目的 检测LPS刺激后小鼠腹腔巨噬细胞HMGB1和相关信号分子P38 MAPK、NF-κB、CBP的表达,探讨脓毒症时巨噬细胞表达和释放HMGB1的信号传导机制.方法 采用LPS刺激RAW264.7细胞,在不同的时间点用免疫细胞化学、激光共聚焦显微镜观察细胞内相关信号分子P38 MAPK、NF-κB、CBP的变化,ELISA检测培养上清HMGB1的含量,Real-time PCR检测培养细胞HMGB1的mRNA水平,Western blot检测胞质和胞核内HMGB1的含量.结果 随着LPs的刺激,胞质内P38 MAPK的绿色荧光逐渐增强,NF-κB的绿色荧逐渐减弱,而胞核内NF-κB绿色荧光逐渐增强,CBP的绿色荧光逐渐增强,3者均于刺激后6h达高峰.LPS刺激后12 ～48 h培养细胞胞质和上清中HMGB1蛋白含量逐渐增加,而12～24 h胞核内HMGB1含量逐渐减少,36h后又逐渐增多,各不同时间点差异具有显著性(P＜0.01)；而细胞内HMGB1 mRNA表达在LPS刺激后0～12 h无明显变化,24、36和48h明显增高,与0h相比差异有显著性(P＜0.01).结论 LPS通过依次激活巨噬细胞内信号分子P38 MAPK、NF-κB及CBP来诱导HMGB1的合成、转位和释放表达的.%Objective To explore the signaling molecules related to HMGB1 expression in murine macrophage-like cell line RAW264. 7 cells induced by lipopolysaccharide, HMGBl, P38 MAPK, NF-kB and CBP were detected. Methods After stimulation by LPS, P38 MAPK, NF-kB and CBP protein in cytoplasm and nucelus were observed by immunocytochemistry and laser confocal scanning microscopy. HMGB1 protein in supernatant was measured by ELJSA, the expression of HMGB1 mRNA in cultured cells was determined by Real-time PCR. Cultured cells cytoplasm and nucleus HMGB1 protein were detected by Western blot. Results With LPS stimulation, the green fluorescence of P38 MAPK was gradually increased and the green fluorescence of NF-kB was gradually weakened in cytoplasm, while in nucleus, the green fluorescence of both NF-kB and CBP was gradually increased, and reached its peak after 6 h. HMGB1 protein in supernatant and cytoplasm increased gradually from 12 to 48 h after stimulated by LPS. HMGB1 protein in nucleus decreased gradually from 12 to 24 h after LPS stimulated, increased gradually after 36 h(P < 0.01). The expression of HMGB1 mRNA was not significantly changed from 0 to 12 h but enhanced significantly from 24 to 48 h after LPS stimulation(P <0.01). Conclusions LPS activates P38 MAPK,NF-kB and CBP signaling pathway, which lead to HMGB1 deacetylated and transferred into cytoplasm, finally released into excellular.
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