K562细胞对树突状细胞线粒体功能和能量代谢状态的影响

K562 cells impair mitochondrial function and energy metabolism of dendritic cells in Transwell coculture system

目的探讨共培养条件下血液肿瘤K562细胞对树突状细胞线粒体功能和能量代谢状态的影响。方法采用免疫磁珠分离方法从健康人外周血中分离纯化出单核细胞,用细胞因子GM-CSF和IL-4将单核细胞诱导分化为未成熟DCs(immature DCs,im DCs),再利用细胞因子TNF-α进一步将im DCs诱导为成熟DCs(mature DCs,m DCs),在Transwell系统中分别将im DCs和m DCs与K562细胞共培养48 h,用流式细胞术检测K562细胞对DCs的线粒体膜电位、细胞内钙离子浓度的影响,采用MTT比色法检测线粒体酶活力变化,用傅立叶变换红外光谱(fourier transform infrared spectroscopy,FTIR)技术检测细胞能量代谢状态。对照组为正常培养和撤细胞因子培养48 h的DCs。结果与K562细胞共培养后,和对照组DCs相比,im DCs和m DCs的线粒体膜电位下降(P<0.05),im DCs和m DCs胞内钙离子浓度增加(P<0.01),im DCs和m DCs的线粒体酶活力下降(P<0.05,P<0.01),im DCs和m DCs的细胞能量代谢减低(P<0.01),而正常培养组和撤细胞因子后再培养48h的DCs组相比无显著差别。结论与K562细胞共培养后DCs的线粒体功能受到损伤,细胞能量代谢受到抑制,K562细胞可能通过损伤DCs的线粒体功能,抑制细胞能量代谢状态来损伤其迁移能力,进而损伤其免疫功能,从而使血液肿瘤细胞逃脱机体免疫监视,实现血液肿瘤细胞的增殖、扩散和转移。

Objective To determine the effect of K562 cells on mitochondrial function and energy metabolism of co-cultured dendritic cells（ DCs）. Methods The monocytes were purified from fresh peripheral blood of healthy voluenteers with immune magnetic beads,and then were cultured in the media containing 100 ng / m L rh GM-CSF and 100 ng / m L IL-4 in RPMI1640 /10% FBS to induce the cells into immature DCs（ im DCs）. And then mature DCs（ m DCs） were developed by adding 20 ng / m L rh TNF-α into the culture medium of im DCs. Then the obtained im DCs and m DCs were respectively co-cultured with K562 cells in Transwell chamber for 48 h,and the cells cultured in normal medium for 48 h and those cultured in the medium without cytokines for 48 h served as controls. Mitochondrial membrane potential and intracellular Ca2 ＋concentration in the cytoplasm of DCs were respectively measured by flow cytometry,mitochondrial enzyme activity were studied by MTT assay,cell energy metabolism were investigated by Fourier transformed infrared spectrum scanning. Results Compared with the control cells,co-cultured with K562 resulted in decreased mitochondrial membrane potentials（ P 〈 0. 05）,increased intracellular Ca2 ＋concentrations（ P 〈0. 01）,and decreased mitochondrial enzyme activities in the im DCs and m DCs（ P 〈 0. 05,P 〈 0. 01）,moreover,the cell energy metabolism were also suppressed. Wherever there were no obvious differences between the 2 groups of control cells. Conclusion Mitochondrial functions and energy metabolism status of DCs were impaired by K562 cells,so migration and immune function of DCs were impaired,this could be one of aspects of tumor immune escape,so as to realize the proliferation,spread and metastasis of leukemia cells.