低氧条件下M2型巨噬细胞外泌体对骨髓间充质干细胞成骨分化的影响机制

Mechanism of M2 macrophage-derived exosomes on osteogenic differentiation of bone marrow mesenchymal stem cells under hypoxic conditions

摘要:

目的 探究低氧条件下M2型巨噬细胞外泌体对骨髓间充质干细胞（BMSCs）成骨分化的影响机制。方法 将鼠源性巨噬细胞系（RAW264.7）与鼠源性BMSCs系CP-M131细胞培养至第3代，用白细胞介素-4诱导巨噬细胞极化为M2型巨噬细胞，提取并鉴定常氧与低氧培养条件下M2型巨噬细胞的外泌体。将CP-M131细胞分为PBS组、N-M2 exo组与H-M2 exo组，分别与PBS、90 ng/mL N-M2 exo及90 ng/mL H-M2 exo共孵育72 h。孵育结束后，通过免疫荧光观察BMSCs系CP-M131细胞对不同氧气条件下M2型巨噬细胞外泌体的摄取情况。采用实时荧光定量聚合酶链反应（qRT-PCR）与Western blotting检测BMSCs中摄取不同实验组外泌体后骨形态发生蛋白-2（BMP-2）、碱性磷酸酶（ALP）、成骨特异性转录因子（Runx）基因和蛋白表达，以及细胞外调节蛋白激酶（ERK）、磷酸化ERK（p-ERK）的蛋白表达。采用ALP染色评估不同氧气条件M2型巨噬细胞外泌体对BMSCs成骨能力的影响。结果 成功诱导巨噬细胞极化为M2型巨噬细胞，且提取到不同处理组的外泌体。qRT-PCR和Western blotting结果表明，与N-M2 exo组比较，H-M2 exo组的CP-M131细胞BMP-2、ALP、Runx基因和蛋白表达均上调（P <0.05）；H-M2 exo组的ERK与p-ERK蛋白表达均上调（P <0.05）。ALP染色观察到低氧条件下M2型巨噬细胞外泌体促进CP-M131细胞成骨分化。结论 低氧条件下M2型巨噬细胞外泌体可以促进BMSCs成骨分化。

Abstract:

Objective To investigate the mechanism by which exosomes derived from M2 macrophages under hypoxic conditions regulate the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).Methods Murine macrophage line RAW264.7 and murine BMSC line CP-M131 were cultured to the third passage. Macrophages were polarized to M2 phenotype using interleukin-4. Exosomes from M2 macrophages cultured under normoxic (N-M2 exo) and hypoxic (H-M2 exo) conditions were extracted and characterized. CP-M131 cells were divided into PBS group, N-M2 exo group (treated with 90 ng/mL N-M2 exo), and H-M2 exo group (treated with 90 ng/mL H-M2 exo) for 72 hours. Exosome uptake by BMSCs was observed via immunofluorescence. Quantitative real-time PCR (qRT-PCR) and Western blotting were used to detect mRNA and protein expression of bone morphogenetic protein-2 (BMP-2), Alkaline phosphatase (ALP), Runt-related transcription factor (Runx), extracellular signal-regulated kinase (ERK), and phosphorylated ERK (p-ERK). ALP staining was performed to assess osteogenic differentiation.Results M2 macrophage polarization and exosome isolation were successfully achieved. Compared to the N-M2 exo group, the H-M2 exo group exhibited significantly upregulated mRNA and protein expression of BMP-2, ALP, and Runx in CP-M131 cells (P < 0.05). ERK and p-ERK protein expression were also elevated in the H-M2 exo group (P < 0.05). ALP staining confirmed enhanced osteogenic differentiation in the H-M2 exo group.Conclusion Hypoxic M2 macrophage-derived exosomes promote osteogenic differentiation of BMSCs through ERK pathway activation.