目的:初步探讨铜绿假单胞菌外膜囊泡(outer membrane vesicles，OMVs)对巨噬细胞炎症反应的调节作用及其分子机制。方法:体外培养铜绿假单胞菌(n P.n aeruginosa)提取OMVs，不同浓度OMVs刺激RAW264.7细胞后，酶联免疫吸附法(enzyme linked immunosorbent assay，ELISA)、实时荧光定量PCR(quantitative real time PCR，qRT-PCR)分别检测细胞上清中白细胞介素(interleukin，IL)-8及其mRNA的表达水平，并通过Western blot法检测细胞内核因子-κB(nuclear factor-κB，NF-κB)抑制蛋白(inhibitor of NF-κB，IκB)磷酸化水平；siRNA沉默Toll样受体4(Toll-like receptor 4，TLR4)后，检测细胞中IL-8的分泌水平；最后通过髓样分化因子(myeloid differentiation factor88，MyD88)抑制剂以及NF-κB抑制剂BAY11-7082预处理后OMVs刺激细胞，并检测细胞中IL-8的变化水平。n 结果:不同浓度的OMVs(1、5、10 μg/mL)刺激处理RAW264.7细胞后，细胞内IL-8及其mRNA表达水平较未刺激组(0 μg/mL OMVs)均明显增高[IL-8(1、5、10 μg/mL OMVs)：(31.54±4.25)pg/mL、(82.04±10.20)pg/mL、(136.30±16.03)pg/mL比(16.42±6.14)pg/mL，n t值分别为7.23，8.92和10.76，n P值均<0.05; mRNA(1、5、10 μg/mL OMVs)：(1.25±0.09)，(2.34±0.12)、(4.25±0.43)比(1.00±0.07)，n t值分别为5.24，7.98和9.63，n P值均<0.05]，且呈一定的剂量依赖性。siRNA沉默TLR4后细胞中IL-8分泌水平较对照组显著降低[(46.07±18.23)pg/mL比(115.50±22.02)pg/mL，n t＝9.25，n P<0.05]。而通过MyD88抑制剂以及NF-κB抑制剂预处理后，RAW264.7细胞内IL-8的分泌水平较处理前也明显减少[MyD88抑制剂预处理后：(54.04±12.02)pg/mL比(134.02±18.01)pg/mL,n t＝9.72，n P<0.05；NF-κB抑制剂预处理后：(51.03±17.01)pg/mL比(142.01±22.02)pg/mL，n t＝7.21，n P<0.05]。n 结论:铜绿假单胞菌OMVs能够通过TLR4以及NF-κB途径诱导巨噬细胞RAW264.7分泌IL-8。“,”Objective:To investigate the effects of n Pseudomonas aeruginosa outer membrane vesicle (OMVs) on the regulation of inflammatory response and its molecular mechanism in macrophages.n Methods:OMVs was extracted from the Pseudomonas aeruginosa after cultivating in vitro.After stimulation with different concentrations of OMVs on RAW264.7 cell, the expression levels of IL-8 in the supernatant and its mRNA were detected by enzyme linked immunosorbent assay (ELISA) and quantitative real time PCR(qRT-PCR) respectively.The phosphorylation level of inhibitor of NF-κB(IκB)was detected by Western blot.The secretion level of IL-8 in cells was detected after silencing Toll-like receptor 4(TLR4) by siRNA.Finally, after pretreatment with myeloid differentiation factor88(MyD88) inhibitor and nuclear factor-κB (NF-κB)inhibitor BAY11-7082, the levels of IL-8 was detected in the cells after stimulation with OMVs.Results:After the treatment with OMVs of 1, 5 and 10 μg/mL in RAW264.7 cells, the levels of IL-8 and mRNA in the cells were significantly increased compared with the unstimulated group(0 μg/mL OMVs)[IL-8(OMVs with 1, 5, 10 μg/mL, respectively): (31.54±4.25)pg/mL, (82.04±10.20) pg/mL, (136.30±16.03) pg/mL vs (16.42±6.14) pg/mL, n t values were 7.23, 8.92 and 10.76 respectively, all n P values<0.05; mRNA(OMVs with 1, 5, 10 μg/mL, respectively): (1.25±0.09), (2.34±0.12), (4.25±0.43) vs (1.00±0.07),n t values were 5.24, 7.98 and 9.63 respectively, all n P values<0.05], which showed a certain dose dependence.After silencing TLR4 with siRNA, the secretion level of IL-8 in the cells was significantly reduced compared with that in the control group [(46.07±18.23)pg/mL vs (115.50±22.02)pg/mL,n t=9.25, n P<0.05]. At the same time, after pretreatment with MyD88 inhibitor and NF-κB inhibitor, the secretion level of IL-8 in RAW264.7 cells were also significantly reduced compared with unstimulated group of [pretreatment with MyD88 inhibitor : (54.04±12.02)pg/mL vs(134.02±18.01) pg/mL,n t=9.72, n P<0.05; pretreatment with NF-κB inhibitor: (51.03±17.01) pg/mL vs (142.01±22.02) pg/mL,n t=7.21, n P<0.05].n Conclusion:Pseudomonas aeruginosa OMVs could induce the secretion of IL-8 via the TLR4 and NF-κB pathway in macrophage RAW264.7.