mercaptoethanol (2-ME), hepes (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid) in DMEM / F12. The results showed that SMH showed a typical fibroblast-like morphology with strong growth and division in culture medium. After passaged for 230 days, SMH was successfully passaged to 58 passages. Transfection of SMH with p EGFP-N3 plasmid with green fluorescent protein (GFP) revealed that GFP was successfully expressed in cells with a transfection efficiency of about 40%. The recombinant plasmid cells were selected using geneticin (G418), and a cluster of cells with high fluorescence expression efficiency was obtained. Chromosomal analysis showed that cells with a normal diploid karyotype (2n = 44) accounted for 64%. The mitochondrial cytochrome oxidase subunitⅠ (COⅠ) gene was identified from the turbot. The establishment of this cell line laid the foundation for the study of functional genes and other cell lines based on the expression of turbot, and had important theoretical significance on the pathogenicity and molecular mechanism of turbot.