肿瘤相关巨噬细胞通过分泌IL-10影响上皮性卵巢癌肿瘤微环境中T细胞亚群Treg/Th17失衡

Impact of tumor-associate macrophages( TAMs) to imbalance of Treg/Th17 in Eoc microenvironment

目的:探讨M2型肿瘤相关巨噬细胞(TAM)是否为上皮性卵巢癌外周血和肿瘤微环境中Treg/Th17比例失衡的影响机制之一。方法:流式细胞术检测M2型细胞与CD4+T细胞共培养后Treg/Th17比例变化,Western blot检测T细胞中的转录因子Foxp3及RORrt水平,ELISA检测共培养上清中IL-10的含量,结晶紫检测上清对肿瘤细胞增殖的影响,Transwell法检测上清对肿瘤细胞迁移能力的影响,分析TAM细胞对Treg/Th17失衡影响的机制。结果:①CD4+T细胞与M2型巨噬细胞共培养后,Treg/Th17比例为0.76±0.33相比Control组0.41±0.25,M0组0.40±0.32,M1组0.31±0.16有显著增高(P<0.05);②共培养上清对Skov3细胞具有显著的促增殖能力,共培养1 d后,M2组为14 942.43±434.19较Control组:12 445.57±179.34,CD3/28组12 470.32±434.18明显上升(P<0.001);共培养2 d后,M2组为30 129.09±520.53较Control组25 622.81±897.07,CD3/28组25 721.62±1 808.60显著提高(P<0.05);③共培养后的T细胞中,Treg特异性转录因子Foxp3上调(P=0.047),Th17特异性转录因子RORrt出现下降趋势但无统计学差异(P=0.294)。④T细胞与M2细胞共培养3 d后上清中IL-10含量为(264.04±75.9)pg/ml,较CD3/28组(60.89±46.54)pg/ml,M0组(44.81±32.93)pg/ml,M1组(42.71±26.09)pg/ml均显著提高(P=0.001)。结论:M2型TAM细胞促进Treg/Th17比例增加,上调Treg特异性转录因子Foxp3,下调Th17特异性转录因子RORrt。同时,TAM与T细胞共培养上清具有显著促进肿瘤细胞增殖和迁移的能力。而这一机制可能是与其促进IL-10分泌增多有关。

Objective：To investigate the impact and mechanisms of TAM to the imbalance of Treg /Th17 in the Eoc microenvi-ronment.Mte hods：Build the in vitro M2 macrophage model ,which was like TAMs .Use flow cytometry to detect the difference of the Treg/Th17 before and after the co-culture of M2 macrophage and CD 4＋T cells.Use Western bolt to detect the change of T cell transcription factor and ELISA to detect the IL-10 levels in the supernatant after co-culture.Use crystal violet methods to detect the influence to the ovarian tumor cell proliferation between the different co-culture supernatants and the Transwell to detect the influence to the ovarian tumor cell migration.Thus to analysis the how TAMs influence the imbalance of Treg/Th17 in Eoc microenvironments.R esults：①After coc-ultured with M2 macrophage ,the ratio of Treg/Th17 was（ 0.76 ±0.33 ） significant increased compared with control （0.41±0.25） ,M0（ 0.40±0.32） and M1（0.31±0.16） （P＆lt;0.05）.②After co-cultured,the supernatant of M2 group has a significant ability to promote the proliferation of Skov-3 cells.After co-cultured for 1 day, the Skov-3 cell number of M2 group was 14 942.43 ±434.19 , which was significantly higher than the control group （ 12 445.57 ±179.34 ） and CD3/28 group （12 470.32±434.18）（P＆lt;0.001）.After co-cultured for 2 days,the Skov-3 cell number of M2 group was 30 129.09±520.53 ,which was significantly higher than the control group （25 622.81±897.07） and CD3/28 group（25 721.62±1 808.60） （P＆lt;0.05）.③After＆nbsp;co -cultured with M2 macrophage , the Treg-specific transcription factor Foxp 3 increased （ P=0.047 ） compared with control and M 1 group .④After co-cultured with M2 macrophage for 3 days,the concentration of IL1-0 in the supernatant was（264.04±75.9）pg/ml, which was significantly higher than CD 3/28 group （ 60.89 ±46.54 ） pg/ml,M 0 group （ 44.81 ±32.93 ） pg/ml, M1 group （ 42.71 ± 26.09）pg/ml（P=0.001）.Conclusion： M2 macrophage induces the increase of the radio of Treg /Th17 as well as the increase of Treg-specific transcription factor Foxp 3 and the decrease of Th17 -specific transcription factor ROR-γt.Meanwhile , the co-culture supernatant of M2 macrophage and CD4＋T cell have the ability to promote the proliferation and migration of ovarian cancer cell ,the mechanism which ,may related to the IL -10 in the supernatant .