【摘要】：目的:研究上皮性卵巢癌細(xì)胞SKOV3分泌的外泌體(exosomes)能否調(diào)控單核巨噬細(xì)胞分化為M2型腫瘤相關(guān)巨噬細(xì)胞(TAMs),并進(jìn)一步參與腫瘤的轉(zhuǎn)移。方法:分離SKOV3細(xì)胞外泌體,透射電鏡觀察形態(tài)。卵巢癌細(xì)胞外泌體、M-CSF+IL-4和空培養(yǎng)基分別與人外周血CD14+單核細(xì)胞共培養(yǎng)3天,觀察細(xì)胞形態(tài)。結(jié)晶紫計(jì)數(shù)單核細(xì)胞貼壁率;流式細(xì)胞儀檢測(cè)共培養(yǎng)后單核細(xì)胞CD206、HLA-DR的表達(dá)情況;ELISA法檢測(cè)共培養(yǎng)后上清中IL-10和IL-12的含量;體外遷移實(shí)驗(yàn)檢測(cè)腫瘤細(xì)胞遷移能力的變化。結(jié)果:透射電鏡顯示,外泌體近似圓形,直徑30~80nm。結(jié)晶紫計(jì)數(shù)顯示,外泌體共培養(yǎng)組和M-CSF+IL-4組的OD值(分別為0.13±0.06,0.16±0.04)較空培養(yǎng)基組(0.04±0.01)增加,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。倒置顯微鏡發(fā)現(xiàn),細(xì)胞貼壁,形態(tài)類似巨噬細(xì)胞。流式結(jié)果顯示,外泌體共培養(yǎng)組和M-CSF+IL-4組的單核細(xì)胞CD206(分別為71.86±5.62、99.27±0.32)表達(dá)水平升高,HLA-DR(分別為12.71±7.22、3.55±0.27)表達(dá)水平降低,與空培養(yǎng)基組比較,差異均有統(tǒng)計(jì)學(xué)意義(P0.05)。ELISA檢測(cè)結(jié)果顯示,外泌體共培養(yǎng)組和M-CSF+IL-4組的上清IL-10含量(分別為71.72±0.81、82.13±2.11)增加,IL-12含量(分別為34.88±4.75、19.71±4.28)減少,與空培養(yǎng)基組比較,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。體外遷移實(shí)驗(yàn)顯示,外泌體刺激單核細(xì)胞上清組的腫瘤細(xì)胞遷移數(shù)(121.58±2.25)明顯增加,分別與空培養(yǎng)基對(duì)照組、單核細(xì)胞上清組比較,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論:卵巢上皮癌細(xì)胞SKOV3的外泌體可誘導(dǎo)單核巨噬細(xì)胞分化極化為卵巢癌腹膜內(nèi)TAMs表型,從而促進(jìn)卵巢癌細(xì)胞的遷移能力。
[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 鹵0.06 鹵0.16 鹵0.04) was higher than that of the empty medium group (0.04 鹵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 鹵5.62 鹵99.27 鹵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 鹵7.223.55 鹵0.27, respectively). The content of IL-10 in supernatant of exocrine co-culture group and M-CSF IL-4 group (71.72 鹵0.81 鹵82.13 鹵2.11, respectively) increased the content of IL-12 (34.88 鹵4.75 鹵19.71 鹵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 鹵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.
【作者單位】： 同濟(jì)大學(xué)附屬第一婦嬰保健院婦科;
【基金】：國(guó)家自然科學(xué)基金資助項(xiàng)目(No:81372787) 上海市衛(wèi)計(jì)委重點(diǎn)基金資助項(xiàng)目(20134033) 浦東新區(qū)重點(diǎn)項(xiàng)目(No:PW2010D-5)資助
【分類號(hào)】：R737.31

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