Chen X Y, Sun R X, Zhang W Y, Liu T, Zheng Y H, Wu Y
Department of Hematology and Research Laboratory, West China Hospital, Sichuan University, Chengdu 610041, China.
Zhonghua Xue Ye Xue Za Zhi. 2017 Jun 14;38(6):480-486. doi: 10.3760/cma.j.issn.0253-2727.2017.06.004.
To investigate the relationship between M2-polarized macrophages and early response in multiple myeloma and its molecular mechanism. Two hundred and forty bone marrow biopsy tissue were collected and M2-polarized macrophages were stained by anti-CD163 monoclonal antibody. In vitro M2-polarized macrophages were derived from human peripheral blood mononuclear cell or THP-1 cells and identified by flow cytometry. Two myeloma cell lines RPMI 8226 and U266 were co-cultured with M2 macrophages using a transwell system. We measured myeloma cells proliferation through CCK-8 method and the pro-inflammatory cytokines expression (TNF-α and IL-6) by ELISA. Real time PCR was applied to measure chemokines (CCL2 and CCL3) , chemokine receptors (CCR2, CCR5) , VEGF and their receptors. In addition, flow cytometry was used to analyze the apoptosis of myeloma cells induced by dexamethasone. ①Patients with high percentage of M2 macrophage involvement in bone marrow showed poorer response (23.9% versus 73.0%, (2)=60.31, <0.001). ② In vitro the proliferation of RPMI 8226 cells (=0.005 at 24 h, =0.020 at 36 h) or U266 myeloma cells (= 0.030 at 24h, =0.020 at 36h) co-cultured with M2-polarized macrophages was higher than control group. ③In vitro the apoptotic rate of RPMI 8226 cells (29.0% versus 71.0%, =4.97, =0.008) or U266 myeloma cells (24.9% versus 67.7%, =6.99, =0.002) co-cultured with M2-polarized macrophages was lower than control group. ④ In vitro M2-polarized macrophages promoted myeloma cells secreting higher level of IL-6, TNF-α and higher expression of CCL2, CCL3, CCR2, CCR5, VEGFA, VEGFR-1,-2 compared with the non-macrophage co-culture system. M2-polarized macrophages promote myeloma cells proliferation and inhibit apoptosis through a very complex mechanism involving pro-inflammatory cytokines IL-6 and TNF-α, chemokines and related receptors such as CCL2, CCL3, CCR2, CCR3, and VEGF as well as related VEGFR.
探讨M2极化巨噬细胞与多发性骨髓瘤早期反应之间的关系及其分子机制。收集240份骨髓活检组织，用抗CD163单克隆抗体对M2极化巨噬细胞进行染色。体外M2极化巨噬细胞来源于人外周血单个核细胞或THP-1细胞，并通过流式细胞术进行鉴定。使用Transwell系统将两种骨髓瘤细胞系RPMI 8226和U266与M2巨噬细胞共培养。我们通过CCK-8法检测骨髓瘤细胞增殖，通过ELISA检测促炎细胞因子表达（TNF-α和IL-6）。应用实时PCR检测趋化因子（CCL2和CCL3）、趋化因子受体（CCR2、CCR5）、VEGF及其受体。此外，使用流式细胞术分析地塞米松诱导的骨髓瘤细胞凋亡。①骨髓中M2巨噬细胞浸润比例高的患者反应较差（23.9%对73.0%，χ² = 60.31，P < 0.001）。②体外，与M2极化巨噬细胞共培养的RPMI 8226细胞（24小时时P = 0.005，36小时时P = 0.020）或U266骨髓瘤细胞（24小时时P = 0.030，36小时时P = 0.020）的增殖高于对照组。③体外，与M2极化巨噬细胞共培养的RPMI 8226细胞（29.0%对71.0%，χ² = 4.97，P = 0.008）或U266骨髓瘤细胞（24.9%对67.7%，χ² = 6.99，P = 0.002）的凋亡率低于对照组。④体外，与非巨噬细胞共培养系统相比，M2极化巨噬细胞促进骨髓瘤细胞分泌更高水平的IL-6、TNF-α以及更高水平表达CCL2、CCL3、CCR2、CCR5、VEGFA、VEGFR-1、-2。M2极化巨噬细胞通过涉及促炎细胞因子IL-6和TNF-α、趋化因子和相关受体如CCL2、CCL3、CCR2、CCR3以及VEGF及其相关VEGFR的非常复杂的机制促进骨髓瘤细胞增殖并抑制凋亡。
