大菱鲆心脏细胞系的建立与鉴定  

Development and Characterization of a Heart Cell Line From Turbot(Scophthalmus maximus)

作　　者：马佳璐[1,2,3] 高进[2,3,4] 孙爱[2] 温海深[1] 刘肖锋 李文龙[2] 陈松林[2,3] 

机构地区：[1]中国海洋大学水产学院,青岛266003 [2]中国水产科学研究院黄海水产研究所,青岛266007 [3]海洋科学与技术国家实验室,海洋渔业科学与食物产出过程功能实验室,青岛266237 [4]南京农业大学无锡渔业学院,无锡214081

出　　处：《农业生物技术学报》2016年第7期1092-1100,共9页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金重点项目(No.31130057);黄海水产研究所级基本科研业务费(No.20603022013034);山东省泰山学者攀登计划专项

摘　　要：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。The development of fish cell line provides an important tool to study virology, immunology, genetics, oncology, developmental biology, toxicology, clinical medicine and biotechnology in aquaculture. Many fish cell lines have been established from freshwater and anadromous species, but few in marine fishes. As a widely cultivated marine fish species with high economic value in Europe and China, the number of cell lines was inadequacies in the research of turbot（Scophthalmus maximus）. To expand quantity of marine fish cell lines, also provide alternative tool for gene function research or the isolation of the virus, a new cell line,Scophthalmus maximus heart cell line（SMH）, was established from turbot heart in the research. Tissue block method was used and the heart tissues were collected aseptically, and washed 3 times with phosphate-bufferedsaline（PBS） and minced into small pieces（1 mm3） by surgical scissors in DMEM- F12 medium（p H 7.2）.Then tissue pieces were washed again with PBS until it was neat without blood. It had been cultured in 25 cm2cell-culture flask which should be inverted at 24 ℃, then returned the flask 4～5 h later. After completion of the above steps, 3 m L DMEM- F12 complete medium was added into the flask, which was supplemented with20% FBS, 50 mmol/L 2- mercaptoethanol（2- Me）, 10 ng/m L basic fibroblast growth factor（b FGF）, 100 IU/m L penicillin and 100 μg/m L streptomycin, 20 mmol/L 4-（2- hydroxyethyl）- 1- piperazineethanesulfonic acid（hepes）. When cells grew into a conflent monolayer, they were subcultured by trypsin solution（0.25% trypsin and 0.03% EDTA in PBS） at a split ratio of 1∶2. The cultured SMH cells, fibroblastic in morphology, had been subcultured to passage 58 in 230 d in a good proliferating state. The SMH cells at passage 23 were cryopreserved in liquid nitrogen. After 3 months, the cells could undergo cryopreservation with proliferated to80% confluency in 5～6 d. The morphology and proliferation ability of SMH cells were the sam

关 键 词：大菱鲆 大菱鲆心脏细胞系(SMH) GFP转染 线粒体细胞色素C氧化酶Ⅰ(COⅠ)基因 

分 类 号：Q233[生物学—细胞生物学]