在LPS刺激的枯否细胞中敲减干扰素调节因子3（IRF3）表达可影响多条信号转导通路  

Effects of inhibited expression of IRF3 in LPS-stimulated Kupffer cells on the activation of signal transduction pathways

作　　者：朱彤[1] 涂文娟 谈志丽 刘亮明[1,3] 

机构地区：[1]南京医科大学附属上海市松江区中心医院,201600 [2]上海市同济医院消化内科 [3]上海交通大学附属第一人民医院松江分院

出　　处：《中华微生物学和免疫学杂志》2015年第8期587-594,共8页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金项目（81070357,30660066）;上海市松江区科学技术攻关项目（14SJGGYY22）

摘　　要：目的：探讨 IRF3 shRNA对脂多糖（ lipopolysaccharide,LPS）刺激枯否细胞（ Kupffer cell,KC） TLR4信号下游 IRF3-IFN-β、NF-κB/p38 MAPK-TNF-α/IL-1β和 IL-10分子的影响。方法采用在体灌注分离培养大鼠原代KC,并以IRF3 shRNA腺病毒体外感染KC。细胞分4组：1组：腺病毒（-）LPS（-）；2组：腺病毒（-）LPS（＋）；3组：腺病毒（＋）LPS（-）；4组：腺病毒（＋）LPS（＋）。 KC培养上清液细胞因子的分泌水平采用ELISA分析；mRNA表达采用real-time PCR检测；KC核蛋白质表达采用 Western blot方法。结果 LPS刺激诱导了原代 KC对 IRF3 mRNA和蛋白质的表达, IRF3 shRNA腺病毒应用后,细胞对IRF3 mRNA的组成性表达及LPS刺激诱导的IRF3 mRNA和蛋白质表达均明显受抑,但对IRF3蛋白质核内组成性表达无明显影响；LPS刺激枯否细胞IFN-β mRNA表达和蛋白质分泌均升高。 IRF3 shRNA应用后,抑制了上述LPS的刺激效应,但对细胞IFN-β的组成性表达和分泌无明显影响；LPS刺激诱导了细胞对前炎细胞因子TNF-α和IL-1β mRNA表达及蛋白质的分泌,IRF3 shRNA腺病毒的应用,抑制了LPS刺激诱导细胞TNF-α和IL-1β蛋白质分泌水平,但对LPS刺激细胞TNF-α和IL-1β mRNA表达以及细胞组成性TNF-α和IL-1β mRNA表达和蛋白质分泌无明显影响；LPS刺激后,细胞IL-10 mRNA表达和培养上清液IL-10蛋白质分泌均显著增加。 IRF3 shRNA腺病毒的应用,促进了LPS刺激诱导KC对IL-10的转录表达和分泌,但对细胞组成性IL-10表达和分泌无明显影响；LPS刺激使核内p-p65和p-p38 MAPK蛋白水平升高,但IRF3 shRNA腺病毒应用对LPS刺激细胞上述分子表达及对细胞组成性表达均无明显影响。结论干扰腺病毒能有效抑制LPS刺激原代枯否细胞IRF3表达及其下游信号转导；IRF3有助于促进LPS刺激KC对TNF-α和IL-1β分泌,但抑制LPS刺激后IL-10的表达；LPS诱导细胞NF-κB和p38 MAPK活化不受IRF3信号影响。Objective To investigate the effects of interferon regulator factor 3 （IRF3） shRNA on the expression of TLR4 downstream signal molecules including IRF3-IFN-β, NF-κB/p38 MAPK-TNF-α/IL-1βand IL-10 in lipopolysaccharide （LPS）-stimulated Kupffer cells （KCs）. Methods KCs were isolated from rats by in situ perfusion. The adenovirus strains carrying IRF3 shRNA were used for the transfection of purified KCs. The isolated KCs were randomly divided into four groups including adenovirus（-） LPS（-） treatment group, adenovirus（-） LPS（＋） treatment group, adenovirus（＋） LPS（-） treatment group and ad-enovirus（＋） LPS（＋） treatment group. The levels of cytokines in the supernatants of KC culture were detec-ted by enzyme-linked immunosorbent assay （ ELISA ） . Real-time PCR and Western blot assay were per-formed to analyze the expression of related cytokines at mRNA and protein levels, respectively. Results The expression of IRF3 at mRNA and protein levels in primary cultured KCs were induced by LPS. The cel-lular constitutive expression of IRF3 at mRNA level and the LPS-induced expression of IRF3 were signifi-cantly inhibited after transfection of KCs with adenovirus strains carrying IRF3 shRNA. However, the nucle-ar constitutive expression of IRF3 protein was not affected by IRF3 shRNA. The expression of IFN-βat mR-NA and protein levels in KCs were induced by LPS, but were suppressed by the interference with IRF3 shR-NA. No significant changes of the cellular constitutive expression of IFN-βat mRNA and protein levels were observed in IRF3 shRNA-treated KCs. Enhanced expression of proinflammatory cytokines including TNF-αand IL-1β at mRNA and protein levels were detected in LPS-stimulated KCs. Transfection of KCs with ade-novirus strains carrying IRF3 shRNA inhibited the LPS-induced secretion of TNF-α and IL-1β, but neither LPS-induced expression of TNF-α and IL-1β at mRNA level nor cellular constitutive expression of TNF-αand IL-1βat mRNA and protein levels were af

关 键 词：干扰素调节因子3 短发夹RNA腺病毒 信号通路 脂多糖 原代枯否细胞 

分 类 号：R392[医药卫生—免疫学]