HMGB1在TNF-α诱导大鼠滑膜细胞株RSC-364增殖中的作用及机制

Role of HMGB1 in the proliferation of rat RSC-364 synoviocytes induced by TNF-α

目的:探讨HMGB1在TNF-α诱导的大鼠滑膜RSC-364细胞增殖中的作用及机制。方法:将常规培养的RSC-364细胞分为正常对照组和10μg.L-1TNF-α刺激组,分别于6 h、12 h、24 h收集细胞。RT-PCR检测HMGB1、STAT1和STAT3 mRNA的表达;免疫细胞化学和流式细胞术检测HMGB1、PCNA、STAT1和STAT3蛋白表达。结果:①TNF-α能显著上调HMGB1 mRNA和蛋白的表达,同时PCNA蛋白表达也增强(P<0.05或P<0.01)。②TNF-α作用12 h后,STAT1 mRNA和蛋白的表达明显增强,24 h表达最高(P<0.01)。③TNFα作用6 h-24 h对STAT3 mRNA和蛋白的表达无明显影响(P>0.05)。④HMGB1蛋白表达与PCNA、STAT1蛋白表达呈正相关;STAT1与PCNA蛋白表达亦呈正相关。结论:TNF-α可能通过诱导RSC-364细胞高度表达HMGB1,促进滑膜细胞增殖;STAT1可能参与了其信号转导及调控过程。

AIM ： To investigate the possible mechanism of proliferation of rat RSC - 364 synoviocyte line stimulated by tumor necrosis factor （TNF-α）. METHODS： RSC -364 cells induced by 10 μg/L TNF-α were collected at 6 h, 12 h, 24 h, respectively, as well as normal control cells in vitro. The expression of mRNA of high mobility group box chromosomal protein 1 （ HMGB1 ）, signal transducer and activator of transcription （STAT） 1 and 3 were detected by reverse transcription polymerase chain reaction （ RT - PCR）. The expression of HMGB1, proliferation cell nuclear antigen （PCNA） , STAT 1 and 3 proteins were measured by immunocytochemistry and flow cytometry （FCM）. RESULTS： （1） Compared with control group, HMGB1 mRNA expression in the cells stimulated by 10 μg · L^-1 TNF-α obviously increased, especially at 24 h. HMGB1 proteins were positively expressed in the nuclear of normal cells by immunocytochemistry as well as PCNA. 10 μg · L^-1 TNF-α markedly up - regulated expression of HMGB1 and PCNA proteins at 6 h, 12 h and 24 h. HMGB1 proteins were observed not only in nuclear but also in cytoplasm （ P 〈 0. 05 or P 〈 0. 01 ）. （2） Both mRNA and protein levels of STAT1 were increased in the RSC -364 cells exposed to 10 μg · L^-1 TNF-α after 12 h （P 〈 0.01 ）, the highest expression was at time point of 24 h. （3） No significant difference in expression of STAT3 mRNA from 6 h to 24 h and its protein was observed （P 〉 0. 05）. （4） There were positive correlations between HMGB1 and PCNA or HMGB1 and STAT1, respectively （r = 0. 586, P 〈 0. 01 ; r = 0. 520, P 〈 0. 01 ）. Moreover the expression of STAT1 and PCNA protein was also positively correlated （r =0. 476, P 〈 0. 05）. CONCLUSION： HMGB1 appears to be an important mediator in the proliferation of RSC - 364 cell line induced by TNF-α and STAT1 might be involved in the regulation of signal transduction.