卵巢上皮癌细胞SKOV3通过分泌外泌体促进单核巨噬细胞分化为肿瘤相关巨噬细胞的研究

Study on differentiation of monocytes into tumor-associated macrophage promoted by epithelial ovarian cancer cells SKOV3 through exosomes secretion

目的：研究上皮性卵巢癌细胞SKOV3分泌的外泌体（exosomes）能否调控单核巨噬细胞分化为M2型肿瘤相关巨噬细胞（TAMs）,并进一步参与肿瘤的转移。方法：分离SKOV3细胞外泌体,透射电镜观察形态。卵巢癌细胞外泌体、M-CSF＋IL-4和空培养基分别与人外周血CD14＋单核细胞共培养3天,观察细胞形态。结晶紫计数单核细胞贴壁率;流式细胞仪检测共培养后单核细胞CD206、HLA-DR的表达情况;ELISA法检测共培养后上清中IL-10和IL-12的含量;体外迁移实验检测肿瘤细胞迁移能力的变化。结果：透射电镜显示,外泌体近似圆形,直径30~80nm。结晶紫计数显示,外泌体共培养组和M-CSF＋IL-4组的OD值（分别为0.13±0.06,0.16±0.04）较空培养基组（0.04±0.01）增加,差异有统计学意义（P〈0.05）。倒置显微镜发现,细胞贴壁,形态类似巨噬细胞。流式结果显示,外泌体共培养组和M-CSF＋IL-4组的单核细胞CD206（分别为71.86±5.62、99.27±0.32）表达水平升高,HLA-DR（分别为12.71±7.22、3.55±0.27）表达水平降低,与空培养基组比较,差异均有统计学意义（P〈0.05）。ELISA检测结果显示,外泌体共培养组和M-CSF＋IL-4组的上清IL-10含量（分别为71.72±0.81、82.13±2.11）增加,IL-12含量（分别为34.88±4.75、19.71±4.28）减少,与空培养基组比较,差异有统计学意义（P〈0.05）。体外迁移实验显示,外泌体刺激单核细胞上清组的肿瘤细胞迁移数（121.58±2.25）明显增加,分别与空培养基对照组、单核细胞上清组比较,差异有统计学意义（P〈0.05）。结论：卵巢上皮癌细胞SKOV3的外泌体可诱导单核巨噬细胞分化极化为卵巢癌腹膜内TAMs表型,从而促进卵巢癌细胞的迁移能力。

Objective： To investigate whether exosomes secreted from ovarian cancer cells have ability to activate monocytes to a tumor associated macrophages-like phenotype characteristics in ovarian cancer. Methods： Exosomes isolated from SKOV3 cells were identified by transmission electron microscopy. Freshly isolated monocytes from peripheral human blood underwent treatment with exosomes. Cellular morphology were detected and Cellular adherence was measured using crystal violet. Phenotypes of monocytes treated with different groups were analyzed using APC-labeled anti-CD206,PE-labeled anti-HLA DR. The cell culture supernatants after co-culture were collected and IL-10、IL-12 levels was measured by ELISA. Use transwell to detect the influence to the ovarian tumor cell migration between the different co-culturesupernatants. Results： Using electron microscopy,exosomes secreted by SKOV3 cells display a round-shaped appearance,ranging 30 ~ 80 nm in diameter. After co-cultured 3 days,both exosomes from SKOV3 cells or M-CSF ＋ IL-4 induced naive monocytes adherence as measured by crystal violet staining,while RPMI 1640 treatment did not（ 0. 13 ± 0. 06 vs 0. 16 ± 0. 04 vs 0. 04± 0. 01,P 0. 05）. Monocytes treated with exosomes or M-CSF ＋ IL-4 became stretched and elongated. Treating naive monocytes with exosomes from SKOV3 cells or M-CSF ＋ IL-4 significantly increased the surface expression of CD206（ 71. 86 ± 5. 62、99. 27 ± 0. 32） and reduced the surface expression of HLA-DR（ 12. 71 ± 7. 22、3. 55 ± 0. 27） compared with vehicle-treated samples as quantified by flow cytometry（ P 0. 05）. The results of ELISA showed that monocytes stimulated with exosomes or M-CSF ＋ IL-4 has higer IL-10（ 71. 72 ± 0. 81、82. 13 ± 2. 11）pg / ml and lower IL-12（ 34. 88 ± 4. 75、19. 71 ± 4. 28） pg / ml compared with control group（ P 0. 05）. In exosomes group,the migration SKOV3 cells was enhanced significantly in contrast with blank control and monocytes group（ P 0. 05）. Conclusion： Exosomes secreted by SKOV3 cells activate monocytes to a TAM-like phenotype,thus promoting the migration of ovarian cancer cells.