摘要
目的 探讨蟾毒灵(BU)抑制M2型巨噬细胞介导结直肠癌转移的作用.方法 用佛波酯(PMA)诱导人急性白血病单核细胞(THP-1)分化为M0 型巨噬细胞,48h后用含有白细胞介素4(IL-4)和IL-13的培养基处理M0型巨噬细胞,通过酶联免疫吸附试验(ELISA)、形态学、实时荧光定量(RT-qPCR)实验观察其表面标志物和形态变化,RT-PCR和ELISA实验检测M2型巨噬细胞的表面标志物转化生长因子-β(TGF-β)、IL-10.通过ELISA实验比较M2型巨噬细胞和结直肠癌细胞HCT116上清液中IL-6分泌水平,通过Tr-answell 实验、划痕实验、RT-qPCR和Western blot实验检测条件培养基对结直肠癌细胞HCT116的的影响.在条件培养基中加入BU后,通过Western blot、Transwell实验、划痕实验、和RT-qPCR实验观察可以HCT116中 AKT/PI3K 蛋白以及迁移和上皮间质转化能力的变化.结果 将THP-1成功诱导成为M2型巨噬细胞.M2型巨噬细胞通过分泌IL-6激活了 HCT116中AKT/PI3K蛋白磷酸化,促进了其迁移和上皮间质转化能力.BU可以抑制M2巨噬细胞介导的HCT116迁移和上皮间质转化.结论 M2型巨噬细胞释放IL-6激活了结直肠癌细胞AKT/PI3K信号通路,促进了其迁移和上皮间质化.此外,BU可以抑制其促迁移和上皮间质化作用.
Abstract
Objective To investigate the role of bufalin(BU)in inhibiting M2-type macrophage-mediated colorec-tal cancer metastasis.Methods Human acute leukemia mononuclear cells(THP-1)were differentiated into M0 macrophages using phorbol ester induction(PMA)for 48 hours.The M0 macrophages were then treated with IL-4 and IL-13 medium.Surface markers and morphological changes were observed through ELISA,morphology,and RT-qPCR experiments.RT-PCR and ELISA experiments were conducted to detect the surface markers TGF-β and IL-10 of M2 macrophages.The secretion level of IL-6 in the supernatant of M2 macrophages and colorectal cancer cells HCT116 was compared using ELISA.Additionally,the effect of conditioned medium on colorectal cancer cell HCT116 was assessed through Transwell,Wound healing,RT-qPCR,and Western blot experiments.Subsequent-ly,bufalin was added to the conditioned medium and the changes in AKT/PI3K protein,migration,and epithelial-mesenchymal transition ability in HCT116 were observed using Western blot,Transwell,Wound healing and RT-qPCR experiments.Results THP-1 were successfully differentiated into M2 macrophages.The activation of AKT/PI3K protein in HCT116 cells was induced by the secretion of IL-6 from M2 macrophages,which in turn promoted the migration and epithelial-mesenchymal transition ability of the HCT116 cells.The migration and epithelial-mes-enchymal transition mediated by M2 macrophages in HCT116 cells were effectively inhibited by Bufalin.Conclu-sion The release of IL-6 from M2 macrophages activates the AKT/PI3K signaling pathway in colorectal cancer cells,thereby promoting their migration and epithelial-mesenchymal transition capacity.Moreover,bufalin exhibits inhibitory effects on this effect.
基金项目
国家自然科学基金面上项目(81973625)
上海市青年科技启明星计划(22YF1441400)
安徽医科大学研究生科研与实践创新项目(YJS20230098)
上海市中医医院中医传承人才项目(第三届)(2023zycc02)
国家科技期刊平台
NSTL
万方数据