三氧化矿物聚合物对年轻恒牙牙髓干细胞体外增殖分化的影响

Effects of mineral trioxide aggregate on proliferation and differentiation of dental pulp stem cells from young permanent teeth in vitro

目的 探讨不同浓度三氧化矿物聚合物（MTA）对年轻恒牙牙髓干细胞（DPSCs）体外增殖分化的影响.方法 从年轻恒牙中分离出牙髓组织,用组织贴块法培养DPSCs,并通过检测干细胞表面标志物STRO-1进行鉴定.制备浓度为0.02、0.20、2.00、20 g/L的MTA浸提液.采用四甲基偶氮噻唑蓝比色法（MTT）检测不同浓度MTA对DPSCs存活率及增殖的影响;加入适宜浓度MTA培养4周后,茜素红染色检测矿化结节形成情况;荧光定量聚合酶链反应（PCR）分别测定适宜浓度MTA培养12、24、36、48 h后碱性磷酸酶（ALP）、骨涎蛋白、骨钙素、牙本质涎蛋白（DSP）mRNA的表达.结果 免疫荧光显示干细胞表面标志物表达呈阳性.浓度为0.20 g/L的MTA促进DPSCs增殖的活力较强,培养4周后茜素红染色可见有矿化结节形成.作用于DPSCs不同时间后,骨钙素和DSP mRNA的表达水平呈时间依赖性,在48 h内随着时间的增长而上升.而ALP和BSP mRNA的表达水平则是先上升后下降.结论 0.02、0.20、2.00 g/L浓度的MTA能有效地促进年轻恒牙DPSCs增殖,且0.20 g/L MTA能有效地诱导DPSCs向成牙本质细胞分化.

Objective To investigate the effects of the different concentration of mineral trioxide aggregate （MTA） on the proliferation and differentiation of dental pulp stem cells （DPSCs） from the young permanent teeth.Methods DPSCs were isolated from the young permanent teeth and cultured by tissue explant method.The expression of STRO-1 was detected by using immunofluorescence technology.DPSCs were cultured with different concentrations of MTA （0.02,0.20,2.00,20.00 g/L）.Cell proliferation was detected by MTT array.Cells were cultured in the appropriate concentration of MTA for 4 weeks,and then stained by Alizarin red to detect their mineralized nodule formation capacity.The cells were cultured with the appropriate concentration of MTA and collected after 12,24,36,48 h.The mRNA expression of ALP,BSP,OC and DSP after the treatment of MTA were detected by quantitative PCR.Results DPSCs were positive for STRO-1.The capacity of 0.20 g/L MTA promoting the proliferation of DPSCs was stronger than other concentrations.After 4 weeks,the mineralized nodules of DPSCs were observed after alizarin red staining.The PCR showed that with increasing induction time,the expression levels of DSP and OC were up-regulated.But that of ALP and BSP was increased first and then decreased.Conclusions In this study,MTA can promote the proliferation of DPSCs at 0.02,0.20,2.00 g/L concentration.It can induce odontoblast differentiation effectively by 0.20 g/L MTA.