液泡膜ATP酶亚基的N末端结构域与巨噬细胞集落刺激因子协同极化巨噬细胞影响胃癌细胞的增殖能力

Effect of synergistic polarization macrophage modulated by N-terminal domain of a2 vacuolar ATPase and macrophage colony stimulating factor on proliferation of gastric cancer cells

目的探讨液泡膜ATP酶亚基的N末端结构域（a2NTD）和巨噬细胞集落刺激因子（M-CSF）对巨噬细胞极化的协同作用，以及极化的巨噬细胞对胃癌细胞增殖能力的影响。 方法分离健康人外周血单个核细胞，诱导培养巨噬细胞后分为4组：对照组（RPMI1640），实验Ⅰ组（M-CSF 100 ng/ml），实验Ⅱ组（a2NTD 500 ng/ml）和实验Ⅲ组（a2NTD 500 ng/ml加M-CSF 100 ng/ml），刺激48 h后应用双重免疫荧光标记法检测巨噬细胞膜表面抗原的表达，用ELISA法检测细胞因子IL-10和IL-12的分泌情况，并用CCK-8法检测巨噬细胞对胃癌SGC-7901细胞增殖能力的影响。 结果巨噬细胞膜表面抗原CD68在4组巨噬细胞膜上均有表达（＋），平均吸光度值（MD值）分别为：对照组0.092±0.005，实验Ⅰ组0.095±0.006，实验Ⅱ组0.094±0.005，实验Ⅲ组0.094±0.005 ，4组比较，差异无统计学意义（P〉 0.05）。CD206在对照组弱表达（-），MD值为0.025±0.004；在实验Ⅰ组和实验Ⅱ组均有表达（＋），MD值分别为0.191±0.012和0.197±0.136，在实验Ⅲ组超强表达（＋＋＋），MD值为0.285±0.011；除实验Ⅰ组与实验Ⅱ组比较差异无统计学意义（P〉 0.05），其余各组两两比较，差异均有统计学意义（均P〈 0.01）。实验Ⅰ组和实验Ⅱ组细胞分泌IL-10水平分别为（85.65±13.64） ng/L和（87.77±14.25）ng/L，均高于对照组[（71.67±7.56）ng/L，P 〈 0.01] ；分泌IL-12水平分别为（9.91±1.50）ng/L和（10.15±1.80）ng/L，均低于对照组[（16.87±1.10）ng/L，P〈 0.01]。实验Ⅲ组分泌IL-10水平为（116.98±14.27）ng/L，高于其余各组（均P〈 0.01）；分泌IL-12水平为（5.31±0.88）ng/L，低于其余各组（均P〈 0.01）。析因分析显示，在对IL-10和IL-12分泌量的影响上，a2NTD和M-CSF具有交互效应（均P 〈0.05）。各组巨噬细胞均能加快胃癌SGC-7901细胞的增殖，且实验Ⅲ组巨噬细胞对胃癌SGC-7901细胞增殖速度的促进作用明显强于其他各组（均P〈 0.01）。 结论a2NTD与M-CSF在促使巨噬细胞极化及细胞因子分泌上具有协同作用，协同极化的巨噬细胞能明显增强胃癌细胞增殖能力。

ObjectiveTo investigate the synergistic effect between the N-terminus domain of the a2 isoform of vacuolar ATPase （a2NTD）and macrophage colony-stimulating factor （M-CSF）on modulating macrophage polarization and the impact of polarized macrophages on proliferation of gastric cancer cells. MethodsPeripheral blood mononuclear cells were derived from healthy donor and induced into macrophages. Then macrophages were randomly divided into four groups： the control group （RPMI 1640）, the experimental group Ⅰ （M-CSF 100 μg/L）, the experimental group Ⅱ （a2NTD 500 μg/L）and the experimental group Ⅲ （a2NTD 500 μg/L plus M-CSF 100 μg/L）. After stimulation for 48 hours, double color immunofluorescence cytochemistry was adopted to detect the expression of cell membrane molecules on macrophages; ELISA was used to measure the secretion of cytokines IL-10 and IL-12; CCK-8 assay was used to evaluate the impact of macrophages on proliferation ability of gastric cancer cell strain SGC-7901. ResultsThe expression of CD68, also known as macrophage surface antigen, was detected on macrophage membrane in all four groups （＋）. The mean absorbance （A）was 0.092±0.005 in control group, 0.095±0.006 in group Ⅰ, 0.094±0.005 in group Ⅱ, 0.094±0.005 in group Ⅲ, and no significant differences were observed among 4 groups （all P 〉 0.05）. Meanwhile, the expression of CD206, which mainly exists on M2 macrophage membrane, was hard to detect in control group （-）with A 0.025±0.004; it was normal in group Ⅰ and group Ⅱ（＋）with A 0.191±0.012 in group Ⅰ and 0.197±0.136 in group Ⅱ（P = 0.212）, and it was up-regulated significantly in group Ⅲ （＋＋＋）with A 0.285±0.011. There were significant differences between either two groups except group Ⅰ and group Ⅱ （all P 〈 0.01）. Secretion of IL-10 in group Ⅰ and group Ⅱ [（85.65±13.64）ng/L and （87.77±14.25）ng/L] was significantly higher compared with control group [（71.67±7.56）ng/L, P 〈 0.01]. Secretion of IL-12 in group Ⅰ and group Ⅱ[（9.91±1.50）ng/L and （10.15±1.80）ng/L] was significantly lower compared with control group [（16.87±1.10）ng/L, P 〈 0.01]. Secretion of IL-10 in group Ⅲ [（116.98±14.27）ng/L] was the highest, and secretion of IL-12 [（5.31±0.88）ng/L] was the lowest （all P 〈 0.01）. There was a synergistic effect between a2NTD and M-CSF on the secretion of both IL-10 and IL-12. Elevated proliferation of gastric cancer cell strain SGC-7901 was detected in all four groups, in which group Ⅲ showed the greatest impact compared with other 3 groups （P 〈 0.01）. Conclusionsa2NTD and M-CSF show a synergistic effect in modulating macrophage phenotype and the secretion of IL-10 and IL-12. The polarized macrophage can significantly enhance proliferation of gastric cancer cell strain SGC-7901.