M2型巨噬细胞调控卵巢癌腹膜内肿瘤血管生成的实验研究

目的:探讨上皮性卵巢癌(epithelial ovarian cancer,EOC)腹水中M2型巨噬细胞(M2 macrophage)调控腹膜内肿瘤血管生成的机制。方法:分离卵巢癌患者腹水中M2型巨噬细胞,体外无血清刺激后,收集上清M2作为条件培养基(M2 macrophage conditional medium,M2 CM),分别采用结晶紫染色实验、Transwell小室迁移实验和小管样结构形成实验,检测共培养刺激后其对人脐静脉血管内皮细胞系EA.hy926增殖、迁移以及小管样结构形成的影响;酶联免疫吸附试验(ELISA)检测刺激后EA.hy926细胞分泌促血管蛋白因子——白细胞介素8(IL-8)以及血管内皮生长因子(VEGF)的影响。结果:①经M2 CM刺激后,EA.hy926细胞的增殖能力提高,结晶紫染色后检测OD值为0.192 6±0.002(P<0.05)。②细胞迁移能力提高,迁移细胞数为84.81±2.04(P<0.001)。③M2 CM可显著促进内皮细胞小管样结构形成,与对照组差异有统计学意义(P<0.05)。④ELISA显示经M2 CM刺激后,EA.hy926分泌IL-8为(1 570.45±118.64)ng/L,VEGF分泌量也显著升高,为(502.21±133.61)ng/L,与对照组差异有统计学意义(P<0.001)。结论:EOC腹水中M2型巨噬细胞可能通过上调内皮细胞分泌VEGF及IL-8等促血管生成因子,增加血管内皮细胞增殖、迁移能力及小管样结构形成,从而促进卵巢癌腹膜内血管生成。

M2 Macrophages Modulate Peritoneal Angiogenesis in Ovarian Cancer

Objective:The aim of this study is to investigate whether M2 macrophages from ascites could induce angiogenesis in epithelial ovarian cancer(EOC).Methods:Separated M2 macrophages from ascites,and then it was cultured and stimulated without FBS for 24 h,collected the conditional medium(M2 CM).To evaluate the effects of M2 CM on the growth of EA.hy926,cell numbers was measured by crystal violet staining method after it co-culture with M2 CM.The effect of M2 CM on the migration of EA.hy926 cells was examined by using a transwell migration assay in 24-well Boyden chambers system(tm).Production of tubular structures was an important process in angiogenesis,we investigated the effect of different conditional medium on EA.hy926 tube formation by capillary-like network formation assay.The supernatants of EA.hy926 cells co-cultured with different conditional medium were collected,then,the expressions of two cytokines(IL-8 and VEGF) were measured by ELISA.Results:① Incubation of EA.hy926 cells with M2 CM increased endothelial cell generation compared to untreated cells(P<0.05).②In migration assay,EA.hy926 cells were shown more invasive cell numbers when the supernatant of M2 CM was used as chemoattractant(84.81±2.04),which was significantly more than that of negative control group(P<0.001).③EA.hy926 cells treated with M2 CM resulted in a more extensive network of interconnecting tubes compared to control medium(P<0.05).④Both IL-8 and VEGF secretion were up regulated after EA.hy926 cell co-cultured with M2 CM.After cultured with M2 CM for 48 hours,a significant increased amount of IL-8(1 570.45±118.645)ng/L were found in an ELISA in the culture supernatant,compared with that of the control medium(P<0.001);the amount of VEGF protein secreted(502.21±133.615)ng/L was also up-regulated(P<0.001),determined in the same supernatant samples.Conclusions:The M2 macrophages from EOC ascites may induced EA.hy296 cell proliferation,migration and tube capillary-like formation via up-regulated the secretion of pro-angiogenic cytokines(eg.IL-8,VEGF).