小鼠原代骨髓单核细胞分离与培养方法的改进

Improvement of isolation and culture method of primary mouse bone marrow monocytes

目的探索更好的原代小鼠骨髓单核细胞的分离与培养方法。方法将小鼠股骨取出置入预冷的PBS中修剪、冲出骨髓组织至预冷的DMEM培养基中,吹打混匀制成单细胞悬液后过滤。应用差速贴壁方法培养6~8 h后留取未贴壁的细胞,加入巨噬细胞集落刺激因子(M-CSF)培养7 d,获得小鼠骨髓单核巨噬细胞。光镜下观察细胞形态;流式细胞术检测骨髓单核巨噬细胞表面抗原CD11b、F4/80的表达,评估细胞纯度;应用转化生长因子(TGF)-β_(1)和M-CSF培养将其诱导分化为肌成纤维细胞,免疫印迹检测其标志物α-平滑肌肌动蛋白(α-SMA)的表达。结果冰上预冷缩短热缺血时间方法分离的骨髓单核细胞损伤小,细胞形态均一,纯度高(可达95%以上),较对照组有所升高,但差异无统计学意义(P>0.05);加入TGF-β_(1)共培养后可见细胞呈细长纺锤形,与对照组相比,α-SMA的表达明显升高,差异有显著统计学意义(P<0.01)。结论本方法分离提取的原代小鼠骨髓单核细胞纯度高,状态良好,表型稳定,满足做进一步实验研究的要求。

Objective To explore a better method for the isolation and culture of primary mouse bone marrow monocytes(BMMC). Methods The mouse femurs were taken out and placed in pre-cooled phosphate buffered saline(PBS) for trimming, and the bone marrow tissue was flushed out and placed into pre-cooled DMEM medium, and the single-cell suspension was prepared by pipetting and mixing, and then filtered. After culturing for 6-8 hours by the differential adhesion method, the non-adherent cells were collected, with macrophage colony-stimulating factor(M-CSF) added,and cultureed for 7 days to obtain mouse bone marrow mononuclear macrophages. The cell morphology was observed by light microscope. The expression of surface antigen CD11b and F4/80 of bone marrow mononuclear macrophages was detected by flow cytometry, and the cell purity was evaluated. The BMMC was cultured with transforming growth factor(TGF)-β_(1)and M-CSF, to induce myofibroblasts. The expression of its marker α-smooth muscle actin(α-SMA) was detected by western blotting. Results The bone marrow monocytes isolated by the method of precooling on ice to shorten the warm ischemia time have little damage, uniform cell morphology, high purity(up to more than 95%), which was slightly higher than that of the control group(P>0.05). After co-culture with TGF-β_(1), the cells were shown to be elongated and spindle-shaped, compared with the control group, and the expression of α-SMA was significantly increased, with statistically significant difference(P<0.01). Conclusion The primary mouse bone marrow monocytes isolated by this method have high purity, good state and stable phenotype, which are suitable for further experimental research.