草鱼肠巨噬细胞的分离培养与鉴定

Isolation, cultivation and identification of Ctenopharyngodon idella intestinal macrophages

为了建立草鱼肠巨噬细胞的分离培养与鉴定方法,本研究在采用刮除法结合胶原酶IV消化法制备肠黏膜固有层单细胞悬液的基础上,使用鱼类脏器单核细胞分离试剂盒分离肠巨噬细胞,再经差异贴壁法纯化肠巨噬细胞,最后,用含10%胎牛血清和5%草鱼血清的RPMI 1640完全培养液,在28°C、5%CO2条件下原代培养肠巨噬细胞,并通过细胞形态学检查、分子标记检测和功能验证实验加以鉴定。结果显示,每尾鱼(约250 g)肠巨噬细胞的获得量约为3×107个,细胞活率为99.6%,纯度达到95%以上;倒置显微镜观察发现,原代培养4~5 d的贴壁细胞多呈圆形或多边形,体积明显增大,细胞汇合度达到95%;光镜和电镜观察到染色后的贴壁细胞具有巨噬细胞的形态结构特征,贴壁细胞经荧光定量PCR在m RNA水平检测到巨噬细胞特异性标志分子(草鱼巨噬细胞集落刺激因子受体),功能实验证实贴壁细胞具有吞噬功能,脂多糖能够显著提高其呼吸暴发活性。本研究首次成功建立了草鱼肠巨噬细胞的分离培养与鉴定方法,为开展口服疫苗诱导的草鱼肠黏膜免疫应答研究提供细胞模型。

In order to establish the method for primary isolation,culture and identification of grass carp(Ctenopharyngodon idella)intestinal macrophages,in the study,the intestinal mucosal lamina propria mononuclear cells(LPMC)were firstly prepared by curettage combined with collagenase IV digestion.Then,the intestinal macrophages were isolated from LPMC suspension using fish organ mononuclear cell isolation kit and purified by differential adherence method.The intestinal macrophages after purification were cultured primarily with RPMI 1640 complete medium supplemented with 10%FBS and 5%serum of C.idella at 28°C in a 5%CO2 incubator,and these cells were identified by morphological examination,molecular marker detection and function validation test.The results showed that the yield of intestinal macrophages was about 3×107 cells per fish(about 250 g in weight),with a survival rate of 99.6%and a purity of more than 95%,respectively.The adherent cells in primary culture for 4–5 days were round or polygonal,the cell volume increased obviously,and the confluence degree of cell layer reached 95%observed under inverted microscope.After staining,the adherent cells had the morphological and structural characteristics of macrophages observed under light and electron microscopes,respectively.The expression of macrophage-specific marker molecule(macrophage colony stimulating factor receptor of C.idella)was detected by RT-PCR.It was also confirmed that the adherent cells in primary culture had phagocytosis,and LPS could significantly improve their respiratory burst activity.The above results indicated that the method for isolation,primary culture and identification of C.idella intestinal macrophages was successfully established for the first time,which might provide a cell model for research on the intestinal mucosal immune response of C.idella induced with oral vaccine.