三氧化二砷抑制TNF-α诱导大鼠滑膜细胞株增殖及机制

Arsenic trioxide inhibits proliferation of rat synoviocyte line stimulated with TNF-α and its mechanism

目的探讨三氧化二砷(As2O3)抑制TNF-α诱导大鼠滑膜细胞株增殖作用及机制。方法将常规培养的RSC-364细胞株分为对照组、10μg/LTNF-α组及TNF-α加不同浓度As2O3组。MTT法检测细胞增殖;RT-PCR检测高迁移率族蛋白1(HMGB-1)mRNA表达;流式细胞术(FCM)检测细胞增殖指数及周期分布,免疫细胞化学和FCM检测HMGB-1、PCNA蛋白表达。结果(1)As2O3可显著抑制滑膜细胞增殖,并呈时间和剂量依赖性(P<0.05,或P<0.01);(2)与正常组相比,TNF-α显著上调PCNA蛋白表达及HMGB-1 mNA及蛋白的表达(P<0.05,或P<0.01),HMGB-1蛋白不仅表达于细胞核,且表达于胞质;As2O3显著降低PCNA蛋白及HMGB-1蛋白和mRNA的表达(P<0.01),并呈剂量依赖性;(3)与TNF-α组相比,As2O3可使细胞周期G0/G1期增加,G2/M期减少,增殖指数减低(P<0.01);(4)HMGB-1与PCNA蛋白表达呈正相关,二者均与细胞增殖指数呈正相关(r=0.946,P<0.001)。结论As2O3抑制TNF-α诱导的滑膜细胞增殖,可能与其抑制HMGB-1的表达及细胞周期进程有关。

Objective To investigate the effect and possible mechanism of arsenic trioxid （ As2O3 ） on proliferation ot RSC-364 synovioeyte lines stimulated with TNF-α. Methods RSC-364 synoviocytes were cultured with standard medium as control group or medium supplemented with 10 μg/LTNF-α and different concentrations of As2O3 respectively. MTT assay were carried out to study cell proliferation. Proliferation index （PI） and cell cycle were detected by flow eytometry （FCM）. RT-PCR was used to detect the mRNA expression of High mobility group box chromosomal protein （HMGB）1. HMGB-1 and proliferation cell nuclear antigen （PCNA） proteins were detected by immunoeytoehemistry and FCM. Results （ 1 ） As2O3 inhibited proliferation of cell lines stimulated by TNF-α time-dependently and dose-dependently. （2）Compared with normal group, TNF-α up-regulated HMGB-1 protein and mRNA as well as PCNA protein. HMGB-1 protein was not only in nuclear but also in cytoplasm by immunocy-tochemistry. As2O3 down-regulated mRNA and protein of teins （ P 〈 0. 01 ）. （ 3 ） Compared with TNF-α stimulation HMGB-1 in a dose,dependent manner; so did PCNA pro- group, G0/G1 phase cell percentage increased and G2/M phase decreased with the increasing As2O3 concentrations. PI also remarkably decreased （P 〈 0. 01 ）. （4）There was a positive correlation between HMGB-1 and PCNA（ r =0. 946, P 〈0. 001 ）. Moreover both of them were also , with positive correlation to PI （r=0.731, P〈0.001; r =0.706 P〈0.001）. Conclusion Arsenic trioxide may inhibit synoviocyte proliferation stimulated with TNF-α. which maybe explained by down-regulating expression of HMGB-1 and blocking cell cycle.