【摘要】 目的研究15-脱氧-前列腺素J2(15 d-PGJ2)对小鼠单核巨噬细胞中巨噬细胞移动抑制因子(MIF)表达的影响及作用机制。方法以小鼠单核巨噬细胞系J774A.1为研究对象,分别给予以下处理:(1)脂多糖(LPS)组:1μg/ml LPS孵育1 h;(2)正常对照组:PBS孵育1 h;(3)阴性对照组:5μmol/L 15 d-PGJ2孵育1 h;(4)15 d-PGJ2组:5μmol/L 15 d-PGJ2预孵育1 h,1μg/ml LPS孵育1 h;(5)GW9662组:10μmol/L GW9662预孵育1 h,5μmol/L 15d-PGJ2孵育1 h,1μg/ml LPS孵育1 h;(6)Vehicle组:即GW9662对照组,使用GW9662的溶剂DMSO替代GW9662。采用免疫荧光和琼脂糖凝胶电泳技术检测小鼠单核巨噬细胞系J774A.1中MIF的表达,RT-q PCR和Western blot技术检测15 d-PGJ2对LPS诱导的J774A.1炎症反应模型中MIF mRNA和蛋白水平变化情况。观察GW9662对15 d-PGJ2调节MIF作用的影响,采用高内涵分析技术检测15 d-PGJ2是否调节巨噬细胞炎症反应时过氧化物酶体增殖物激活受体γ(PPAR-γ)的核转位。结果 J774A.1在基因和蛋白水平均表达MIF。LPS组细胞中MIF mRNA表达上调到正常对照组的1.75倍(P=0.037),15 d-PGJ2组细胞中MIF mRNA表达上调被抑制,下调至LPS组的50%(P=0.026),MIF蛋白水平变化情况与mRNA水平相一致。GW9662逆转了15 d-PGJ2对MIF mRNA表达的下调(P=0.016)。高内涵自动成像系统分析结果显示,15 d-PGJ2处理组细胞中PPAR-γ的核质比上调至LPS组的1.39倍(P=0.003)。结论 15 d-PGJ2可抑制单核巨噬细胞中MIF的表达,该作用可能依赖于PPAR-γ。更多还原

【Abstract】 Objective To investigate the effect of 15-Deoxy-Δ12,14-prostaglandin J2( 15 d-PGJ2) on the expression of macrophage migration inhibitory factor( MIF) and its underlying mechanism in J774 A. 1.Methods The murine monocyte / macrophage cell line J774 A. 1 were divided into six groups: lipopolysaccharide( LPS) group,incubated with 1 μg/ml LPS for 1 h; normal control group,incubated with PBS for 1 h; nega-tive control group,incubated with 5 μmol / L 15 d-PGJ2 for 1 h; 15 d-PGJ2 group,incubated with 5 μmol / L 15d-PGJ2 for 1 h followed by 1 μg / ml LPS for 1 h; GW9662 group,incubated with 5 μmol / L 15 d-PGJ2 for 1 h following GW9662 10 μmol / L for 1 h,and then incubated with 1 μg / ml LPS for 1 h; and Vehicle group,control of GW9662,GW9662 was replaced by its solvent DMSO. The expression of MIF was detected via immunofluorescence and agarose gel electrophoresis. RT-q PCR and Western blotting were used to test whether 15 d-PGJ2 could regulate mRNA and protein expression of MIF in J774 A. 1 upon LPS challenge. The effect of peroxisome proliferator-activated receptor-γ( PPAR-γ) antagonist GW9662 on the regulation of MIF by 15 d-PGJ2 was observed. The effects of 15 d-PGJ2 on the nuclear translocation of PPAR-γ upon LPS challenge were detected via high content screening analysis. Results MIF DNA and protein expressions were detected in J774 A. 1. MIF mRNA expression was up-regulated( 1. 75 ± 0. 09,P = 0. 037) when challenged with LPS and 15 d-PGJ2 inhibited its upregulation( 0. 84 ± 0. 08,P = 0. 026) in J774 A. 1. The protein level was consistent with the mRNA level. PPAR-γ antagonist GW9662 reversed the effect of 15 d-PGJ2( mRNA,1. 48 ± 0. 06,P = 0. 016; protein,1. 28). Furthermore,nuclear translocation of PPAR-γ was regulated by 15 d-PGJ2 in J774A. 1 upon LPS challenge( 1. 39 ± 0. 02 vs. 1. 01 ± 0. 03,P = 0. 003). Conclusion 15 d-PGJ2 may down-regulate the MIF expression in J774 A. 1 in a PPAR-γ-dependent manner.更多还原