低氧对巨噬细胞外泌体的表达及其对骨肉瘤细胞的化疗抗性影响的体外研究  

Hypoxia enhances chemoresistance of osteosarcoma cells by promoting exosomes secretion of macrophages

作　　者：刘洲[1] 刘路平[1] 李伟强[1] LIU Zhou;LIU Luping;LI Weiqiang(Department of Orthopaedics,Second Affiliated Hospital of Kunming Medical University,Kunming 650000,China)

机构地区：[1]昆明医科大学第二附属医院骨科,650000

出　　处：《免疫学杂志》2021年第3期230-238,共9页Immunological Journal

摘　　要：目的在体外探究低氧对巨噬细胞外泌体分泌的影响及对骨肉瘤细胞顺铂耐药性的改变。方法Transwell侵袭实验检测不同氧浓度(1%O2的低氧与常氧)条件下骨肉瘤细胞MG63对巨噬细胞的趋化能力;在低氧与常氧条件下将MG63与巨噬细胞进行共培养,流式细胞术及RT-PCR检测巨噬细胞的分化;分离纯化来源于不同氧浓度条件下培养的M2型巨噬细胞外泌体,利用透射电镜、纳米粒径、Western blot进行鉴定,双免疫荧光进行示踪MG63对外泌体的摄取;克隆形成实验及流式细胞术检测来源于不同氧浓度下的巨噬细胞外泌体对MG63的增殖、凋亡影响;CCK-8检测来源于不同氧浓度下的巨噬细胞外泌体对MG63顺铂耐药性的影响。结果低氧能够促进骨肉瘤细胞对巨噬细胞的趋化能力,并进一步诱导巨噬细胞向M2型分化;成功分离不同氧浓度下培养的M2巨噬细胞外泌体,且低氧能够显著上调巨噬细胞对外泌体的分泌;低氧能够明显上调巨噬细胞外泌体对骨肉瘤细胞增殖能力的促进作用,抑制骨肉瘤细胞发生凋亡,并上调骨肉细胞对顺铂的耐药性。结论在体外低氧能够促进骨肉瘤细胞对巨噬细胞的趋化能力,并进一步通过诱导巨噬细胞向M2型分化及提高M2型巨噬细胞外泌体的分泌,进而上调骨肉瘤细胞的增殖能力,抑制其凋亡,最终导致骨肉瘤细胞产生顺铂耐药性。This study was performed to investigate the effect of hypoxia on the secretion of macrophage exosomes and the cisplatin resistance of osteosarcoma cells. Transwell invasion test was used to detect the chemotaxis of osteosarcoma cell MG63 to macrophages under different oxygen concentrations(1%O2 hypoxia and normoxia). MG63 macrophages were cocultured under hypoxia or normoxia conditions, and the differentiation of macrophages was detected by flow cytometry and RT-PCR. The exosomes of M2 type macrophages were isolated and purified under different oxygen concentrations, and then identified by projection electron microscopy, nanoparticle sizing and Western blotting. The uptake of MG63 was traced by double immunofluorescence. The effects of exosomes on the proliferation and apoptosis of MG63 were detected by clonogenesis and cell flow cytometry, while CCK-8 was used to detect the effect of exosomes on cisplatin resistance of MG63 cells. Data showed that the recruitment ability of osteosarcoma cells to macrophages was significantly enhanced under hypoxia, and macrophages were further induced to differentiate into M2 type;the exosomes of M2 macrophages cultured in different oxygen concentrations were successfully isolated, and hypoxia could significantly promote the secretion of exosomes. Furthermore,exosomes produced by macrophages under hypoxia can significantly promote the proliferation of osteosarcoma cells,inhibit their apoptosis, and up regulate the resistance of osteosarcoma cells to cisplatin. Taken together, hypoxia can promote the chemotaxis of osteosarcoma cells to macrophages, and further increase the secretion of M2 macrophage exosomes by inducing the differentiation of macrophages to M2, and then up regulate the proliferation of osteosarcoma cells, inhibit their apoptosis, and finally lead to cisplatin resistance of osteosarcoma cells.

关 键 词：低氧 M2型巨噬细胞 外泌体 化疗抗性 

分 类 号：R783.1[医药卫生—口腔医学]