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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 explore the effect of microenvironment created by soluble cytokines secreted by hepatocellular carcinoma cells (HCCs) on mitochondrial functional status of dendritic cells (DCs), so as to further understand the mechanism of tumor immune escape. Methods CD14 + monocytes were isolated from human peripheral blood by immunomagnetic beads and monocytes were induced to differentiate by adding rhGM-CSF (rhGM-CSF) and interleukin-4 (IL-4) To immature DCs (imDCs), imDCs were induced to mature DCs (mDCs) by using recombinant human tumor necrosis factor-α (rhTNF-α). The imDCs and mDCs were incubated with hepatocellular carcinoma cells DCs incubated with Transwell for 48h and normal and withdrawal of growth factors were used as controls. The effects of HCCs on mitochondrial membrane potential, enzyme activity and intracellular calcium concentration were studied by immunofluorescence and MTT assay. Results After co-culture with HCCs, the results of flow cytometry showed that the mitochondrial membrane potential of imDCs and mDCs decreased from (659.991 ± 17.052) and (473.741 ± 11.676) to (482.681 ± 7.935) and (407.189 ± 5.051), respectively (P <0.05). The intracellular calcium concentration in imDCs and mDCs increased from (427.983 ± 3.358) and (232.587 ± 3.815) to (517.308 ± 0.249) and (413.062 ± 2.981), respectively The results of colorimetric assay showed that the enzyme activities of imDCs and mDCs decreased from (0.764 ± 0.089) and (0.708 ± 0.019) to (0.291 ± 0.019) and (0.218 ± 0.019), respectively (P <0.01). Conclusion HCCs may impair the immune function by inhibiting the mitochondrial function of DCs.