Department of Joint Surgery, Qinghai University Affiliated Hospital, Xining, 810000, Qinghai Province, China.
State Key Laboratory of Military Stomatology & National Clinical Research Center for Oral Diseases & Shaanxi Key Laboratory of Stomatology, Department of Out-Patient, School of Stomatology, Fourth Military Medical University, Xi'an, 710032, China.
J Orthop Surg Res. 2022 Mar 4;17(1):137. doi: 10.1186/s13018-022-03029-0.
Bone marrow mesenchymal stem cells have always been a heated research topic in bone tissue regeneration and repair because of their self-renewal and multi-differentiation potential. A large number of studies have been focused on finding the inducing factors that will promote the osteogenic differentiation of bone marrow mesenchymal stem cells. Previous studies have shown that macrophage exosomes or miRNA-26a-5p can make it work, but the function of this kind of substance on cell osteogenic differentiation has not been public.
M2 macrophages are obtained from IL-4 polarized bone marrow-derived macrophages. Exosomes were isolated from the supernatant of M2 macrophages and identified via transmission electron microscopy (TEM), western blotting, and DLS. Chondrogenic differentiation potential was detected by Alcian blue staining. Oil red O staining was used to detect the potential for lipogenic differentiation. And MTT would detect the proliferative capacity of cells. Western blot was performed to detect differential expression of osteogenic differentiation-related proteins.
The results showed that M2 macrophage exosomes will promote bone differentiation and at the same time inhibit lipid differentiation. In addition, M2 macrophage-derived exosomes have the function of promoting the expression of SOX and Aggrecan suppressing the level of MMP13. The exosome inhibitor GW4689 suppresses miRNA-26a-5p in M2 macrophage exosomes, and the treated exosomes do not play an important role in promoting bone differentiation. Moreover, miRNA-26a-5p can enable to promote bone differentiation and inhibit lipid differentiation. miRNA-26a-5p can promote the expression of ALP (alkaline phosphatase), RUNX-2 (Runt-related transcription factor 2), OPN(osteopontin), and Col-2(collagen type II). Therefore, it is speculated that exosomal miRNA-26a-5p is indispensable in osteogenic differentiation.
The present study indicated that M2 macrophage exosomes carrying miRNA-26a-5p can induce osteogenic differentiation of bone marrow-derived stem cells to inhibit lipogenic differentiation, and miRNA-26a-5p will also promote the expression of osteogenic differentiation-related proteins ALP, RUNX-2, OPN, and Col-2.
骨髓间充质干细胞因其自我更新和多向分化潜能，一直是骨组织再生和修复的热门研究课题。大量研究集中于寻找促进骨髓间充质干细胞成骨分化的诱导因子。先前的研究表明，巨噬细胞外泌体或 miRNA-26a-5p 可以发挥作用，但这种物质对细胞成骨分化的功能尚未公开。
从 IL-4 极化的骨髓来源的巨噬细胞中获得 M2 巨噬细胞。从 M2 巨噬细胞的上清液中分离出外泌体，并通过透射电子显微镜（TEM）、western blot 和 DLS 进行鉴定。通过茜素红 S 染色检测软骨分化潜能。油红 O 染色用于检测脂肪生成分化的潜力。MTT 用于检测细胞的增殖能力。Western blot 用于检测成骨分化相关蛋白的差异表达。
结果表明，M2 巨噬细胞外泌体促进骨分化，同时抑制脂分化。此外，M2 巨噬细胞衍生的外泌体具有促进 SOX 和 Aggrecan 表达、抑制 MMP13 水平的功能。外泌体抑制剂 GW4689 抑制 M2 巨噬细胞外泌体中的 miRNA-26a-5p，经处理的外泌体在促进骨分化方面不起重要作用。此外，miRNA-26a-5p 可以促进骨分化，抑制脂分化。miRNA-26a-5p 可以促进 ALP（碱性磷酸酶）、RUNX-2（Runt 相关转录因子 2）、OPN（骨桥蛋白）和 Col-2（胶原 II）的表达。因此，推测外泌体 miRNA-26a-5p 在成骨分化中不可或缺。
本研究表明，携带 miRNA-26a-5p 的 M2 巨噬细胞外泌体可诱导骨髓来源干细胞的成骨分化，抑制脂生成分化，miRNA-26a-5p 还可促进成骨分化相关蛋白 ALP、RUNX-2、OPN 和 Col-2 的表达。
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