| Vital pulp therapy which includes pulp capping and pulpotomy, is mainlyused for curing reversible injury of dental pulp, aiming to maintain vitality andfunction of dental pulp-dentin complex. In order to achieve a satisfactorytreatment effect of vital pulp therapy, proper pulp capping material is needed toisolate the pulp from the infection, and to provide biological environment forpulp restoration. MTA can protect pulp tissue from outside stimulation, provideappropriate micro-environment for pulp restoration and play an important role onpromotion of dental pulp cells differentiation as well as dentin bridgeformation. Human dental pulp stem cells (hDPSCs) are undifferentiatedmesenchymal cells present in dental pulp tissue. They have multilineagedifferentiation abilities including differentiate into odontoblast and formingdentin. Recently, MAPKs signal pathway, which is of hot concerned researchareas in cell proliferation and differentiation as an evolutionarily conserved cellinteraction mechanism, play a role on various cell proliferation, differentiationand apoptosis.The article is based on vitro isolation, cultivation and identification of thehuman dental pulp stem cells. Moreover, MTT, Real-time PCR and Western blotare used to study effect of MTA on proliferation and differentiation of dental pulp stem cells and effects on total protein of MAPKs pathways andphosphorylated protein. Furthermore, inhibitors are used to block specificpathways of MAPKs to observe their impacts on MTA-induced hDPSCsdifferentiation. The study is supposed to detect the role of MAPKS pathway inthe process which MTA promotes differentiation related gene expression ofhuman dental pulp stem cells. Also, it is supposed to provide theoretical evidencefor further molecular mechanism study of MTA-induced hDPSCs differentiationduring dental pulp injury and self-restoration.This study is divided into three parts as follows.Part I The effects of MTA on the proliferation and differentiation of humandental pulp stem cellsObjectives To isolate and cultivate human dental pulp stem cells and toobserve the effect of MTA on survival, proliferation and differentiation of the cells.Materials and methods Dental pulp tissue of healthy third molars wascollected and the stem cells were isolated and identificated in vitro. MTA ofdifferent concentration was used to treat hDPSCs for different long time and itseffects on hDPSCs survival, proliferation and mRNA expressions of alp, dspp,bsp, ocn and colI were detected by MTT, Real-time PCR and Western blotmethods.Results HDPSCs are identified through cell morphologic observation andimmunocytochemistry test. MTA of high concentrations (20mg/ml and 10mg/ml)decrease survival rate of the cells obviously. MTA of lowconcentrations(2mg/ml and less than 2mg/ml) have no toxicity on cells and canpromote hDPSCs proliferation. Moreover, MTA of low concentrations can increasemRNA expressions of ALP,DSPP,BSP,OCN COLI .Conclusions HDPSCs were isolated and cultured successfully. MTA canpromote the proliferation and differentiation of hDPSCs.Part II The effects of MTA on protein expression of MAPKs in human dental pulpulp stem cellsObjectives To observe the protein expression of each MAPK pathway inhDPSCs treated with MTA alone and MTA with inhibitors.Materials and methods HDPSCs were randomly divided into MTAtreated groups for different treating time and control group. Western blot wasperformed to detect respective expression of total ERK1/2, JNK1/2, p38MAPKsand active form (phosphorylated protein). Then hDPSCs were randomly dividedinto blank control group, MTA group, and experiment group, in which the cellswere treated with both MAPKs inhibitors and MTA. Then respectively wedetected total protein and phosphorylated protein expression of the threepathways.Results As hDPSCs exposed to MTA with or without inhibitors for 2hours, the total protein levels of MAPKs remained constant, but threephosphorylated proteins changed obviously. MTA stimulated phos-ERK, phosp38and phos-JNK expression of cells treated with MTA for 15 mins. However,after having been treated with inhibitors of ERK, JNK and p38, allphosphorylated protein expression decreased greatly. The difference hasstatistical significance compared with control group .Conclusions MTA can not promote total protein expression of MAPKs,but it can increase expression of phos-MAPKs protein obviously. MAPKsinhibitors can inhibit phosphorylation of MAPKs specially.Part III The role of MAPKs pathway in MTA-induced differentiation ofhuman dental pulp stem cellsObjectives To observe the role of MAPKs in hDPSCs differentiation bydetecting mRNA expression of alp, dspp, bsp, ocn and colI after cells have been treated with inhibitors and MTA.MaterialMaterials and methods HDPSCs were randomly divided into blankcontrol group, masculine control group and experimental group. Cells weretreated with inhibitors for 1h and MTA for 12h. Real-time PCR was used todetect mRNA expression of alp, dspp, bsp, ocn and colI.Results Compared with the control group, mRNA expression of alp, dspp,bsp, ocn and colI in hDPSCs decreased obviously after treated with U0126.However, mRNA expression showed no changes after SB203580 and SP600125were added.Conclusions ERK pathway play an important role in the process of MTAinducedhDPSCs differentiation, while JNK and p38MAPK may not participatein the process.In conclusion, it is indicated that MTA could promote proliferation anddifferentiation of human dental pulp stem cells and activate the MAPKs pathway,which increased phosphorylation protein expression of MAPKs in hDPSCs.Moreover, ERK MAPK way was the one involved in differentiation of hDPSCsinto odontoblast-like cells induced by MTA. The results provide newexperimental evidence for molecular mechanism study of MTA-inducedhDPSCs differentiation, and for potential new methods of pulpitis therapy. |
PDF Full Text Request