长链非编码 RNA 存在于巨噬细胞衍生的外泌体中，促进骨骨折大鼠模型中骨髓间充质干细胞的成骨作用。
Long Non-Coding RNAs Within Macrophage-Derived Exosomes Promote BMSC Osteogenesis in a Bone Fracture Rat Model.
机构信息
Department of Orthopedics, Beijing Chaoyang Hospital, Capital Medical University, Beijing, 100020, People's Republic of China.
出版信息
Int J Nanomedicine. 2023 Feb 28;18:1063-1083. doi: 10.2147/IJN.S398446. eCollection 2023.
PURPOSE
To investigate the effect of macrophage exosomal long non-coding (lnc)RNAs on bone mesenchymal stem cell (BMSC) osteogenesis and the associated mechanism.
METHODS
Rat BMSCs and spleen macrophages were co-cultured with serum derived from the fracture microenvironment of rat tibia. BMSC osteogenesis was evaluated using Alizarin red staining and the expression of , and mRNA. BMSC osteogenesis was evaluated after co-culture with macrophages stimulated using hypoxic conditions or colony-stimulating factor (CSF). The uptake of macrophage-derived exosomes by BMSCs was evaluated using the exosome uptake assay. High-throughput sequencing and bioinformatics analyses were performed to identify key lncRNAs in the macrophage exosomes. The effect of lncRNA expression levels on BMSC osteogenesis was also assessed using a lncRNA overexpression plasmid and siRNA technology. M1 and M2 macrophages were distinguished using flow cytometry and the key exosomal lncRNA was detected by in situ hybridization.
RESULTS
In the fracture microenvironment, macrophages (stimulated using either hypoxia or CSF) significantly increased the osteogenic ability of BMSCs. We showed that BMSCs assimilated macrophage-derived vesicles and that the inhibition of exosomal secretion significantly attenuated the macrophage-mediated induction of BMSC osteogenesis. The hypoxia condition led to the up-regulation of 310 lncRNAs and the down-regulation of 575 lncRNAs in macrophage exosomes, while CSF stimulation caused the up-regulation of 557 lncRNAs and the down-regulation of 407 lncRNAs. In total, 108 lncRNAs were co-up-regulated and 326 lncRNAs were co-down-regulated under both conditions. We eventually identified LOC103691165 as a key lncRNA that promoted BMSC osteogenesis and was expressed at similar levels in both M1 and M2 macrophages.
CONCLUSION
In the fracture microenvironment, M1 and M2 macrophages promoted BMSC osteogenesis by secreting exosomes containing LOC103691165.
目的
研究巨噬细胞外泌体长链非编码（lnc）RNAs 对骨髓间充质干细胞（BMSC）成骨的影响及其相关机制。
方法
将大鼠 BMSCs 和脾巨噬细胞与大鼠胫骨骨折微环境来源的血清共培养。通过茜素红染色和、 和 mRNA 的表达评估 BMSC 成骨情况。通过缺氧条件或集落刺激因子（CSF）刺激巨噬细胞共培养后评估 BMSC 成骨情况。通过外泌体摄取实验评估巨噬细胞来源的外泌体被 BMSC 摄取的情况。通过高通量测序和生物信息学分析鉴定巨噬细胞外泌体中的关键 lncRNAs。通过 lncRNA 过表达质粒和 siRNA 技术评估 lncRNA 表达水平对 BMSC 成骨的影响。通过流式细胞术区分 M1 和 M2 巨噬细胞，并通过原位杂交检测关键外泌体 lncRNA。
结果
在骨折微环境中，巨噬细胞（通过缺氧或 CSF 刺激）显著增强了 BMSC 的成骨能力。我们表明 BMSC 摄取了巨噬细胞来源的囊泡，并且抑制外泌体分泌显著减弱了巨噬细胞介导的 BMSC 成骨诱导。缺氧条件导致巨噬细胞外泌体中 310 个 lncRNA 上调和 575 个 lncRNA 下调，而 CSF 刺激导致 557 个 lncRNA 上调和 407 个 lncRNA 下调。总共，有 108 个 lncRNA 共同上调，有 326 个 lncRNA 共同下调。最终，我们确定 LOC103691165 为一个关键的 lncRNA，它促进了 BMSC 的成骨，并在 M1 和 M2 巨噬细胞中表达水平相似。
结论
在骨折微环境中，M1 和 M2 巨噬细胞通过分泌含有 LOC103691165 的外泌体促进 BMSC 成骨。
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