骨髓巨噬细胞程序化释放外泌体对干细胞成骨分化的影响  

Effect of programmed release of bone marrow macrophage-derived exosomes on osteogenic differentiation of stem cells

作　　者：杨璠[1] 江哲珍[1] 胡婧[1] 陶圣祥[1] 邓玲珑[1] Yang Fan;Jiang Zhezhen;Hu Jing;Tao Shengxiang;Deng Linglong(Department of Orthopaedic Trauma and Microsurgery,Zhongnan Hospital of Wuhan University,Wuhan 430071,China)

机构地区：[1]武汉大学中南医院创伤与显微骨科,武汉430071

出　　处：《中华实验外科杂志》2024年第10期2297-2300,共4页Chinese Journal of Experimental Surgery

基　　金：湖北省自然科学基金(2023AFB131);武汉大学中南医院医学科技创新平台支撑项目(PTXM2024014)。

摘　　要：目的:探讨骨髓巨噬细胞程序化释放外泌体对骨髓间充质干细胞(BMSCs)成骨分化的影响。方法:建立小鼠股骨及胫骨骨折模型,提取骨髓巨噬细胞(BMDMs),流式细胞术鉴定BMDMs表型。使用15 ng/ml干扰素γ+100 ng/ml脂多糖及20 ng/ml的白细胞介素(IL)-4诱导RAW264.7细胞向M1及M2型极化,并提取巨噬细胞外泌体(DMs-Exos)。分别使用成骨分化诱导培养基(对照组)、含200μg/ml M1型DMs-Exos的培养基(M1-DMs-Exos组)、含200μg/ml M2型DMs-Exos的培养基(M2-DMs-Exos组)及含200μg/ml M1-M2型DMs-Exos程序化的培养基(M1-M2-DMs-Exos组),诱导BMSCs成骨分化21 d。实时荧光定量聚合酶链反应(RT-qPCR)检测BMSCs成骨分化基因碱性磷酸酶(ALP)/OCN/Runx2的mRNA水平。茜素红染色评估BMSCs的钙结节沉积量。结果以均数±标准差(±s)表示。两组间比较采用独立样本t检验。结果:小鼠骨折后的第1天及第3天BMDMs以M1型为主,第5及第7天以M2型为主。TEM显示DMs-Exos为膜结构的杯状或球形囊泡。RT-qPCR结果提示,M1-DMs-Exos组(0.22±0.12、0.24±0.11、0.26±0.08)及M2-DMs-Exos组(0.41±0.11、0.32±0.07、0.51±0.03)中ALP、OCN及Runx2基因的mRNA相对水平低于对照组(1.00±0.07、1.00±0.05、1.00±0.05,t=12.32、14.27、18.92,P<0.05;t=10.16、17.36、19.21,P<0.05);M1-M2-DMs-Exos组ALP、OCN及Runx2基因的mRNA相对水平(0.85±0.10、0.81±0.09、0.86±0.06)略低于对照组(1.00±0.07、1.00±0.05、1.00±0.05),差异有统计意义(t=2.74、4.50、4.11,P<0.05)。茜素红染色显示,M1-DMs-Exos组(0.26±0.05)及M2-DMs-Exos组(0.38±0.03)的钙盐沉积量低于对照组(1.00±0.02,t=23.63、29.34,P<0.05),M1-M2-DMs-Exos组的钙盐沉积量略低于对照组(t=5.51,P<0.05),但高于其他两组(F=23.23,P<0.05)。结论:巨噬细胞程序性释放外泌体介导的免疫反应促进BMSCs成骨分化。Objective:To investigate the effect of programmed release of bone marrow macrophage-derived exosomes on osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs).Methods:The fracture models of femur and tibia in mice were established.Bone marrow macrophages(BMDMs)were extracted and the phenotype of BMDMs was identified by flow cytometry.15 ng/ml interferonγ+100 ng/ml lipopolysaccharide and 20 ng/ml interleukin(IL)-4 were used to induce M1 and M2 polarization of RAW264.7 cells,and macrophage exosomes were extracted.BMSCs were induced to osteogenic differentiation for 21 days by osteogenic differentiation induction medium(control group),culture medium containing 200μg/ml M1 type BMDMs-Exos(M1-DMs-Exos group),culture medium containing 200μg/ml M2 type DMs-Exos(M2-DMs-Exos group)and culture medium containing 200μg/ml M1-M2 type DMs-Exos programmed induction medium(M1-M2-DMs-Exos group),reapectively.Real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR)was used to detect the mRNA level of bone differentiation gene alkaline phosphatase(ALP)/OCN/Runx2,and alizarin red staining was used to evaluate the calcium nodules deposition in BMSCs.Results are presented as mean±standard deviation.Statistical analysis was performed using independent-t test.Results:On day 1 and day 3 after fracture,the BMDM was mainly M1 type,and on day 5 and day 7,the BMDM was mainly M2 type.TEM showed that DMs-Exos was a cup or spherical vesicle with membrane structure.qPCR results suggested thatthe mRNA relative levels of ALP,OCN and Runx2 genes in M1-DMs-Exos group(0.22±0.12,0.24±0.11,0.26±0.08)and M2-DMs-Exos group(0.41±0.11,0.32±0.07,0.51±0.03)were lower than those in control group(1.00±0.07,1.00±0.05,1.00±0.05,t=12.32,14.27,18.92,P<0.05;t=10.16,17.36,19.21,P<0.05);The mRNA relative levels of ALP,OCN and Runx2 genes in M1-M2-DMs-Exos group(0.85±0.10,0.81±0.09,0.86±0.06)were slightly lower than those in control group(1.00±0.07,1.00±0.05,1.00±0.05,t=2.74,4.50,4.11,P<0.05).Alizarin red sta

关 键 词：骨髓巨噬细胞 外泌体 骨髓间充质干细胞 成骨分化 

分 类 号：R68[医药卫生—骨科学]