Effects Of ERα On The Proliferation And Differentiation Of Human Stem Cells From Apical Papilla

Estrogen receptor(ER),known as one of the members of nucleus receptor(NR),includes ERa and ERβ.Mesenchymal stem cells can express both ERα and ERβ in which ERa is preferred.As the key regulator of hard tissue metabolism,ER has been shown to be activated by its main ligand 17β-estradiol to regulate cell growth,proliferation and differentiation.Previous studies have suggested that 17β-estradiol can enhance the differentiation of stem cells from apical papilla(SCAPs).However,the effects of ERa on the proliferation and differentiation of SCAPs and the potential mechanism remain unclear.PartⅠ.Culture and identification of stem cells from apical papillaObjective:To obtain and identify the stem cells from apical papilla.Methods:Non-carious third molars with open apex were collected in Oral Surgery Department of Jiangsu Provincial Stomatological Hospital after the informed consent was obtained.SCAPs were obtained by enzyme-digestion method and primary culture cells were puriified by limited dilution.Cell morphology was observed under the inverted microscope.The origin of isolated SCAPs was determined by flow cytometry(FCM).Results:The isolated SCAPs presented the typical fibroblast-or spindle-like morphology.Flow cytometry results revealed that the expressions of STRO-1,CD73,CD90 and CD 105 were positive,but CD34 and CD45 were negative in these isolated stem cells.Conclusions:The isolated polyclonal apical papilla cells are derived from mesenchymal stem cells.Part Ⅱ.Effects of ERa on the proliferation and odonto/osteogenic differentiation of stem cells from apical papillaObjective:To investigate the effects of over/low-expressed ERa gene on the proliferation and odonto/osteogenic differentiation of SCAPs.Methods:The viruses of low expression(PLKO.1-sh-ERα)and over expression(GFP-ERα)of ERa were transfected into SCAPs respectively,then the protein and mRNA level of ERα were detected.The proliferation of transfected SCAPs by low/over expression viruses was measured by cell counting kit-8(CCK8)and FCM.ALP activity,alizarin red staining and quantitative calcium measurement were investigated.Furthermore,the expression of odonto/osteogenic markers(OSX,DMP1/DMP1,DSPP/DSP,RUNX2/RUNX2,OCN/OCN)was detected by real time RT-PCR and western blot.Results:The protein level and mRNA level of ERα were down-regulated in the ERαlow-expression group and up-regulated in the ERa over-expression group.ERa had no effect on the proliferation of SCAPs.The ERa low-expression significantly decreased the ALP activity and calcified nodules formation of SCAPs,and down-regulated the expression of odonto/osteogenic markers(OSX,DMP1/DMP1,DSPP/DSP,RUNX2/RUNX2,OCN/OCN)at both mRNA and protein levels.However,the results in the ERa over-expression group were opposite.Conclusions:Transfectionof the low/over-expression of ERainto SCAPs were successful.ERa had no effect on the proliferation of SCAPs.Over-expression of ERa enhanced their odonto/osteogenic differentiation,while down-expression of ERα...,suggesting that ERa could modulate the committed differentiation of SCAPs.Part Ⅲ.MAPK pathway involvement in ERα-mediated odonto/osteogenic differentiation of SCAPsObjective:To investigate the mechanism of MAPK pathway involvement in the ERα-based odonto/osteogenic differentiation of SCAPs.Methods:The expression of MAPK pathway-related proteins(p-ERK,ERK,p-p38,p38,p-JNK,JNK)and downstream transcription factors was investigated by western blot in cytoplasm and nucleus of transfected SCAPs by low/over-expression ERa viruses.Results:The level of phosphor-JNK and phosphor-ERK was activated but.the level of phosphor-p38 was not affected.Moreover,the expression of downstream transcription factors wasalso activated.Conclusions:ERa enhanced the odonto/osteogenic differentiation of SCAPs via JNK and ERK MAPK pathway.