M2型巨噬细胞来源外泌体促进人上皮性卵巢癌细胞顺铂耐药性

M2-type macrophage-derived exosomes promote cisplatin resistance in human epithelial ovarian cancer cells

目的 探讨M2型巨噬细胞来源的外泌体对OVCAR-3人上皮性卵巢癌细胞顺铂化疗敏感性的影响。方法 利用佛波酯(PMA)联合白细胞介素4(IL-4)将人单核细胞系THP1细胞诱导为M2型巨噬细胞,实时荧光定量PCR检测肿瘤坏死因子α(TNF-α)、诱导型一氧化氮合酶(iNOS)、巨噬细胞半乳糖型C型凝集素1(Mgl1)、精氨酸酶1(Arg1)的mRNA表达水平。超速离心法分离M2型巨噬细胞来源的外泌体,通过透射电镜、粒径分析仪和Western blot法分别对分离的外泌体进行鉴定。PKH67标记外泌体后与OVCAR-3细胞共培养,免疫荧光细胞染色检测外泌体被细胞的摄取情况。将外泌体与OVCAR-3细胞共培养后,再经过顺铂(DDP)处理,CCK-8实验检测细胞存活率,流式细胞术和Hoechst 33342染色检测细胞凋亡,Western blot法测定细胞中耐药蛋白P-糖蛋白(ABCB1)、乳腺癌耐药蛋白(ABCG2)的表达变化。结果 经PMA和IL-4诱导的细胞TNF-α、iNOS的mRNA水平下降,Mgl1、Arg1的mRNA水平升高,说明诱导获得M2型巨噬细胞;经鉴定成功分离到M2型巨噬细胞来源的外泌体,且PKH67标记的外泌体可被OVCAR-3细胞摄取;M2型巨噬细胞来源的外泌体与OVCAR-3细胞共培养后,能够增强细胞对DDP的耐药,抑制细胞的凋亡,提高耐药蛋白ABCB1、ABCG2的表达水平。结论 M2型巨噬细胞来源的外泌体能够提高人上皮性卵巢癌细胞存活,抑制癌细胞凋亡并促进耐药蛋白表达,增强癌细胞对DDP的耐药性。

Objective To investigate the effect of M2 macrophage-derived exosomes on the chemosensitivity of OVCAR-3 human epithelial ovarian cancer cells to cisplatin.Methods Using phorbol ester(PMA)combined with interleukin 4(IL-4)to induce the human monocyte line THP1 cells into M2 macrophages,Real-time fluorescence quantitative PCR detected the mRNA expression levels of 1macrophage-related genes tumor necrosis factor alpha(TNF-α),inducible nitric oxide synthase(iNOS),macrophage galactose-type C-type lectin 1(Mgl1)and arginine 1(Arg1).The exosomes derived from M2 macrophages were isolated by ultracentrifugation,and the isolated exosomes were identified by transmission electron microscopy,particle size analyzer and Western blot respectively.PKH67-labeled exosomes were co-cultured with OVCAR-3 cells,and immunofluorescence cell staining was used to detect the uptake of exosomes by cells.After the exosomes were co-cultured with OVCAR-3 cells,they were treated with cisplatin(DDP),the CCK-8 experiment was used to detect cell viability,and flow cytometry and Hoechst 33342 staining were used to detect cell apoptosis.Western blot was used to detect the expression changes of drug resistance protein P-glycoprotein(ABCB1)and breast cancer drug resistance protein(ABCG2)in cells.Results The mRNA levels of TNF-αand iNOS in cells induced by PMA and IL-4 decreased,and the mRNA levels of Mgl1 and Arg1 increased,indicating that M2 macrophages were obtained by induction.It was identified that M2 macrophage-derived exosomes were successfully isolated,and PKH67-labeled exosomes could be taken up by OVCAR-3 cells.After M2 macrophage-derived exosomes were co-cultured with OVCAR-3 cells,it can enhance the resistance of cells to DDP,inhibit the apoptosis of cells,and increase the expression levels of drug-resistant proteins ABCB1 and ABCG2.Conclusion M2 macrophage-derived exosomes can improve the survival of human epithelial ovarian cancer cells,inhibit the apoptosis of cancer cells,promote the expression of drug resistance proteins,and enhance the drug resistance of cancer cells to DDP.