目的：探究骨肉瘤来源的外泌体对肿瘤相关巨噬细胞分化的影响及其具体作用机制。方法：收集2018年3月至 2019年10年于川北医学院附属医院骨科及小儿外科行骨肉瘤切除术、病理诊断明确的18例原发性骨肉瘤患者的肿瘤组织及癌 旁组织，Western blotting实验检测Tim-3的表达水平；分离骨肉瘤MG63细胞外泌体（MG63-Exo），并采用透射电镜及纳米粒径分 析进行鉴定，双荧光染色法验证其是否能够被巨噬细胞吞噬，利用qPCR检测MG63-Exo对巨噬细胞分化及IL-10、TGF-β、VEGF 表达的影响，应用Transwell侵袭和迁移实验及Western blotting检测MG63-Exo所诱导分化的巨噬细胞对MG63细胞迁移及侵袭 及EMT相关蛋白表达的影响；应用CRISPR/Cas9技术敲除MG63细胞中的Tim-3，Western blotting实验检测MG63-Exo中Tim-3 表达，再次应用qPCR、Transwell及Western blotting实验检测来源于敲除Tim-3的MG63外泌体对巨噬细胞分化及其处理后的巨 噬细胞对MG63的迁移、侵袭、EMT相关蛋白表达的影响；最后利用骨肉瘤肺转移小鼠模型验证上述不同来源的外泌体对骨肉瘤 肺转移的影响。结果：透射电镜及纳米粒径分析结果证实成功分离了MG63-Exo，荧光共聚焦显微观察结果显示其能够被巨噬 细胞吞噬。与对照组相比，MG63-Exo能够显著促进巨噬细胞的M2型分化（P<0.05）；与对照组相比， 经MG63-Exo诱导的M2巨 噬细胞能够显著促进骨肉瘤细胞的迁移、侵袭与EMT能力（均P<0.05）；应用CRISPR/Cas9技术敲除Tim-3后的MG63细胞中 Tim-3 mRNA及蛋白表达均显著降低（P<0.05）， 且Tim-3能够以外泌体的形式转移至巨噬细胞中； 与MG63-Exo共培养的巨噬细 胞相比，来源于Tim-3敲除细胞的MG63-Exo能显著抑制巨噬细胞的M2型分化（P<0.05）；相比与经MG63-Exo诱导的巨噬细胞 进行共培养的MG63细胞，Tim-3敲除的MG63-Exo诱导的巨噬细胞能显著抑制肿瘤细胞的迁移、侵袭与EMT及促进肿瘤肺转移 （均P<0.05）。结论：骨肉瘤来源的外泌体通过Tim-3诱导巨噬细胞的M2极化并促进肿瘤的侵袭及转移能力。

Objective: To investigate the effect of exosomes derived from osteosarcoma on the differentiation of tumor-related macrophagesanditsmechanism.Methods:FromMarch2018toOctober2019,tumortissuesandcorrespondingnormaltissuesfrom18patients with primary osteosarcoma who underwent osteosarcoma resection and pathological diagnosis in the Departments of Orthopedics and Pediatric Surgery of theAffiliated HospitalofNorthSichuanMedicalCollegewerecollected. The expression level of Tim-3 was detected by Western blotting; Exosomes of osteosarcoma MG63 cells (MG63-Exo) were isolated and identified by transmission electron microscopy and nanoparticle size analysis, and its phagocytosis by macrophages was verified by Dual fluorescent staining; The effects of MG63-Exo on macrophage differentiation and the expression levels of IL-10, TGF-β and VEGF were detected by qPCR; The effects of MG63-Exo induced macrophages on the migration and invasion of MG63 cells and the expression of EMT related proteins were detected by Transwell invasion and migration assay and Western blotting; CRISPR/cas9 was used to knock out Tim-3 in MG63 cells, and its knockout efficiency was verified by Western blotting, and then qPCR, transwell assay and Western blotting were used to detect the effect of MG63-Exo with Tim-3 knock-out on macrophage differentiation, as well as migration, invasion and expression of EMT related proteins in MG63 cells; Finally, the mouse model of osteosarcoma lung metastasis was used to verify the effect of exosomes from different sources on the lung metastasis of osteosarcoma. Results: Transmission electron microscopy and nanoparticle size assay confirmed that MG63-Exo were successfully isolated, and Confocal fluorescence results confirmed that it could be phagocytized by macrophages; qPCR results showed that MG63-Exo significantly promoted M2 differentiation of macrophages compared with PBS (P<0.05); Compared with PBS control group, M2 macrophages induced by MG63-Exo significantly promoted the migration, invasion and EMT of osteosarcoma cells (all P<0.05); The mRNA and protein expressions of Tim-3 in the MG63 cells knocked out by CRISPR/ cas9 (Tim-3-KO) were significantly reduced (all P<0.05), and Tim-3 could be transferred into macrophages in the form of exosomes; Compared with MG63-Exo co-cultured macrophages, the M2 type differentiation of macrophages treated with Tim-3-KO-exo was significantly decreased (P<0.05); Compared with the MG63 cells co-cultured with macrophages induced by MG63-Exo, the migration, invasion and EMT were significantly reduced while the lung metastasis was significantly promoted in MG63 cells co-cultured with macrophages induced by Tim-3-KO-Exo (all P<0.05). Conclusion: Exosomes derived from osteosarcoma can induce M2 polarization of macrophages through Tim-3 and promote the invasion and metastasis of tumor.

四川省科技厅应用基础项目资助（No. 2018JY0250）