背景:本研究旨在研究各种Toll样受体(TLR)和C型凝集素受体(CLR)配体对人牙髓干细胞(hDPSC)成骨分化的影响。
方法:将hDPSC培养并用各种浓度(0.01、0.1、1.0和10µg/mL)的TLR或CLR激动剂(PG-LPS,大肠杆菌,LPS,聚(I:C),Pam3CSK4,Furfurman,和Zymosan)。通过MTT测定法测定细胞活力。通过碱性磷酸酶(ALP)活性检测TLR和CLR激动剂对hDPSCs成骨分化的影响,茜素红S染色,和VonKossa染色。此外,成骨相关基因的mRNA表达(ALP,COL1A1,RUNX2,OSX,通过RT-qPCR检查OCN和DMP1)。非参数分析用于统计分析。当p<0.05时,认为统计学上的显著差异。
结果:用TLR和CLR激动剂治疗与hDPSC集落形成单位能力的增加相关。与对照组相比,TLR和CLR激动剂通过降低ALP活性显著抑制hDPSCs的成骨分化,矿化结节形成,和成骨相关基因的mRNA表达水平(ALP,COL1A1,RUNX2,OSX,OCN和DMP1)。TRIF而非Akt信号传导的抑制挽救了TLR和CLR激动剂减弱hDPSC矿化的作用。
结论:TLR或CLR的激活通过TRIF依赖性信号通路对hDPSC的成骨分化表现出抑制作用。
BACKGROUND: This study aimed to investigate the effects of various toll-like receptor (TLR) and C-type lectin receptor (CLR) ligands on osteogenic differentiation in human dental pulp stem cells (hDPSCs).
METHODS: hDPSCs were cultured and treated with various concentrations (0.01, 0.1, 1.0, and 10 µg/mL) of TLR or CLR agonists (PG-LPS, E.coli LPS, poly(I:C), Pam3CSK4, Furfurman, and Zymosan). Cell viability was determined by MTT assay. The effects of TLR and CLR agonists on osteogenic differentiation of hDPSCs were measured by alkaline phosphatase (ALP) activity, Alizarin Red S staining, and Von Kossa staining. In addition, the mRNA expression of osteogenesis-related genes (ALP, COL1A1, RUNX2, OSX, OCN and DMP1) was examined by RT-qPCR. A non-parametric analysis was employed for the statistical analyses. The statistically significant difference was considered when p < 0.05.
RESULTS: Treatment with TLR and CLR agonists was associated with an increase in hDPSCs\' colony-forming unit ability. Compared with the control group, TLR and CLR agonists significantly inhibited the osteogenic differentiation of hDPSCs by decreasing the ALP activity, mineralised nodule formation, and mRNA expression levels of osteogenesis-related genes (ALP, COL1A1, RUNX2, OSX, OCN and DMP1). The inhibition of TRIF but not Akt signalling rescued the effects of TLR and CLR agonist attenuating hDPSCs\' mineralisation.
CONCLUSIONS: The activation of TLRs or CLRs exhibited an inhibitory effect on osteogenic differentiation of hDPSCs via the TRIF-dependent signalling pathway.