【摘要】 目的：探讨顺铂（cisplatin,DDP）耐药型人肺腺癌（lung adenocarcinoma,LUAD）细胞株A549/DDP释放的富含胰岛素样生长因子结合蛋白3（insulin-like growth factorbinding protein 3,IGFBP3）的外泌体对诱导巨噬细胞分化及其对DDP耐药的影响。方法：体外培养亲本A549及A549/DDP细胞，用不同浓度的DDP处理48 h后计算IC₅₀值。收集A549或A549/DDP细胞培养上清液，利用超速离心法分离外泌体，分别命名为A-exo或A/D-exo。用终浓度为15μg/m L的PMA诱导THP-1细胞分化为M0型巨噬细胞，与A549细胞按1∶1比例混合后接种于裸鼠腋下；在肿瘤细胞接种当天，在肿瘤细胞接种部位分别注射PBS、A-exo和A/D-exo，同时每4 d 1次腹腔注射DDP进行治疗；于小鼠荷瘤的第35天，经流式细胞法检测移植瘤组织中人源CD11b⁺CD206⁺或CD11b⁺CD86⁺巨噬细胞的募集情况。用抗体芯片筛选A-exo或A/D-exo中携带的蛋白，并用ELISA法检测A-exo和A/D-exo中IGFBP3蛋白的含量进行验证。用不同浓度的rh IGFBP3处理A549或A549/DDP细胞，分别采用MTS法和Transwell小室实验检测rh IGFBP3对细胞增殖或迁移能力的影响。用rh IGFBP3处理M0巨噬细胞4 d，收集培养上清液；采用ELISA法检测不同浓度的rh IGFBP3对M0巨噬细胞产生TGF-β及TNF-α含量的影响；另外，用rh IGFBP3或rh IGFBP3预处理的M0巨噬细胞培养上清液处理A549细胞，并再次检测DDP对A549细胞的IC₅₀值。结果：DDP对A549/DDP细胞的IC₅₀值较A549细胞显著提高（P<0.01）；与PBS及A-exo组比较，A/D-exo可明显促进A549细胞移植瘤的生长（P<0.05），并促进CD11b⁺CD206⁺巨噬细胞募集到肿瘤组织中（P<0.05）。成功收集获得外泌体A-exo和A/D-exo；与A-exo比较，A/D-exo中携带高水平的IGFBP3；分析显示，LUAD患者中IGFBP3的表达水平明显上调，且IGFBP3高表达患者的总生存率较IGFBP3低表达者有所降低。高浓度rh IGFBP3（100 ng/m L）对A549或A549/DDP细胞均有明显的促增殖作用（P均<0.05），但对A549或A549/DDP细胞迁移能力的影响没有统计学意义。高浓度的rh IGFBP3 （100 ng/m L）能诱导M0型巨噬细胞产生TGF-β1 （P<0.05），但不能诱导TNF-α的产生。经IGFBP3预处理的M0型巨噬细胞的培养液上清（而非rh IGFBP3）处理后，DDP对A549细胞的IC₅₀值显著提高（P<0.05）。结论：A549/DDP细胞通过分泌富含IGFBP3的外泌体介导M2型巨噬细胞分化并促进A549细胞的继发耐药。更多还原

【Abstract】 Objective: To investigate the effects of insulin-like growth factor-binding protein 3（IGFBP3）, which is carried in exosomes released by cisplatin（DDP）-tolerant human lung adenocarcinoma（LUAD） A549/DDP cells, on differentiation of macrophages and its effect on DDP resistance of A549 cells.Methods: The parental A549 and A549/DDP cells were cultured in vitro, and the IC₅₀ values were calculated after treatment with different concentrations of DDP for 48 h. The supernatants of A549 or A549/DDP cells culture were collected, and the exosomes were isolated using ultracentrifugation and named A-exo or A/D-exo, respectively. THP-1 cells were induced to differentiate into M0-type macrophages with PMA（15 μg/m L）, mixed with A549 cells at a ratio of 1∶ 1, and then inoculated in the axillae of nude mice; on the day of tumor cell inoculation, the tumor cells were injected with PBS, A-exo, and A/D-exo at the inoculation site of the tumor cells,respectively, and at the same time, the treatment was carried out by intraperitoneal injection of DDP 1 time every 4 d. On the 35th day of the tumor loading in mice, the recruitment of human CD11b⁺CD206⁺ or CD11b⁺CD86⁺ macrophages in transplanted tumor tissues was detected by flow cytometry（FCM）. Antibody microarrays were used to screen for proteins carried by A-exo or A/Dexo and validated by detecting the amount of IGFBP3 protein in A-exo and A/D-exo by ELISA method. A549 or A549/DDP cells were treated with different concentrations of rhIGFBP3, and the effects of rh IGFBP3 on the proliferation or migration ability of the cells were detected by MTS assay and Transwell assay, respectively. M0-type macrophages were treated with rh IGFBP3for 4 d, and the culture supernatant was collected; the effects of different concentrations of rh IGFBP3 on the production of TGF-β and TNF-α content by M0-type macrophages were detected by ELISA; in addition, A549 cells were treated with rhIGFBP3 or culture supernatant of M0-type macrophages pretreated with rhIGFBP3, and again detected the IC₅₀ value of DDP on A549 cells.Results: The IC₅₀ value of DDP on A549/DDP cells was significantly higher than that of A549 cells（P < 0.01）; A/D-exo significantly promoted the growth of A549 cells xenograft tumors（P < 0.05） and facilitated the recruitment of CD11b⁺CD206⁺ macrophages into tumor tissues（P < 0.05）, compared with PBS and A-exo groups. Exosomes A-exo and A/D-exo were successfully obtained; high levels of IGFBP3 were carried in A/D-exo compared with A-exo.The analysis showed that the expression level of IGFBP3 was significantly up-regulated in patients with LUAD, and the overall survival rate of patients with high expression of IGFBP3was reduced compared with those with low expression of IGFBP3. High concentration of rh IGFBP3（100 ng/mL） had a significant pro-proliferative effect on either A549 or A549/DDP cells（both P < 0.05）, but there was no statistically significant effect on the migratory ability of A549 or A549/DDP cells. High concentrations of rhIGFBP3（100 ng/mL） induced TGF-β1 production by M0-type macrophages（P < 0.05）, but not TNF-α production. The IC₅₀value of DDP on A549 cells was significantly increased（P < 0.05） after treatment with culture supernatant of M0-type macrophages pretreated with IGFBP3（but not rhIGFBP3）.Conclusion: A549/DDP cells mediate M2-type macrophage differentiation and promote secondary drug resistance in A549 cells by secreting IGFBP3-rich exosomes.更多还原