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

发布时间：2018-10-09 18:30

【摘要】：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(P0.05)。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(P0.05)。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(P0.05)。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(P0.05)。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。
[Abstract]:Aim: to investigate whether the exocrine (exosomes) secreted by epithelial ovarian cancer cells (SKOV3) can regulate the differentiation of mononuclear macrophages into type M2 tumor-associated macrophages (TAMs),) and further participate in tumor metastasis. Methods: the exocrine bodies of SKOV3 cells were isolated and the morphology was observed by transmission electron microscope. Human peripheral blood CD14 monocytes were co-cultured with M-CSF IL-4 and empty medium for 3 days to observe the morphology of ovarian cancer cells. The adherent rate of monocytes was counted by crystal violet, the expression of CD206,HLA-DR was detected by flow cytometry and the contents of IL-10 and IL-12 in supernatant of co-cultured monocytes were detected by Elisa, and the migration ability of tumor cells was detected by migration assay in vitro. Results: transmission electron microscopy showed that the exocrine was approximately round, with a diameter of 30 ~ 80 nm. The crystal violet count showed that the OD value of the exocrine co-culture group and M-CSF IL-4 group (0.13 卤0.06 卤0.16 卤0.04) was higher than that of the empty medium group (0.04 卤0.001), the difference was statistically significant (P0.05). Inverted microscope found that the cells adhered to the wall, similar to the shape of macrophages. The results of flow cytometry showed that the expression level of monocyte CD206 (71.86 卤5.62 卤99.27 卤0.32) in exocrine coculture group and M-CSF IL-4 group was significantly lower than that in blank medium group (P 0.05), and the expression level of HLA-DR was decreased (12.71 卤7.223.55 卤0.27, respectively). The content of IL-10 in supernatant of exocrine co-culture group and M-CSF IL-4 group (71.72 卤0.81 卤82.13 卤2.11, respectively) increased the content of IL-12 (34.88 卤4.75 卤19.71 卤4.28), which was significantly higher than that in blank medium group (P0.05). In vitro migration test showed that the number of tumor cell migration increased significantly (121.58 卤2.25) in the exocrine stimulated monocyte supernatant group, and the difference was statistically significant compared with the control group and the monocyte supernatant group (P0.05). Conclusion: the exocrine of ovarian epithelial cell line SKOV3 can induce the differentiation of mononuclear macrophages into TAMs phenotype in peritoneal of ovarian cancer cells, thus promoting the migration ability of ovarian cancer cells.
【作者单位】：同济大学附属第一妇婴保健院妇科;
【基金】：国家自然科学基金资助项目(No:81372787) 上海市卫计委重点基金资助项目(20134033) 浦东新区重点项目(No:PW2010D-5)资助
【分类号】：R737.31

【参考文献】