论文部分内容阅读
鱼类细胞系作为一种重要的研究工具已经被广泛地应用于病毒学、环境毒理学、发育生物学以及生理学等研究中。大菱鲆(Scophthalmus maximus)作为一种重要的海水养殖鱼类,尚未见其脑细胞系建立的报道。本研究通过组织块法,以大菱鲆脑组织为材料,建立了大菱鲆脑细胞系(S.maximus brain cell line,SMB)。培养细胞系所用的培养基是在DMEM/F12基本培养基中添加了4-羟乙基哌嗪乙磺酸(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid,Hepes)、抗生素(青链霉素)、胎牛血清(fetal bovine serum,FBS)、碱性成纤维生长因子(basic fibroblast growth factor,b FGF)和β-巯基乙醇(2-mercaptoethanol,2-ME)所配成的完全培养基。SMB细胞由上皮样细胞和成纤维样细胞构成,生长分裂旺盛,至今已传40余代。探究温度对其生长的影响,结果表明,SMB细胞在18~30℃均能正常生长,最适生长温度为24℃。核型分析显示,SMB细胞出现了染色体非整倍性,正常二倍体核型(2n=44t)约占43%。通过脂质体转染法成功的将增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)报告基因转入SMB细胞中,并成功地获得了表达,转染率约为30%。该细胞系的建立将为大菱鲆致病性病毒的感染机制、基因功能分析等研究提供新的实验材料。
As an important research tool, fish cell line has been widely used in virology, environmental toxicology, developmental biology and physiology. Scophthalmus maximus has not been reported as a brain cell line as an important mariculture fish. In this study, S.maximus brain cell line (SMB) was established by tissue block method using the turbot brain tissue as material. The culture medium used to culture the cell line was obtained by adding 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (Hepes), antibiotics Fetal bovine serum (FBS), basic fibroblast growth factor (b FGF) and 2-mercaptoethanol (2-ME) . SMB cells by the epithelial cells and fibroblast-like cells, strong growth and split, has been handed down more than 40 generations. To investigate the effect of temperature on its growth, the results showed that SMB cells could grow normally at 18-30 ℃ with the optimal growth temperature of 24 ℃. Karyotype analysis showed that there was chromosome aneuploidy in SMB cells. The normal diploid karyotype (2n = 44t) accounted for about 43%. The EGFP reporter gene was successfully transfected into SMB cells by lipofection and successfully expressed. The transfection rate was about 30%. The establishment of this cell line will provide a new experimental material for the research on the mechanism of infection and gene function analysis of causative virions of turbot.