周期性流体静压力对牙周膜干细胞分化的影响

摘要

目的 观察周期性流体静压力对人牙周膜干细胞(human periodontal ligament stem cells,hPDLSCs)分化的影响.方法 酶消化法分离人牙周膜细胞,采用单克隆法分离hPDLSCs.流式细胞仪检测细胞表面标志物的表达,结晶紫染色观察hPDLSCs克隆形成.hPDLSCs成骨、成脂诱导分化后采用茜素红、油红O染色,观察hPDLSCs的多向分化能力.利用自主研发的力学加载装置,对PDLSCs加载0~120 kPa的周期性流体静压力7 d,采用Real-time PCR检测hPDLSCs中过氧化物酶体增殖物活化受体-γ(PPAR-γ)、抗Runt相关转录因子2(Runx2)、碱性螺旋-环-螺旋转录因子(Scleraxis)及牙骨质蛋白1(CEMP1)的表达量.结果 单克隆法分离的hPDLSCs高表达干细胞表面标志物CD90(95.2%)、CD105(95.5%),而低表达CD45(1.3%)、CD34(1.36%).hPDLSCs具有克隆形成能力,并且成骨、成脂诱导后细胞出现红色钙结节和脂滴.经周期性力学刺激后,hPDLSCs中Scleraxis的表达显著高于对照组,而PPAR-γ、Runx2及CEMP1的表达与对照组相比无显著差异.结论 周期性力学刺激可以维持hPDLSCs向牙周膜韧带成纤维细胞分化的潜能.%Objective To observe the effect of mechanical stimulation on the differentiation of periodontal ligament stem cells in vitro.Methods Human periodontal ligament cells were isolated and cultured by enzymatic digestion.Monoclonal method was used to separate hPDLSCs.The surface marker of hPDLSCs was detected by flow cytometry.Crystal violet staining was used to observe the cell clones of hPDLSCs, and then induced into osteoblast and adipose cells.The potential differentiation was identified by Alizarin red and oil red O staining.Cyclic hydrostatic pressure was performed on hPDLSCs using independently developed mechanical loading device, the pressure is 0~120kPa and the frequency of load is 0.1Hz, and cells were treated with pressure 1h/d for consecutive 7 days.The mRNA expression levels of peroxisome proliferators-activated receptor-γ (PPAR-γ), runt-related transcription factor 2 (Runx2), Scleraxis and Cementum Protein 1(CEMP1) were measured by Real-time PCR.Results The hPDLSCs obtained by the limited dilution were positive for CD90 (95.2%), CD105 (95.5%) and negative for CD45 (1.3%), CD34 (1.36%).Single colonies were observed after crystal violet staining.Alizarin red-positive mineral deposits and Oil Red O-positive cells indicated that the cultured cells have osteogenic and adipogenic potential.After mechanical stimulation, the expression of Scleraxis was significantly higher than that in control group, whereas the expression of PPAR-γ, Runx2 and CEMP1 had no significant difference compared with the control group.Conclusion Cyclic hydrostatic pressure can maintain the potentials of hPDLSCs for periodontal ligament fibroblasts differentiation.