【摘要】 目的：观察泛素调控蛋白A20在慢性乙肝患者外周血中的变化及其对CD14+单核细胞功能的影响。方法：选取慢性乙肝患者47例和健康对照者26例，纯化外周血CD14+单核细胞和CD4+T细胞，流式细胞术检测CD14+单核细胞中A20水平，实时定量PCR检测CD14+单核细胞中A20 mRNA相对表达量。A20 siRNA或对照siRNA转染慢性乙肝患者纯化的CD14+单核细胞，ELISA检测上清中炎症细胞因子水平，实时定量PCR检测FasL和TRAIL mRNA相对表达量。CD14+单核细胞与HepG2.2.15细胞共培养，检测CD14+单核细胞诱导靶细胞死亡的比例。CD14+单核细胞与CD4+T细胞共培养，流式细胞术检测CD4+T细胞分泌IFN-γ和IL-17的比例。结果：慢性乙肝患者外周血CD14+单核细胞中A20+细胞比例高于健康对照者（P=0.000 4），慢性乙肝患者外周血CD14+单核细胞中A20平均荧光强度和A20 mRNA相对表达量亦高于健康对照者（P<0.001）。A20 siRNA转染慢性乙型肝炎患者纯化的CD14+单核细胞后，分泌炎症细胞因子水平高于未转染细胞和对照siRNA转染细胞（P<0.05），但FasL和TRAIL mRNA未见明显变化（P>0.05）。A20 siRNA转染的CD14+单核细胞诱导靶细胞死亡的比例高于未转染细胞和对照siRNA转染细胞（P<0.05），诱导CD4+T细胞分泌IFN-γ和IL-17的比例亦高于未转染细胞和对照siRNA转染细胞（P<0.05）。结论：慢性乙肝患者CD14+单核细胞中高表达的A20有抑制细胞毒性及CD4+T细胞活化的作用，可能与导致乙型肝炎病毒感染慢性化相关。更多还原

【Abstract】 Objective:To investigate the change of ubiquitin-editing protein A20 and its influence on CD14+monocytes func?tion in chronic hepatitis B patients.Methods:Forty-seven chronic hepatitis B patients and twenty-six healthy controls were enrolled.Peripheral CD14+monocytes and CD4+T cells were purified. A20 level in CD14+monocytes was assessed by flow cytometry,while A20 mRNA relative level was semi-quantified by real-time PCR. Purified CD14+monocytes from chronic hepatitis B patients were transfected by A20 siRNA or control siRNA. Levels of proinflammatory cytokines in cultured supernatants were measured by ELLISA.FasL and TRAIL mRNA relative levels were semi-quantified by real-time PCR. CD14+monocytes were co-cultured with HepG2.2.15cells,and percentage of target cell death was investigated. CD14+monocytes were also co-cultured with CD4+T cells,and percentage of IFN-γ and IL-17-secreting CD4+T cells were assessed by flow cytometry.Results:Percentage of A20-positive cells in CD14+mono?cytes was elevated in chronic hepatitis B patients when compared with healthy controls(P=0.000 4). Mean fluorescence intensity and mRNA relative level of A20 in CD14+monocytes was also increased in chronic hepatitis B patients when compared with healthy con?trols(P<0.001). Proinflammatory cytokine levels were increasingly secreted by chronic hepatitis B patients derived CD14+monocytes with A20 siRNA transfection when compared with untransfection and control siRNA transfection(P<0.05),however,there were no significant differences of FasL or TRAIL mRNA relative level(P>0.05). Percentage of target cell death induced by CD14+monocytes was elevated in A20 siRNA transfected cells when compared with untransfected and control siRNA transfected cells(P<0.05),per?centage of IFN-γ-secreting and IL-17-secreting CD4+T cells,which induced by CD14+monocytes were also increased when compared with untransfected and control siRNA transfected cells(P<0.05).Conclusion:Increased level of A20 in CD14+monocytes from chronic hepatitis B patients suppressed cytotoxicity and activation of CD4+T cell,which might induce persistent hepatitis B virus infection.更多还原