MAPK在介导人肺泡巨噬细胞模型噬菌中的作用

Role of MAPK in phagocytosis of bacteria by human alveolar macrophage

目的 探讨MAPK信号传导途径在介导人肺泡巨噬细胞吞噬细菌中的作用。方法使用Ficoll—Hypaque密度梯度法将外周血单核细胞分离自13名健康志愿者外周血，经2μg／L粒细胞-巨噬细胞集落刺激因子诱导培养12d成单核细胞源性巨噬细胞（GM-MO），即肺泡巨噬细胞研究替代细胞模型。用共聚焦荧光显微镜检测GMMO吞噬荧光标记的金黄色葡萄球菌；使用多功能酶标仪检测p38MAPK、ERK、JNK特异性抑制剂对GM-MO吞噬金黄色葡萄球菌量的影响。以细胞吞噬抑制剂细胞松弛素D为阳性对照。结果GM—MD吞噬金黄色葡萄球菌呈时间依赖，4h后呈逐渐饱和状态。以吞噬反应4h为观测终点，共聚焦荧光显微镜检测显示绝大部分金黄色葡萄球菌均被吞噬进细胞内。使用细胞松弛素D后，GM-MO对金黄色葡萄球菌的吞噬几乎全部被抑制，其荧光值为（4259士869）RFU；p38aMAPK特异性抑制剂Gw856553随着浓度的升高可抑制GM—MD对金黄色葡萄球菌的吞噬，并呈浓度依赖性：浓度为10μmol／L时荧光值为（18290±5491）RFU，浓度为10μmol／L时荧光值为（16802±6440）RFU，与空白对照[（19489士5450）RFU3相比差异均有统计学意义。而ERK抑制剂U0126和JNK抑制剂SP600125在各实验浓度下（10^-8～10mol／L）均对GM-M0吞噬金黄色葡萄球菌无影响。结论p38MAPK途径可能参与了介导人肺泡巨噬细胞对细菌的吞噬作用，而ERK及JNK信号传导途径与此无关。

Objective To investigate the role of mitogen-activated protein kinase （MAPK） pathway in the phagocytosis of bacteria by human alveolar macrophage. Methods Peripheral blood mononuclear cells were isolated from venous blood of 13 healthy volunteers using Ficoll-Hypaque density gradients. Monocytes were incubated with media containing 2 μg/L GM-CSF for 12 days to allow full differentiation into macrophage, a functionally equivalent model of human alveolar macrophage （GM-MO）. Confocal microscopy was used to detect the phagocytosis of heat-killed Staphylococcus aureus Alexa-Fluor 488 conjugate by GM-MO. The effects of p38 MAPK inhibitor, ERK inhibitor and JNK inhibitor on the phagocytosis of Staphylococcus aureus by GM-MO were measured using a Fluostar Optima fluorimeter. Data were expressed as relative fluorescent units （RFU）. Phagocytosic inhibitor, cytochalasin D was set up as positive control. Results Phagocytosis of Staphylococcus aureus by GM-Mφ was time-dependent, after four hours phagocytosis was coming to saturation. The phagocytic reaction for four hours as the observation point, confocal fluorescence microscopy showed that the vast majority of Staphylococcus aureus were phagocytized into cells. GM-MO phagocytosis of Staphylococcus aureus was almost inhibited by cytochalasin D, the fluorescence value was （4 259 ±869） RFU. p38a MAPK specific inhibitor GW856553 inhibited GM-MO phagocytosis of Staphylococcus aureus with the increase of concentration, showing concentration-dependence：the fluorescence value was （18 290± 5 491） RFU at the concentration of 10-6 mol/L,the fluorescence value was （16 802±6 440） RFU at the concentration of 105 mol/L,there was statistically significant difference compared with blank control [（19 489± 5 450） RFU] While the ERK inhibitor UO126 and JNK inhibitor SP600125 in all the experimental concentration （10-8- 10.5 mol/L） had no impact on the phagocytosis of Staphylococcus aureus by GM-MO. Conclusions p38 MAPK pathway may play a key role in the phagocytosis of bacteria by human alveolar macrophage, nevertheless the ERK and JNK pathways do not take part in this process.