The Regulation of Exosome Derived from Human Bone Marrow Mesenchymal Stem Cells on the Polarization of Glioma-associated Macrophages
-
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
-
[1] 陈忠平,周旺宁. 我国胶质瘤诊断治疗现状和努力方向[J].中国肿瘤,2005,14(2):78-81. doi: 10.3969/j.issn.1004-0242.2005.02.003 [2] 吴劲松,毛颖. 脑胶质瘤手术理念和研究热点[J].中国神经精神疾病杂志,2009,35(6):376-379. doi: 10.3969/j.issn.1002-0152.2009.06.005 [3] 张姗姗,沈云天,范秋虹,等. 胶质瘤干细胞放射抗拒和放射增敏蛋白质组学研究进展[J].中华放射肿瘤学杂志,2020,29(2):150-153. [4] Furnari F B,Lin H Huang,H - J S,et al. Growth suppression of glioma cells by PTEN requires a functional phosphatase catalytic domain[J]. Proc Natl Acad Sci U S A,1997,94(23):12479-12484. doi: 10.1073/pnas.94.23.12479 [5] 孙涛,葛春林. 肿瘤相关巨噬细胞的研究进展[J].临床肿瘤学杂志,2013,12(6):228-231. [6] 赵银龙, 肿瘤相关巨噬细胞与胶质瘤关系的研究进展[D]. 石家庄: 河北医科大学（硕士学位论文）, 2016. [7] 吴婷,周武雄. 肿瘤微环境中肿瘤相关巨噬细胞的研究进展[J].细胞与分子免疫学杂志,2016,32(02):265-267. [8] 杨黎,张毅. 肿瘤相关巨噬细胞研究进展[J].中国免疫学杂志,2020,36(2):129-135. doi: 10.3969/j.issn.1000-484X.2020.02.001 [9] Toh W S, Lai R C, Zhang B, et al. MSC exosome works through a protein-based mechanism of action[J]. Biochem Soc Trans,2018,46(4):843-853. [10] Wu P,Zhang B,Shi H,et al. MSC-exosome:A novel cell-free therapy for cutaneous regeneration[J]. Cytotherapy,2018,20(3):291-301. [11] 宋玉仙,张东亚,许玉君,等. 人脐带间充质干细胞来源外泌体可调控巨噬细胞的极化[J].中国组织工程研究,2019,23(13):2002-2008. [12] Tj iu,Jeng-Wei,Ch en,et al. Tumor-associated macrophage-induced invasion and angiogenesis of human basal cell carcinoma cells by cyclooxygenase-2 induction[J]. J Invest Dermatol,2009,129(4):1016-25. doi: 10.1038/jid.2008.310 [13] Miller Julie J,Shih Helen A,Andronesi Ovidiu C,et al. Isocitrate dehydrogenase-mutant glioma:Evolving clinical and therapeutic implications[J]. Cancer,2017,123(23):4535-4546. doi: 10.1002/cncr.31039 [14] 段国升. 脑胶质瘤临床治疗的进展[J].中华神经外科杂志,2004,20(2):85-87. [15] Onishi M,Ichikawa T,Kurozumi K,et al. Angiogenesis and invasion in glioma[J]. Brain Tumor Pathology,2011,28(1):13-24. doi: 10.1007/s10014-010-0007-z [16] Xia Z,Zhang C,Zeng Y,et al. Transplantation of BMSCs expressing hVEGF165/hBD3 promotes wound healing in rats with combined radiation‐wound injury[J]. International Wound Journal,2014,11(3):293-303. doi: 10.1111/j.1742-481X.2012.01090.x [17] Harding C V,Heuser J E,Stahl P D. Exosomes:Looking back three decades and into the future[J]. Journal of Cell Biology,2013,201(4):485-485. [18] Zhou Haibo,Xiao Ji ng,Wu Ni ng,et al. MicroRNA-223 regulates the differentiation and function of intestinal dendritic cells and macrophages by targeting C/EBPβ[J]. Cell Reports,2015,13(6):1149-1160. doi: 10.1016/j.celrep.2015.09.073 [19] 储以微. 脑胶质瘤免疫微环境的研究进展[J].中国肿瘤生物治疗杂志,2014,21(5):485-492. [20] Reina-Campos M,Moscat J,Diaz-Meco M. Metabolism shapes the tumor microenvironment[J]. Current Opinion in Cell Biology,2017,48(01):47-53. [21] 解杨,童鹿青,易立,等. 酸性肿瘤微环境对胶质瘤细胞侵袭 迁移能力的影响及其机制研究[J].中华神经医学杂志,2019,018(003):217-224. [22] 叶显宗. 肿瘤相关巨噬细胞促进胶质瘤干细胞侵袭及其机制研究[J]. 重庆: 第三军医大学（博士学位论文）, 2012, [23] 韩尚容,潘燕,李学军. 肿瘤相关巨噬细胞靶向治疗研究进展[J].中国临床药理学杂志,2015,31(6):478-481. [24] 宋瑞卉,张宝刚,李洪利,等. 肿瘤相关巨噬细胞通过EMT促进乳腺癌的浸润转移[J].中国细胞生物学学报,2013,35(5):609-614. [25] 孙耀辉,鲁煜,杨雷,等. 肿瘤相关巨噬细胞与食管癌关系的研究进展[J].国际免疫学杂志,2020,43(1):103-108. doi: 10.3760/cma.j.issn.1673-4394.2020.01.020 [26] 孙鹏,王艳林. 肿瘤相关巨噬细胞在肿瘤发展及治疗中的作用[J].肿瘤防治研究,2014,41(7):829-833. [27] 李一明,卢亦成. 神经干细胞与胶质瘤微环境[J].中华神经外科杂志,2009,25(6):574-576. doi: 10.3760/cma.j.issn.1001-2346.2009.06.034 [28] 彭娟, 丁童, 郑利民, 等. 肿瘤相关巨 噬细胞对鼻咽癌进展和预后的影响[J].癌 症,2006,25(11):1340-1345. [29] 张琳. M2样肿瘤相关巨噬细胞对胶质瘤血管拟态的影响及机制研究[D]. 济南: 山东大学（博士学位论文）, 2017. [30] 郭秋均,李杰. 肿瘤相关巨噬细胞在重塑肿瘤免疫微环境中的作用[J].肿瘤,2013,33(10):922-927. [31] Lv Longhui,Huang Xiaoque,Xiong Xiaoming. Exosome-derived miR-20a inhibit apoptosis of TAM by targeting BCL2L11 in nasopharyngeal carcinoma[J]. Chongqing Medicine,2017,46(6):721-724,728. [32] Yin Z,Han Z,Hu T,et al. Neuron-derived exosomes with high miR-21-5p expression promoted polarization of M1 microglia in culture[J]. Brain Behavior and Immunity,2019,83(1):270-282. [33] 彭一琳,倪煦东,曹帆,等. 前列腺癌细胞来源的外泌体诱导巨噬细胞极化[J].中国生物化学与分子生物学报,2019,35(2):179-186. -
下载:
点击查看大图
图(5)
计量
- 文章访问数: 3465
- HTML全文浏览量: 2193
- PDF下载量: 36
- 被引次数: 0
