Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, VA, USA.
C. Kenneth and Dianne Wright Center for Clinical and Translational Research, Virginia Commonwealth University, Richmond, VA, USA.
Methods Mol Biol. 2022;2455:85-91. doi: 10.1007/978-1-0716-2128-8_8.
The rapid increase in the incidence of obesity contributes to a parallel increase in nonalcoholic steatohepatitis (NASH). Monocyte-derived macrophages, recruited from the bone marrow to the liver, promote NASH-related inflammation and fibrosis. In addition, adipose tissue macrophages (ATMs) release pro-inflammatory cytokines (PICs) which stimulate adipose tissue lipolysis liberating free fatty acids (FFAs) that can accumulate in the liver as triglycerides (TGs), thereby inducing steatosis. As such, bone marrow-derived macrophages (BMDMs) function as an essential tool to study the pathogenesis of NASH. BMDMs are primary bone marrow-derived cells which are differentiated into macrophages in vitro in the presence of growth factors. Macrophage colony-stimulating factor (M-CSF) is required for the proliferation and differentiation of committed myeloid progenitors into cells of the macrophage/monocyte lineage. Here, we describe a protocol for the isolation of mouse bone marrow cells and subsequent macrophage differentiation in which bone marrow cells are cultured in the presence of M-CSF, supplemented either by conditioned medium from L929 cells or in purified form. The efficiency of the differentiation is confirmed by immunofluorescent staining of macrophage surface antigen F4/80. The BMDMs serve as an excellent ex vivo model for a variety of studies, including hepatocyte-macrophage and adipocyte-macrophage cross-talk regulating NASH.
肥胖发病率的迅速增加导致非酒精性脂肪性肝炎 (NASH) 的发病率也相应增加。单核细胞衍生的巨噬细胞从骨髓招募到肝脏，促进 NASH 相关的炎症和纤维化。此外，脂肪组织巨噬细胞 (ATMs) 释放促炎细胞因子 (PICs)，刺激脂肪组织脂肪分解，释放游离脂肪酸 (FFAs)，这些脂肪酸可以在肝脏中积聚为甘油三酯 (TGs)，从而导致脂肪变性。因此，骨髓来源的巨噬细胞 (BMDMs) 是研究 NASH 发病机制的重要工具。BMDMs 是主要的骨髓来源细胞，在存在生长因子的情况下在体外分化为巨噬细胞。巨噬细胞集落刺激因子 (M-CSF) 是髓系前体细胞增殖和分化为巨噬细胞/单核细胞谱系细胞所必需的。在这里，我们描述了一种从小鼠骨髓中分离细胞并随后进行巨噬细胞分化的方案，其中在 M-CSF 的存在下培养骨髓细胞，通过 L929 细胞的条件培养基或纯形式进行补充。通过巨噬细胞表面抗原 F4/80 的免疫荧光染色来确认分化的效率。BMDMs 作为一种出色的体外模型，可用于多种研究，包括肝细胞-巨噬细胞和脂肪细胞-巨噬细胞相互作用调节 NASH。
