The Regulation of Exosome Derived from Human Bone Marrow Mesenchymal Stem Cells on the Polarization of Glioma-associated Macrophages
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摘要:
目的 探究人骨髓间充质干细胞分泌的外泌体对肿瘤相关巨噬细胞极化的影响，并初步探讨其对胶质瘤发生发展的影响。 方法 利用试剂盒提取人骨髓间充质干细胞分泌的外泌体，通过扫描电镜及Western blot 鉴定外泌体。将胶质瘤细胞分为SHG449：SHG449细胞不做特殊处理；SHG449+M0组：SHG449细胞与M0型巨噬细胞共培养；SHG449+M0+exosome组：SHG449细胞与M0型巨噬细胞共培养后，加入标记有PKH-67的外泌体使其进入M0型巨噬细胞。 结果 （1）人骨髓间充质干细胞分泌的外泌体成功提取，且进入M0型巨噬细胞；（2）外泌体进入胶质瘤细胞SHG449诱导极化的巨噬细胞后细胞形态转化，且M2型巨噬细胞活化标志物Arginase、CD206以及CCL22，TGF-β表达下降（Arginase：P < 0.01，CD206： P < 0.05， CCL22： P < 0.05，TGF-β： P < 0.01），M1型活化标志物iNOS，CD68以及IL-1β，TNF-α表达上升（iNOS： P < 0.01，CD68： P < 0.01，L-1β： P < 0.000 1，TNF-α： P < 0.001）；（3）被外泌体诱导极化的巨噬细胞导致胶质瘤细胞增殖能力下降（ P < 0.05），凋亡能力上升（ P < 0.000 1）。 结论 人骨髓间质细胞分泌的外泌体可抑制肿瘤相关巨噬细胞向M2表型转化，并诱导其向M1表型转化，调节肿瘤免疫微环境，从而达到抑癌的作用。 Abstract:Objective To investigate the effect of exosomes divided from human bone marrow mesenchymal stem cells on the polarization of tumor-associated macrophages, and to preliminarily discuss their influence on glioma cell lines. Methods Exosomes divided from human bone marrow mesenchymal stem cells were extracted and identified by scanning electron microscopy and western blot. The glioma cell line SHG449 was divided into three groups: SHG449 group: SHG449 cells without special treatment; SHG449+M0 group: SHG449 cells co-cultured with M0 macrophages; SHG449+M0+exosome group: SHG449 cells co-cultured with M0 macrophages, and then exosomes labeled with PKH-67 were added to the M0 cells. The mRNA expression of M1 biomarkers（iNOS and CD68）and M2 markers（Arginase and CD206）were detected by qPCR, and the content of IL-1β, TNF-α, CCL22 and TGF-β in the supernatant of SHG449 cells were detected by ELISA. The proliferation and apoptosis of SHG449 cell line were detected by CCk8 and flow cytometry. Results （1）Exosomes secreted by human bone marrow mesenchymal stem cells were successfully extracted and entered into M0-type macrophages.（2）Macrophage morphology was transformed after exosome was intaken by SHG449-induced polarized macrophages, and the expression of M2 macrophages markers Arginase, CD206, CCL22 and TGF-β were decreased（Arginase: P < 0.01, CD206: P < 0.05, CCL22: P < 0.05, TGF-β: P < 0.01）, while the expression of M1 macrophages markers iNOS, CD68, IL-1β and TNF-α increased（iNOS: P < 0.01, CD68: P < 0.01, L-1β: P < 0.000 1, TNF-α: P < 0.001）.（3）Macrophage polarization induced by exosomes resulted in decreased proliferation（ P < 0.05） and increased apoptotic（ P < 0.000 1） of glioma cells. Conclusion Exosomes derived from human bone marrow stromal cells can inhibit the polarization of tumor-associated macrophages to the M2 phenotype and induce their polarization to the M1 phenotype, and regulate the tumor immune microenvironment, thereby suppressing the development of glioma. -
图 1 人骨髓间充质干细胞鉴定
A-B：光学显微镜下观察第5代人骨髓间充质干细胞形态（×100）；C：流式细胞仪检测第5代人源骨髓间充质干细胞阳性表达CD29，CD90，阴性表达CD34，CD45。
Figure 1. Identification of human bone marrow mesenchymal stem cells
图 2 外泌体鉴定
A：扫描电镜观察外泌体呈囊泡样形态（×60 000）；B：Western blot 检测外泌体常见标志物Alix，CD63，CD81以及TSG101的蛋白表达。
Figure 2. Identification of exosomes
图 3 巨噬细胞对外泌体的摄入并促进巨噬细胞向M1型极化
A：大量PKH-67标记的外泌体被巨噬细胞摄取（40×，PKH-67为绿色，DAPI为蓝色）；B：光学显微镜下观察各组细胞形态（×100）；C-F：qPCR检测M1型巨噬细胞活化标志物iNOS，CD68 以及M2型巨噬细胞活化标志物Arginase、CD206。两两组间比较，* P < 0.05，** P < 0.01，*** P < 0.001。
Figure 3. Exosomes were intaken by macrophage and macrophage was polarized to M1 type
图 4 ELISA检测胶质瘤细胞SHG449上清液中IL-1β，TNF-α，CCL22，TGF-β 的含量
A：IL-1β 含量；B：TNF-α含量；C：CCL22含量；D：TGF-β含量。两两组间比较，* P < 0.05，** P < 0.01，*** P < 0.001，**** P < 0.0001。
Figure 4. The content of IL-1β，TNF-α，CCL22，and TGF-β in the supernatant of glioma cells SHG449 by ELISA
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