牙鲆头肾巨噬细胞的分离培养与鉴定

Isolation,Culture,and Characterization of Macrophages from the Head Kidney of Japanese Flounder(Paralichthys olivaceus)

本研究使用密度梯度离心法从牙鲆(Paralichthys olivaceus)头肾中分离得到了巨噬细胞,通过差速贴壁法对获得的细胞进行纯化,后续经过优化培养条件和培养过程,采用L-15培养基(Gibco)、5%胎牛血清、1%青–链霉素、1%非必需氨基酸、30%L929细胞培养基在24℃、无CO2的条件下进行培养。显微镜观察及吉姆萨染色结果显示,贴壁细胞具备巨噬细胞形态特征,使用巨噬细胞特异性基因标记mpeg1基因对细胞进行鉴定,在分离得到的细胞中成功扩增出该基因。本研究为体外研究巨噬细胞功能、深入了解硬骨鱼先天性免疫奠定了基础。

The innate immune system of the Japanese flounder plays a vital role in resisting the invasion of pathogens.A macrophage is a type of leukocyte found in body tissues,which are part of the innate immune system.Macrophages are crucial to the immune response and play an important role in the clearance and phagocytosis of pathogens and abnormal cells.Macrophages can not be subcultured,so it is necessary to establish an efficient technique for macrophage isolation and culture.In this study,macrophages were isolated from the head kidney of the Japanese flounder,and the obtained cells were purified by differential adherence.Trypan blue staining showed that the cell survival rate was 99.62%.Subsequently,L-15 medium(Gibco),5%fetal bovine serum,1%mycillin,1%nonessential amino acids,and 30%L929 cell culture medium were used to culture the cells at 24℃.We compared the macrophages of Japanese flounder from different culture media and different serum concentrations.The results showed that the L-15 medium(Gibco)and 5%serum culture were the best.Cells cultured under this condition had a higher adherence rate,remaining above 85%after seven days of culture.Microscopic observations and Giemsa staining showed that the adherent cells had similar morphological characteristics to the macrophages,including a round shape and oval cell nuclei that were biased toward the side of the cell.Cells were identified via the macrophage-specific marker mpeg1,and the results showed that the gene was successfully amplified in the isolated cells.In this study,techniques to isolate and culture Japanese flounder macrophages were established,providing a basis to study macrophage functioning and to better understand the innate immunity of teleost fish in vitro.