CAJ | 학술논문

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。
목적：탐토 IRF3 shRNA대지다당( lipopolysaccharide,LPS)자격고부세포( Kupffer cell,KC) TLR4신호하유 IRF3-IFN-β、NF-κB/p38 MAPK-TNF-α/IL-1β화 IL-10분자적영향。방법채용재체관주분리배양대서원대KC,병이IRF3 shRNA선병독체외감염KC。세포분4조：1조：선병독(-)LPS(-)；2조：선병독(-)LPS(+)；3조：선병독(+)LPS(-)；4조：선병독(+)LPS(+)。 KC배양상청액세포인자적분비수평채용ELISA분석；mRNA표체채용real-time PCR검측；KC핵단백질표체채용 Western blot방법。결과 LPS자격유도료원대 KC대 IRF3 mRNA화단백질적표체, IRF3 shRNA선병독응용후,세포대IRF3 mRNA적조성성표체급LPS자격유도적IRF3 mRNA화단백질표체균명현수억,단대IRF3단백질핵내조성성표체무명현영향；LPS자격고부세포IFN-β mRNA표체화단백질분비균승고。 IRF3 shRNA응용후,억제료상술LPS적자격효응,단대세포IFN-β적조성성표체화분비무명현영향；LPS자격유도료세포대전염세포인자TNF-α화IL-1β mRNA표체급단백질적분비,IRF3 shRNA선병독적응용,억제료LPS자격유도세포TNF-α화IL-1β단백질분비수평,단대LPS자격세포TNF-α화IL-1β mRNA표체이급세포조성성TNF-α화IL-1β mRNA표체화단백질분비무명현영향；LPS자격후,세포IL-10 mRNA표체화배양상청액IL-10단백질분비균현저증가。 IRF3 shRNA선병독적응용,촉진료LPS자격유도KC대IL-10적전록표체화분비,단대세포조성성IL-10표체화분비무명현영향；LPS자격사핵내p-p65화p-p38 MAPK단백수평승고,단IRF3 shRNA선병독응용대LPS자격세포상술분자표체급대세포조성성표체균무명현영향。결론간우선병독능유효억제LPS자격원대고부세포IRF3표체급기하유신호전도；IRF3유조우촉진LPS자격KC대TNF-α화IL-1β분비,단억제LPS자격후IL-10적표체；LPS유도세포NF-κB화p38 MAPK활화불수IRF3신호영향。
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 affected by IRF3 shRNA. The LPS-induced expression of IL-10 at mRNA and protein levels were enhanced in IRF3 shRNA-treated KCs. However, the cellular constitutive expression of IL-10 at mRNA and protein levels were not affected by the adenovirus. The levels of phosphor-NF-κB p65 subunit and phosphor-p38 MAPK protein in the nuclei of KCs were increased upon the stimula-tion with LPS. Treatment of KCs with IRF3 shRNA showed no significant effects on nuclear phosphor-NF-κB p65 subunit and phosphor-p38 MAPK. Conclusion Transfection of LPS-stimulated primary KCs with ade-novirus strains carrying IRF3 shRNA could effectively inhibit the expression of IRF3 and the transduction of downstream signals. IRF3 enhanced the secretion of TNF-αand IL-1β, but inhibited the expression of IL-10 in LPS-treated KCs. The LPS-induced activation of NF-κB and p38 MAPK in KCs were not affected by IRF3 signal.