中国组织工程研究

中国组织工程研究 ›› 2026, Vol. 30 ›› Issue (13): 3233-3241.doi: 10.12307/2026.087

• 骨髓干细胞 bone marrow stem cells • 上一篇    下一篇

C57BL/6小鼠骨髓来源巨噬细胞分离、培养、鉴定及M1/M2的极化诱导

谭宇航1，2，李  波1，2，唐铭宏1，2，孙泽宇2，罗  旭2   

  1. 1贵州医科大学，贵州省贵阳市   550001；2贵州省人民医院骨科，贵州省贵阳市   550002
  • 收稿日期:2025-03-24 修回日期:2025-06-06 接受日期:2025-07-03 出版日期:2026-05-08 发布日期:2025-12-24
  • 通讯作者: 李波，主任医师，教授，贵州医科大学，贵州省贵阳市 550001；贵州省人民医院骨科，贵州省贵阳市 550002
  • 作者简介:谭宇航，男，1999年生，贵州医科大学在读硕士，医师，主要从事脊柱外科方向研究。
  • 基金资助:
    国家自然科学基金项目(82160419)，项目负责人：李波；贵州省中医药管理局中医药、民族医药科学技术研究项目(QZYY-2024-114)，项目负责人：孙泽宇

Isolation, cultivation, identification, and induction of M1/M2 polarization in bone marrow-derived macrophages from C57BL/6 mice

Tan Yuhang1, 2, Li Bo1, 2, Tang Minghong1, 2, Sun Zeyu2, Luo Xu2   

  1. 1Guizhou Medical University, Guiyang 550001, Guizhou Province, China; 2Department of Orthopedics, Guizhou Provincial People’s Hospital, Guiyang 550002, Guizhou Province, China
  • Received:2025-03-24 Revised:2025-06-06 Accepted:2025-07-03 Online:2026-05-08 Published:2025-12-24
  • Contact: Li Bo, Chief physician, Professor, Guizhou Medical University, Guiyang 550001, Guizhou Province, China; Department of Orthopedics, Guizhou Provincial People’s Hospital, Guiyang 550002, Guizhou Province, China
  • About author:Tan Yuhang, Master candidate, Physician, Guizhou Medical University, Guiyang 550001, Guizhou Province, China; Department of Orthopedics, Guizhou Provincial People’s Hospital, Guiyang 550002, Guizhou Province, China
  • Supported by:
    National Natural Science Foundation of China, No. 82160419 (to LB); Guizhou Provincial Administration of Traditional Chinese Medicine Science and Technology Research Project of Traditional Chinese Medicine and Ethnic Medicine, No. QZYY-2024-114 (to SZY) 

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摘要：

文题释义：

巨噬细胞极化：是指巨噬细胞在特定微环境刺激下，通过改变其表型和功能特性，分化为不同亚型的过程。巨噬细胞极化主要分为M1型(经典激活型)和M2型(替代激活型)。M1型巨噬细胞由脂多糖或干扰素γ诱导，高表达促炎因子，在清除病原体和抗肿瘤免疫中发挥重要作用；而M2型巨噬细胞由白细胞介素4或白细胞介素13诱导，高表达抗炎因子，参与组织修复和免疫调节。
骨髓来源巨噬细胞：是从小鼠股骨和胫骨中提取的骨髓细胞在体外培养分化而成的巨噬细胞，广泛用于研究巨噬细胞的生物学功能。与直接从组织中分离的巨噬细胞相比，骨髓来源巨噬细胞具有来源稳定、可大量获取、易于操控等优势。

