摘要目的:观察并分析泛素编辑蛋白A20对呼吸机相关性肺炎（ventilator associated pneumonia, VAP）患者单核细胞活性的影响。方法:入选2019年2月至9月在郑州大学第一附属医院诊断为VAP的患者24例（VAP组）和健康对照12例（对照组）。分离VAP组外周血单个核细胞（peripheral blood mononuclear cells, PBMCs）、感染部位和非感染部位的支气管肺泡灌洗液（bronchial alveolar lavage fluid, BALF）及对照组PBMCs，检测CD14 +单核细胞中A20水平。纯化VAP患者CD14 +单核细胞和CD4 +T细胞，使用A20 siRNA转染CD14 +单核细胞。转染的CD14 +单核细胞与自体CD4 +T细胞直接/间接接触共培养，检测CD4 +T细胞分泌干扰素-γ（interferon-γ, IFN-γ）和白细胞介素-17（interleukin-17, IL-17）水平。转染的CD14 +单核与NCI-H889细胞直接/间接共培养，检测细胞毒性、分泌细胞因子、颗粒酶B水平及肿瘤坏死因子相关凋亡诱导配体（tumor necrosis factor-related apoptosis inducing ligand, TRAIL）、FasL配体（Fas ligand, FasL）水平。组间比较采用成组 t检验或SNK- q检验。 结果:VAP组外周血CD14 +A20 +细胞比例高于对照组[（62.61±15.33）% vs. （47.13±11.82）， P<0.05]，A20平均荧光强度（mean fluorescence intensity, MFI）亦高于对照组[（227.9±64.18） vs.（178.2±58.62）， P<0.05]。VAP组感染部位BALF中的CD14 +A20 +细胞比例高于未感染部位[（66.14±19.62）% vs. （52.52±13.71）%， P<0.05]，A20 MFI亦高于未感染部位[（268.0±72.56) vs. (197.4±60.01)， P<0.05]。A20 siRNA转染的VAP患者外周血和BALF中的CD14 +单核细胞与自体CD4 +T细胞直接接触共培养后，CD4 +T细胞分泌IFN-γ和IL-17的细胞比例均高于未转染和siRNA转染（ P<0.05），但间接接触共培养中，CD4 +IFN-γ +和CD4 +IL-17 +细胞比例在未转染、对照siRNA转染和A20 siRNA转染中的差异无统计学意义（ P>0.05）。A20 siRNA转染的VAP患者外周血和BALF中的CD14 +单核细胞与NCI-H889细胞直接/间接接触共培养后，靶细胞死亡比例均高于未转染和siRNA转染（ P<0.05），肿瘤坏死因子-α、IL-6、颗粒酶B水平及TRAIL MFI亦高于未转染和siRNA转染（ P<0.05），但靶细胞死亡比例在直接接触和间接接触共培养之间的差异无统计学意义（ P>0.05）。 结论:VAP患者单核细胞中A20水平升高，可能发挥抑制单核细胞活性的作用。

abstractsObjective:To investigate and analyze the effect of ubiquitin-editing protein A20 on monocytes activity in patients with ventilator-associated pneumonia (VAP).Methods:Twenty-four VAP patients (VAP group) and twelve healthy controls (control group) were included from the First Affiliated Hospital of Zhengzhou University between February 2019 and September 2019. Peripheral blood mononuclear cells (PBMCs) and bronchial alveolar lavage fluid (BALF) (both infection site and non-infection site) were collected from VAP patients, while PBMCs were collected from healthy controls. A20 level in CD14 + monocytes were measured. CD14 + monocytes and CD4 + T cells were purified from VAP patients. CD14 + monocytes were transfected by A20 siRNA. Transfected CD14 + monocytes were directly/indirectly co-cultured with autologous CD4 + T cells. The secretion of interferon-γ (IFN-γ) and interleukin-17 (IL-17) by CD4 + T cells was investigated. Transfected CD14 + monocytes were directly/indirectly co-cultured with NCI-H889 cells. Cytotoxicity, and cytokines/granzyme B level, and tumor necrosis factor-related apoptosis inducing ligand (TRAIL)/Fas ligand (FasL) level was assessed. Student t test or SNK-q test was used for comparison. Results:VAP group had elevated percentage of circulating CD14 +A20 + cells than control group [(66.14±19.62)% vs. (52.52±13.71)%, P<0.05], and also had increased A20 mean fluorescence intensity (MFI) than control group [(268.0±72.56) vs. (197.4±60.01), P<0.05]. The percentage of CD14 +A20 + cells in BALF from infection site was higher than from non-infection site in VAP group [(66.14±19.62)% vs. (52.52±13.71)%, P<0.05], while A20 MFI in infection site was also up-regulated compared with non-infection site [(268.0±72.56) vs. (197.4±60.01), P<0.05]. In direct contact co-culture, A20 siRNA transfected CD14 + monocytes, which were purified from peripheral blood and BALF of VAP patients, induced elevated percentage of IFN-γ and IL-17 secreting CD4 + T cells than un-transfection or control siRNA transfection ( P<0.05). However, there were no significant differences of CD4 +IFN-γ + or CD4 +IL-17 + percentages among un-transfection, control siRNA transfection, and A20 siRNA transfection ( P>0.05). A20 siRNA transfected CD14 + monocytes, which were purified from peripheral blood and BALF of VAP patients, induced increased target cell death in both direct and indirect contact co-culture than un-transfection or control siRNA transfection ( P<0.05). Tumor necrosis factor-α, IL-6, granzyme B level and TRAIL MFI was also up-regulated ( P<0.05). There was no remarkable difference of target cell death between direct and indirect contact co-culture ( P>0.05). Conclusions:A20 was increasingly expressed in monocytes of VAP patients, and might dampen the activity of monocytes.