Effeects And Mechanism Of HDAC Inhibitor LMK-235 On Odontoblast Differentiation Of Dental Pulp Cells

BackgroundDental pulp cells might differentiate into osteoblasts,lipocytes,chrondrocytes and especially odontoblasts.In recent years,epigenetic regulation has been proven to play an important role in the differentiation of many cells.Acetylation and deacetylation controlled by the activities of histone acetyltransferases(HATS)and histone deacetylases(HDACs)are the common methods of post-translational histone modification.However,the effects of class Ⅱ HDAC,such as HDAC4,HDAC5 and their specific inhibitor on odontoblast differentiation of DPCs,and the precise molecular and epigenetic mechanisms behind this process remain unclear.LMK-235 is a human specific HDAC4 and HDAC5 inhibitor and in this study,we aimed to investigate how LMK-235 affects the proliferation and differentiation of the DPCs in vitro and identify the possible molecular mechanisms behind this process to provide new ideas for odontoblast differentiation of dental pulp derived cells and dentin regeneration.Research contentsChapter 1 The isolation and identification of DPCsMethods1.Tissue explants collagenase method was used to isolate and culture DPCs.2.Stem cell marker(CD29,CD44,CD90,CD45,CD31 and CD34)were tested by flow cytometry.Then multiple differentiation potential were also tested soon afterwards.Results1.Cells presented with long spindle-shaped fibrocyte-like and adherent growth.2.CD29,CD44,CD90 expression of DPCs were strongly positive while the expression of CD45,CD31 and CD34 were negative.3.DPCs could differentiate into osteoblasts,lipocytes,chrondrocytes detected by Alizarin red S staining,Nil red staining and Alcian blue staining,respectively.Chapter 2 Effects of LMK-235 on biological characteristics of DPCsMethods1.MTT was used to test effects of LMK-235 with different concentration on proliferation of DPSC..2.After cultured for 3 days,ALP,Runx2 and DSPP mRNA expression of DPCs were tested by qRT-PCR.3.HDAC4 and 5 protein expression were also detected by Western blot.Results1.LMK-235 at the concentration of 100nM did not affect the proliferation of DPCs.2.LMK-235 at the concentration of 100nM could upregulate ALP,Runx2,DSPP mRNA expression of DPCs.3.HDAC4 protein expression was downregulated by LMK-235.Chapter 3 Effects of LMK-235 on odontoblast differentiation of DPCsMethods1.Cells were devided into 4 groups:the control group,the LMK-235 group,the MI group and the MI+ LMK-235 group.ALP activity and Alizarin red S staining were carried out after cultured for 7 and 21 days,respectively.2.ALP,Runx2,DSPP and OCN mRNA and protein expression was tested by qRT-PCR and Western blot in DPCs cultured for 7,14,21 days,respectively.Results1.ALP activity and mineralized nodules formation of DPCs were increased by the effects of LMK-235.2.LMK-235 promoted the ALP,Runx2,DSPP and OCN mRNA and protein expression at the concentration of 100nM.Chapter 4 Mechanism of LMK-235 on odontoblast differentiation of DPCsPart 1 Histone acelytational mechanism of LMK-235 on odontoblast differentiation of DPCsMethods1.DPCs were devided into 4 groups and cultured for 10 days:the control group,the LMK-235 group,the MI group,the MI+LMK-235group.2.Histone expression:Histone H3,H3Ac,Histone H4 and H4Ac protein expression was tested by Western blot.3.The combination of H3K27Ac and DSPP promoter was detected by ChIP.Results1.Histone H3 acetylation was upregulated after LMK-235 treatment and mineralized induction.The H3Ac protein expression level was highest in MI+LMK-235 though the 4 groups.2.The combination of H3K27Ac and DSPP promoter was also highest in MI+LMK-235 compared with either the MI group or the LMK-235 group.Part 2 VEGF/AKT pathway took part in odontoblast differentiation of DPCs induced by LMK-235Methods1.DPCs were devided into 4 groups:the control group,the LMK-235 group,the MI group and the MI+LMK-235 group.VEGF,AKT,mTOR mRNA expression were tested by qRT-PCR and AKT、p-AKT protein level were detected by Western blot after cultured for 7,14,and 21 days,respectively.2.DPCs were devided into 3 groups afterwards:MI group、MI+LMK-235 groupand MI-LMK-235+Wortmannin group.ALP activity and Alizarin red S staining were carried out after cultured for 7 and 21 days,respectively,Gene and protein expressions of ALP,DSPP,Runx2,AKT,p-AKT and OCN were tested by qRT-PCR and Western blot after cells were treated for 7,14,and 21 days,respectively.Results1.VEGF,AKT,mTOR mRNA in DPCs were upregulated after mineralized induction and LMK-235 might promote this process.2.Wortmannin inhibited ALP activity and mineralized nudoles formation promoted by LMK-235.3.Wortmannin inhibited gene and protein expression of ALP,DSPP,Runx2 which might be promoted by LMK-235.Conclusions1.DPCs were successfully isolated,cultured and the multiple differentiation potential was also detected.2.LMK-235 at the concentration of 100nM promoted odontoblast differentiation of DPCs by upregulating ALP,Runx2,DSPP and OCN mRNA and protein expression.ALP activity and mineralized nodules were tested to be increased during this process.3.LMK-235 might promote odontoblast differentiation of DPCs via upregulating the acetylation of histone H3 and H4,while the combination of H3K27Ac and the promoter region of DSPP gene.In addition,VEGF/AKT signaling pathway was tested to take part in DPCs odontoblast differentiation promoted by LMK-235.