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目的建立小鼠3种不同部位巨噬细胞体外分离培养的方法,并对其进行鉴定。方法分别从小鼠腹腔、脾脏、骨髓中分离获取巨噬细胞,经台盼蓝染色计数后于含10%胎牛血清的DMEM培养液、1%小牛血清的RPMI-1640培养基中培养;通过活细胞形态观察;HE染色光镜观察;吞噬鸡红细胞功能检测;酸性磷酸酶及非特异性酯酶染色测定细胞内酶的表达等生物学技术鉴定培养的贴壁细胞性质。结果获得高纯度的巨噬细胞,具有巨噬细胞的形态特征,具备良好的吞噬功能,鸡红细胞吞噬率分别为腹腔94.68%,脾脏92.47%,骨髓93.11%;酸性磷酸酶染色阳性率为腹腔89.43%,脾脏85.21%,骨髓88.55%;α-醋酸奈酚酯酶染色阳性率为腹腔85.32%,脾脏81.81%,骨髓85.76%。。结论小鼠3个不同部位均可简便实用的分离出巨噬细胞,可以满足不同的研究目的。
Objective To establish a method for the isolation and culture of macrophages from three different sites in mice and to identify them. Methods Macrophages were isolated from the peritoneal, spleen and bone marrow of mice, respectively. After counting by trypan blue staining, they were cultured in RPMI-1640 medium containing 10% fetal bovine serum and DMEM medium supplemented with 1% fetal bovine serum. The morphology of living cells was observed by HE staining, the function of erythrocytes was detected by phagocytosis of chicken, the expression of intracellular enzymes was detected by acid phosphatase and nonspecific esterase staining, and other biological techniques were used to identify the adherent cells. RESULTS: Macrophages with high purity were obtained with morphological characteristics of macrophages and good phagocytosis. The phagocytosis rates of erythrocytes were 94.68%, 92.47%, 93.11%, respectively. The positive rate of acid phosphatase was 89.43 %, Spleen 85.21%, bone marrow 88.55%. The positive rate of α-acetate N-acetyl-esterase staining was 85.32% in abdominal cavity, 81.81% in spleen and 85.76% in bone marrow. . Conclusions Macrophages can be easily and practically isolated from 3 different parts of mice, which can meet different research purposes.