L929细胞条件培养基诱导培养的小鼠原代巨噬细胞生物学特性对比

Characteristic comparison of mouse primary macrophages cultured in L929 cell conditioned medium

摘要:

本研究旨在对比分析小鼠骨髓源巨噬细胞 (BMDM) 和腹腔巨噬细胞 (PM) 原代培养及巨噬细胞生物学特性差异，为合理选择原代巨噬细胞培养方法提供参考依据。原代培养小鼠BMDM和PM，细胞计数仪测定细胞数量，倒置显微镜观察形态学变化，流式细胞仪检测细胞纯度，CCK-8法测定细胞增殖，中性红吞噬实验测定细胞吞噬功能，实时荧光定量PCR分析巨噬细胞表型变化。结果显示，BMDM原代培养获取的细胞数量显著高于PM (P<0.01)，PM贴壁及伸展时间早于BMDM。BMDM中F4/80+CD11b+百分比 (98.30%±0.53%) 高于PM (94.83%±1.42%)，但无统计学差异 (P>0.05)。在L929细胞条件培养基培养体系中，BMDM增殖能力显著高于PM (P<0.001)。吞噬实验发现，基础状态下BMDM吞噬能力显著高于PM (P<0.01)，经LPS刺激24 h后，除低剂量LPS (0.1 μg/mL) 外，BMDM吞噬能力均显著高于PM (P<0.01或P<0.001)，提示BMDM吞噬能力在基础和激活状态下均强于PM。巨噬细胞极化实验发现，基础状态下Tnfα在BMDM中表达显著高于PM (P<0.001)，Arg1和Ym1在BMDM中表达显著低于PM (P<0.001)，这一差异在极化诱导剂 (LPS+IFN-γ或IL-4) 处理后依然存在。上述结果表明，原代培养BMDM较PM可获取更多细胞，且两种细胞在吞噬功能和极化状态上存在一定生物学差异，应谨慎合理选择巨噬细胞原代培养方法。

Abstract:

The purpose of this study is to provide a culture for mouse bone marrow-derived macrophages (BMDM) and peritoneal macrophages (PM) and to characterize their molecular and cellular biology. The cell number and purity from the primary culture were assessed by cell counter and flow cytometry, respectively. Morphological features were evaluated by inverted microscope. Phagocytosis by macrophages was detected by the neutral red dye uptake assay. Phenotypic markers were analyzed by real-time fluorescent quantitative PCR. Our results show that the cell number was much higher from culture of BMDM than PM, while there was no significant difference regarding the percentage of F4/80+CD11b+ cells (98.30%±0.53% vs. 94.83%±1.42%; P>0.05). The proliferation rate of BMDM was significantly higher than PM in the presence of L929 cell conditioned medium, by using CCK-8 assay. However, PM appeared to adhere to the flask wall and extend earlier than BMDM. The phagocytosis capability of un-stimulated BMDM was significantly higher than PM, as well as lipopolysaccharide (LPS)-stimulated BMDM, except the BMDM stimulated by low dose LPS (0.1 μg/mL). Furthermore, Tnfα expression was significantly higher in un-stimulated BMDM than PM, while Arg1 and Ym1 mRNA expression were significantly lower than PM. The expression difference was persistent if stimulated by LPS+IFN-γ or IL-4. Our data indicate that bone marrow can get larger amounts of macrophages than peritoneal cavity. However, it should be aware that the molecular and cellular characteristics were different between these two culture systems.