广西卵形鲳鲹小脑来源细胞系的建立及特征分析

Establishment and Characterization of a Novel Cell Line from Cerebellum of Golden Pompano (Trachinotus ovatus) Cultured in Guangxi

关键词:  卵形鲳鲹  小脑来源细胞系  细胞特征  细胞毒性

Key words:  Golden pompano (Trachinotus ovatus)  cerebellum cell line  cell characteristics  cytotoxicity

摘要:

[目的]卵形鲳鲹(Trachinotus ovatus)是广西重要的海水经济鱼类,但近年来随着养殖规模扩大,各种病害侵染引起的疾病频繁暴发,给广西卵形鲳鲹养殖产业造成重大损失。建立卵形鲳鲹小脑来源的细胞系(Cell line from cerebellum of Trachinotus ovatus,TOCC),将有利于广西卵形鲳鲹养殖中病毒性病害的分离、鉴定和病害的侵染致病分子机理的研究,以及环境污染和变化的动态监测。[方法]采用胰蛋白酶消化法分离得到卵形鲳鲹小脑组织的原代细胞,并进行原代培养。待原代细胞铺满单层后进行传代培养。对稳定传代的TOCC进行18S rDNA基因测序分析,然后进行生长速率分析和染色体分析,并开展病原菌(溶藻弧菌Vibrio alginolyticus)胞外产物对TOCC的毒性分析。[结果]稳定传代的TOCC为成纤维样细胞,18S rDNA基因分析结果显示其来源于卵形鲳鲹;染色体核型分析发现TOCC具有二倍体特征,特征染色体数目为62条;不同培养条件下TOCC细胞的生长速率不同,28℃生长速度最快;病原菌胞外产物对TOCC具有明显的毒性。[结论]本研究成功建立广西卵形鲳鲹小脑组织来源的细胞系(TOCC)。细菌胞外产物对TOCC细胞毒性结果表明,该细胞系在一定范围内能够替代活体检测,实现体外细胞水平对广西海水养殖中病害侵染和环境污染物的快速检测,为养殖病害的有效预防提供技术支持。

Abstract:

[Objective] Golden pompano (Trachinotus ovatus) is a commercially important marine fish species in Guangxi, but in recent years with the expansion of breeding scale, the frequent outbreaks of pathogens caused by various diseases and infections have caused great losses to the aquaculture industry of Trachinotus ovatus in Guangxi.The establishment of a cell line from cerebellum of Trachinotus ovatus (TOCC) would be conducive to the isolation and identification of viral pathogens in Guangxi oval salamander breeding and the pathogenic molecular mechanism of disease infection, as well as the dynamic monitoring of environmental pollution and changes.[Methods] The primary TOCC cells were cultured by trypsinization, and then subcultured after reaching 100% confluence.18S rDNA gene sequence analysis was applied to identify the origin of TOCC.We further evaluated TOCC cells' characteristics of growth, chromosome, and toxicity of bacterial extracellular products on TOCC cells.[Results] TOCC cells were fibroblast-like cells, the 18S rDNA gene sequence analysis showed that it derived from Trachinotus ovatus.Chromosome karyotype analysis found that TOCC had diploid features, the number of characteristic chromosomes was 62.The growth rates of TOCC cell were different in different culture conditions.The optimal culture temperature for TOCC cells growth was 28℃.ECPs exhibited toxicity to TOCC cells.[Conclusion] A novel cell line derived from the cerebellum tissue of Trachinotus ovatus (TOCC) was successfully established in this study.Cytotoxicity results of bacterial extracellular products to TOCC showed that within a certain range the cell line could replace the in vivo detection and realize in vitro cell level rapid detection of the disease infection and environmental pollutants in Guangxi marine culture and provide a technical support for the effective prevention of aquaculture disease.