| The maintenance and regulation of normally quiescent stem cell populations is tightly controlled by the local microenvironment, according to the requirements of the host tissue. It remains unclear that the change of biological behaviour and regeneration capacity in DPSCs under inflammatory microenvironment.The mechanism of such change remains to be studied.Part ⅠThe biology behavior of the normal dental pulp stem cells (n DPSCs) and the dental pulp stem cells from early inflammation environment(i DPSCs)Aim:To test the role of inflammatory microenvironment played on the DPSCs.Methods:l)The cells from the normal dental pulp and the inflammation dental pulp were isolated and purified by limited dilution technique.2)We measured the expression of TNF-α and IL-1β by western blot and real time RT-PCR.3)The expression of mesenchymal stem cell markers of n DPSCs and i DPSCs were measured by FCM and immunofluorescence assay.4)We tested the proliferation ability of cells by MTT and CFU. Results:The expression of TNF-α and IL-1β in iDPSCs was significantly up-regulated in i DPSCs (P<0.05), i DPSCs expressed the same surface markers as mesenchymal stem cells.The proliferation rate of iDPSCs was higher than that of n DPSCs (P<0.05).As compared with the nDPSCs.Conclusion:In this study we successfully isolated DPSCs from the inflammation dental pulp,and DPSCs have higher proliferation potential in the inflammation environment.Part ⅡThe research of odonto/osteogenic differentiation capacity of n DPSCs and i DPSCs.Aim:To investigate the change of odonto/osteogenic differentiation capacity of DPSCs in the inflammation microenvironment.Methods:The expression of RUNX2/RUNX2, DSP/DSP, DMP1/DMP1, OSX/OSX, OPWOPN, BSP/BSP, OCN/OCN in both n DPSCs and i DPSCs was tested by real time RT-PCR and Western blot.Results:Real-time RT-PCR and western blot showed the expression of RUNX2/RUNX2, DSP/DSP, DMP1/DMP1, OSX/OSX, OPN/OPN, BSP/BSP, OCN/OCN was significantly enhanced in i DPSCs,as compared with nDPSCs.The expression was was down-regulated gradually with the passaging of iDPSCs.Conclusion:The differentiation potential of DPSCs was increased in the inflammatory microenvironment. Part ⅢThe roles of Wnt signaling pathway in the odonto/osteogenic of n DPSCs and i DPSCsAim:To explore the mechanism of Wnt signaling pathway on the odonto/osteogenic differentiation of DPSCs in the inflammatory microenvironments.Methods:l) The expression of β-catenin, GSK3β as well as CaMKII, NLK was tested by western blot following culturing in osteogenic medium for7d.2) The cytoplasm, nucleoprotein or whole-cell protein were extracted following culture in LiCl or DKK1for7d.The expression of β-catenin, CaMKII, NLK, LEF-1and Cyclin D1was investigated through Western Blot.3)And the RUNX2, OCN and DSPP mRNA expression level was detected simultaneously.Results:1) The inflammatory microenvironments could induce the activation of Wnt signaling pathway.2) The expression of the β-catenin, CaMKⅡ, NLK, LEF-1and Cyclin Dl was increased following LiCl treatment,as well as the osteogenic differentiation potential.3)DPSCs exposed to DKK-1for7d showed decreased expression levels of β-catenin, CaMKII, NLK, LEF-1, Cyclin D1in the group of i DPSCs. The expression of RUNX2, OCN, DSPP mRNA was decreased following DKK1treatment.Conclusion:Wnt signaling pathway regulated the odonto/osteogenic differentiation potential of DPSCs in inflammatory microenvironment. |
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