永生化尼罗罗非鱼巨噬细胞系的建立及鉴定

Establishment and identification of immortalized macrophage cell line of tilapia(Oreochromis niloticus)

为获得具有单一克隆特性的能稳定传代培养的罗非鱼巨噬细胞系,本研究从尼罗罗非鱼腹腔中分离纯化巨噬细胞,采用EB病毒(Epstein-Barr virus,EBV)感染,筛选单克隆细胞的方法建立了尼罗罗非鱼巨噬细胞系,并对其进行了EBV感染鉴定、电镜观察、端粒酶活性检测、致癌性评估、核型分析以及分子生物学鉴定。研究表明,EBV已整合到尼罗罗非鱼巨噬细胞中且稳定表达,经30代稳定传代,该细胞系仍维持较好的增殖状态;该细胞系表面不平滑,有明显的钝圆形突起和细长的伪足,表现为典型的巨噬细胞形态;端粒酶活性显著高于未经感染的巨噬细胞,而与He La细胞差异不显著,且该细胞系不具有致癌性,说明永生化细胞系构建成功。核型分析结果发现,该细胞系具有44条染色体,其核型公式为2 n=2 x=44=4 sm+17 st+1 t。PCR检测发现,该细胞系存在CD33和CD205的转录本,这些都是单核巨噬细胞的标志物,经18S r RNA检测证明该细胞系来自尼罗罗非鱼巨噬细胞。永生化尼罗罗非鱼巨噬细胞系已被成功建立,该细胞系为研究罗非鱼链球菌HSP70-肽疫苗的高保护率,以及罗非鱼的免疫防御机制提供了工具。

To obtain a continuous monoclonal Oreochromis niloticus macrophage cell line, macrophages were isolated and purified from peritoneal cavity of O. niloticus. The O. niloticus macrophage cell line was established and EBV infection identification, electron microscopy, telomerase activity assay, carcinogenicity evaluation, karyotype analysis, and molecular biology identification were then carried out. The results showed that EBV had been integrated into O. niloticus peritoneal macrophages and stably expressed. After 30 generations of stable passage,the cell line still maintained a good proliferative state. The cell line surface was not smooth and had obvious blunt round protrusion and slender pseudopodia, which was the typical morphology performance of the macrophages.Telomerase activity of this cell line was significantly higher than that of untreated macrophages（P〈0.05）, but the difference was not significant with He La cells, and the cell line was not carcinogenic, which indicated that immortalized cell lines were successfully constructed. Karyotype analysis showed that the cell line had 44 chromosomes,and the karyotype formula was 2 n=2 x=44=4 sm＋17 st＋1 t. PCR detection revealed that CD33, and CD205 transcripts were found in this cell line, all of which were markers of monocytes/macrophages. 18 S r RNA detection showed that the cell line was from O. niloticus macrophages. Immortalized tilapia macrophage cell lines were successfully established. The establishment of this cell line laid the foundation for the further study of the immunological function of peritoneal macrophages in O. niloticus and also provided a tool for studying the mechanism of high protection rate of O. niloticus Streptococcus HSP70-peptide vaccine.