摘要
背景：巨噬细胞极化在疾病治疗中展现出巨大的应用潜力，尤其是在癌症、炎症和自身免疫性疾病等领域。通过构建体外标准模型，可以为深入研究巨噬细胞极化机制奠定基础。
目的：观察C57BL/6小鼠骨髓来源巨噬细胞的体外生长特征以及构建M1和M2型巨噬细胞极化标准化体外模型。
方法：无菌分离C57BL/6小鼠股骨和胫骨，收集骨髓腔内容物，通过筛网过滤并进行红细胞裂解后，用含20 ng/mL巨噬细胞集落刺激因子的高糖DMEM培养基重悬，按照实验需求接种于6孔板中，在第7天分化为成熟的小鼠骨髓来源巨噬细胞(M0型)，然后用100 ng/mL脂多糖诱导M1型巨噬细胞极化，20 ng/mL白细胞介素4诱导M2型巨噬细胞极化。使用流式细胞术和RT-qPCR检测不同极化状态下巨噬细胞相应标志物的表达，Western blot检测M1型巨噬细胞标志性通路蛋白p-STAT1、STAT1和M2型巨噬细胞标志性通路蛋白p-STAT6、STAT6的表达。
结果与结论：①20 ng/mL巨噬细胞集落刺激因子刺激7 d，流式细胞术检测巨噬细胞表面标志物F4/80的阳性染色率达到98.1%；②骨髓来源巨噬细胞用100 ng/mL脂多糖刺激6 h后，F4/80和CD86的阳性染色率约为35%，RT-qPCR检测M1型巨噬细胞标志物诱导型一氧化氮合酶、白细胞介素6、巨噬细胞炎性蛋白1α和单核细胞趋化蛋白1 mRNA表达均显著高于对照组(P < 0.01)；③骨髓来源巨噬细胞用20 ng/mL白细胞介素4刺激24 h后，CD206平均荧光强度明显升高，RT-qPCR检测M2型巨噬细胞标志物Chi3l3(Ym1)、白细胞介素10和精氨酸酶1 mRNA表达均显著高于对照组(P < 0.01)；④Western blot检测结果显示，脂多糖诱导的M1型巨噬细胞标志性通路蛋白p-STAT1显著激活；白细胞介素4诱导的M2型巨噬细胞标志性通路蛋白p-STAT6显著激活。以上结果表明，脂多糖和白细胞介素4分别有效诱导了骨髓来源巨噬细胞向M1型和M2型巨噬细胞极化。
https://orcid.org/0009-0004-6074-5351 (谭宇航) 

关键词: 骨髓来源巨噬细胞, BMDMs, 巨噬细胞极化, 脂多糖, 白细胞介素4, M1型巨噬细胞, M2型巨噬细胞

Abstract: BACKGROUND: Macrophage polarization demonstrates significant potential in disease treatment, particularly in areas such as cancer, inflammation, and autoimmune diseases. Establishing standardized in vitro models can lay the groundwork for in-depth research into the mechanisms of macrophage polarization.
OBJECTIVE: To observe the in vitro growth characteristics of bone marrow-derived macrophages from C57BL/6 mice and to establish a standardized in vitro model for M1 and M2 macrophage polarization.
METHODS: Femurs and tibias of C57BL/6 mice were aseptically separated, and the contents of the bone marrow cavity were collected. After filtering through a mesh and lysing erythrocytes, the contents were resuspended in high-glucose DMEM containing 20 ng/mL macrophage colony-stimulating factor and inoculated in 6-well plates according to experimental requirements. On day 7, they were differentiated into mature mouse bone marrow-derived macrophages (M0 type). Then, 100 ng/mL lipopolysaccharide was used to induce polarization of M1 macrophages, and 20 ng/mL interleukin-4 was used to induce polarization of M2 macrophages. Flow cytometry and RT-qPCR were used to detect the expression of corresponding markers of macrophages in different polarization states. Western blot assay was used to detect the expression of M1 macrophage marker pathway proteins p-STAT1 and STAT1 and M2 macrophage marker pathway proteins p-STAT6 and STAT6.
RESULTS AND CONCLUSION: (1) After stimulation with 20 ng/mL macrophage colony-stimulating factor for 7 days, the positive staining rate of macrophage surface marker F4/80 by flow cytometry reached 98.1%. (2) After bone marrow-derived macrophages were stimulated with 100 ng/mL lipopolysaccharide for 6 hours, the positive staining rates of F4/80 and CD86 were approximately 35%, and the mRNA expressions of M1 macrophage markers inducible nitric oxide synthase, interleukin-6, macrophage inflammatory protein-1α, and monocyte chemoattractant protein-1 by RT-qPCR were significantly higher than those in the control group (P < 0.01). (3) After bone marrow-derived macrophages were stimulated with 20 ng/mL interleukin-4 for 24 hours, the mean fluorescence intensity of CD206 was significantly increased, and the mRNA expressions of M2 macrophage markers Chi3l3 (Ym1), interleukin-10, and arginase-1 by RT-qPCR were significantly higher than those in the control group (P < 0.01). (4) Western blot assay results showed that lipopolysaccharide-induced M1 macrophages' signature pathway p-STAT1 was significantly activated; interleukin-4-induced M2 macrophages' landmark pathway p-STAT6 was significantly activated. The above results indicate that lipopolysaccharide and interleukin-4 effectively induce bone marrow-derived macrophages to polarize into M1 and M2 macrophages, respectively.

Key words: bone marrow-derived macrophage, BMDMs, macrophage polarization, lipopolysaccharide, interleukin-4, M1 macrophage, M2 macrophage 

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