肝癌细胞对树突状细胞线粒体功能的抑制作用

Hepatocellular carcinoma cells suppress mitochondrial function of dendritic cells in vitro

目的探索肝癌细胞(hepatocellular carcinoma cells,HCCs)分泌的可溶性细胞因子营造的微环境对树突状细胞(dendritic cells,DCs)线粒体功能状态的影响,以进一步理解肿瘤的免疫逃逸机制。方法用免疫磁珠从人外周血分离CD14+单核细胞,加入粒-巨噬细胞集落刺激因子(recombinant human granulocyte-macrophage CSF,rhGM-CSF)、白介素4(IL-4)将单核细胞诱导分化为未成熟DCs(immature DCs,imDCs),利用肿瘤坏死因子α(recombinant human tumor necrosisfactor-α,rhTNF-α)将imDCs诱导为成熟DCs(mature DCs,mDCs),分别将imDCs和mDCs与肝癌细胞在Transwell中共培养48h,正常培养和撤生长因子培养的DCs作为对照,利用免疫荧光和MTT比色法研究HCCs对DCs的线粒体膜电位、酶活力和胞内钙离子浓度的影响。结果与HCCs共培养后,流式细胞仪的检测结果显示:imDCs和mDCs的线粒体膜电位分别从(659.991±17.052)和(473.741±11.676)下降到(482.681±7.935)和(407.189±5.051)(P<0.05),imDCs和mDCs胞内钙离子浓度分别从(427.983±3.358)和(232.587±3.815)增加到(517.308±0.249)和(413.062±2.981)(P<0.05,P<0.01),MTT比色法的检测结果显示imDCs和mDCs的酶活力分别从(0.764±0.089)和(0.708±0.019)下降到(0.291±0.019)和(0.218±0.019)(P<0.01)。结论HCCs可能通过抑制DCs的线粒体功能来损伤其免疫功能。

Objective To study the influence of microenvironment created by dissoluble cytokines derived from hepatocellular carcinoma cells （HCCs） on the functional status of mitochondria in dendritic cells （DCs） in order to investigate the mechanisms of tumor immune escape.Methods CD14＋ monocytes,which were isolated with immune magnetic beads from fresh peripheral blood of healthy human,were then cultured in RPMI1640/10% FBS supplemented with 100 ng/ml rhGM-CSF and 100 ng/ml rhIL-4 for 7 d to develop into immature DCs （imDCs）.Maturation was induced by addition of 20 ng/ml rhTNF-α to imDCs for another 3 d of culture.Acquired imDCs and mDCs were respectively co-cultured with human HCCs for 48 h in Transwell chamber.These treated cells were investigated for mitochondrial membrane potential,enzyme activity and intracellular Ca2＋ concentration （[Ca2＋]in） in the cytoplasma by immune fluorescence and MTT assay.Those untreated cells served as control.Results After co-cultured with HCCs,flow cytometry showed that the mitochondrial membrane potentials of imDCs and mDCs were respectively decreased from （659.991±17.052）and（473.741±11.676）to（482.681±7.935）and（407.189±5.051）（P0.05）,[Ca2＋]in of imDCs and mDCs were respectively increased from （427.983±3.358）and（232.587±3.815）to（517.308±0.249）and（413.062±2.981）（P0.05,P0.01）.MTT assay showed that the enzyme activities of mitochondrions of imDCs and mDCs were respectively decreased from （0.764±0.089） and （0.708±0.019）to（0.291±0.019）and（0.218±0.019）（P0.01）.Conclusion HCCs might suppress immune function of DCs by inhibiting functional status of mitochondrion.