高迁移率族蛋白B1在人牙髓细胞成牙本质分化中的表达

目的:检测人牙髓细胞(human dental pulp cells,hDPCs)诱导矿化过程中高迁移率族蛋白B1(high-mobility group box 1,HMGB1)的表达变化,探讨HMGB1在人牙髓细胞损伤修复中的可能作用。方法:组织块法分离培养hDPCs,收集矿化诱导0、3、7、11、14d后hDPCs的mRNA、蛋白质及细胞爬片,实时荧光定量反转录聚合酶链反应(RT-PCR)和蛋白质印迹法(western blot)分别检测HMGB1、牙本质涎磷蛋白(dentin sialophosphoprotein, DSPP)、牙本质基质蛋白1(dentin matrix protein 1, DMP1)及碱性磷酸酶(alkaline phosphatase, ALP)的mRNA及蛋白表达水平;并检测ALP活性,免疫荧光检测HMGB1在hDPCs矿化过程中的表达。结果:hDPCs矿化诱导后,DMP1、DSPP、ALP及HMGB1的mRNA表达显著性上调,DMP1、DSPP和ALP的mRNA以及碱性磷酸酶活性在诱导7 d、11 d和14 d后与对照组间差异有统计学意义(P<0.05),HMGB1mRNA在矿化诱导11d和14d后与对照组的差异具有统计学意义(P<0.05)。蛋白印迹法检测示细胞内各矿化标记的蛋白表达均较对照组上调,而细胞内HMGB1的蛋白表达较对照组下调。免疫荧光结果显示hDPCs矿化过程中,HMGB1逐渐从胞核转移至胞浆。结论:在hDPCs诱导成牙本质细胞分化过程中,HMGB1在mRNA水平上与DMP1、DSPP和ALP的表达变化趋势相似,而细胞内HMGB1蛋白水平表达下调,且HMGB1在hDPCs细胞内出现转位,提示HMGB1与hDPCs的成牙本质分化有关,可能在牙髓细胞损伤修复中发挥作用。

Expression of High-mobility Group Box B1 in Human Dental Pulp Cells during OdontoblasticDifferentiation.

Objective: To investigate the expression of high-mobility group box B1 (HMGB1) in human dental pulp cells (hDPCs) during odontoblastic differentiation and explore the role of HMGB1 in dental repair. Methods: hDPCswere isolated and cultured in odontoblastic induction medium for 0, 3, 7, 11 and 14 days respectively. Important mineralization-related genes such asdental matrix protein-1 (DMP-1), dental sialophosphoprotein (DSPP) and alkaline phosphatase (ALP) were tested by real-time polymerase chain reaction. Thelevels of mRNA and protein of HMGB1 and the ALP activity were also detected. The expression pattern of HMGB1 during the hDPCsodontoblastic differentiation was detected by immunoflurescence staining. Results: The mRNA levels of DMP1 and DSPP and ALP activity were up-regulated during the hDPCsodontoblastic differentiation on days 7, 11 and 14 (P<0.05). The mRNA level of HMGB1 was significantly increased on days 11 and 14 (P<0.05). The intracellular protein level of HMGB1 was down-regulated and HMGB1 was translocated from nucleus to the cytoplasm during the procedure of odontoblastic differentiation. Conclusion: HMGB1, DMP1, DSPP/DSP and ALP showed analogously expression trend in mRNA level during the hDPCsodontoblastic differentiation. HMGB1 may play an important role in the odontogenic differentiation of hDPCs and dental repair.