摘要：
Objective:To investigate the inhibitory effect of high mobility group box-1 protein (HMGB1) on T cell proliferation and the regulation mechanisms thereof.Methods:Cultured Jurkat cells were divided into 4 groups:control group,HMGB1 treatment (100 ng/ml) for 12,24 and 48 hours groups.Cells were also divided into control group,10,100 and 1 000 ng/ml HMGB1 treated groups,and then stimulated for 24 hours.Proliferation rates of cells were measured using cell counting kit (CCK-8).The expression levels of p53 mRNA,phosphorylated p53 and p53 protein were determined by real-time polymerase chain reaction (RT-PCR) and Western blot respectively.Jurkat cells were transfected with lentivirus containing p53 shRNA,p53 mRNA sequence expressing plasmids or control vector.The cells were afterwards stimulated with HMGB1 (100 ng/ml) for 24 hours,and subjected to cell proliferation assay with the same methods mentioned above.In addition,Jurkat cells were divided into 3 groups:control group,HMGB1 group and HMGB1+SB203580 group,and stimulated with indicated reagents for 24 hours.The expression levels of p53 mRNA,phosphorylated p53 and p53 protein were detected by RT-PCR and Western blot.Results:Compared to the control group,the proliferation rates of cells were decreased after HMGB1 (100 ng/ml) stimulation for 24 and 48 hours (P＜0.05).In dose-dependent stimulations,proliferation rates were reduced in treatment with 100 or 1 000 ng/ml HMGB1 groups (P＜0.05).The expression levels of p53 mRNA,phosphorylated p53 and p53 protein increased in cells after stimulation with 100 ng/ml HMGB1 for 24 and 48 hours (P＜0.05).The expression levels increased obviously in 10 and 100 ng/ml HMGB1 treated cells 24 hours later,but the level was not changed significantly in HMGB1 1 000 ng/ml group.After transfection,the proliferation rates of vector-expressing cells decreased obviously after HMGB1 stimulation.The proliferation of p53 shRNA-expressing cells was basically normal,while of p53 mRNA over expression cells showed lower proliferation rate comparing to vector group (P＜0.05).Treatment with p38 MAPK inhibitor (SB203580) and HMGB1 markedly down-regulated the expression levels of p53 mRNA,phosphorylated and total p53 protein (P＜0.05).Conclusion:Extracellular HMGB1 may inhibit the proliferation of T cells by activation of p53 through p38 MAPK pathway.%目的:探讨高迁移率蛋白族B1 (HMGB1)对T细胞的增殖抑制作用及其分子机制.方法:将培养的Jurkat细胞分为对照组、HMGB1 (100 ng/ml) 12、24、48 h组,HMGB1组加入HMGB1培养相应时间;将细胞分为对照组、HMGB1 10、100、1 000 ng/ml组,予不同浓度HMGB1培养24 h;采用细胞计数试剂盒(CCK-8)法检测各组细胞增殖率,采用实时荧光定量-聚合酶链反应(RT-PCR)和Western blot法检测各组细胞中p53mRNA、磷酸化p53及p53总蛋白的表达水平.将载有p53 shRNA、p53 mRNA表达序列及空白质料的病毒转染入Jurkat细胞中,并选择100 ng/ml HMGB1刺激24 h,采用同样方法检测细胞增殖率.最后,将细胞分为正常组、HMGB1组、SB203580[p38丝裂原活化蛋白激酶(p38MAPK)抑制剂]+HMGB1组进行相应处理.收集各组细胞,RT-PCR和Western blot法检测p53 mRNA、磷酸化p53及p53总蛋白的表达水平.结果:HMGBl (100ng/ml)刺激细胞24、48 h后细胞增殖率降低,较正常组差异有显著性(P＜0.05);100、l 000 ng/ml HMGB1刺激细胞24 h后,增殖率明显下降,与正常组相比差异具有统计学意义(P＜0.05).HMGB1 (100 ng/ml)刺激细胞后,p53 mRNA、磷酸化及总蛋白表达水平在24、48 h逐步上升,且有明显统计学意义(P＜0.05);HMGB1刺激24 h,10、100 ng/ml HMGB1刺激组细胞p53 mRNA、p53磷酸化及总蛋白表达水平较正常组显著上升,但1 000 ng/mlHMGB1没有明显变化;在不同转染组中,予HMGB1刺激细胞,空载组增殖率降低,而p53 shRNA表达组细胞增殖趋于正常,p53 mRNA过表达组增殖率较空载组下降更为明显.Jurkat细胞在p38 MAPK阻断剂和HMGB1共同作用后,p53 mRNA、磷酸化及总蛋白表达水平比HMGB1刺激组明显下降(P＜0.05).结论:胞外HMGB1可通过诱导p38 MAPK信号通路激活p53蛋白,抑制T细胞增殖.
