Stathmin Regulating The Proliferation And Osteo-/odontogenic Differentiation Of Human Dental Pulp Stem Cells Through Shh Signaling Pathway

Background and objectionDental pulp stem cells are considered to be one of the key cells of tooth regeneration.When suffering from irritants caused by caries,abrasion,infection or other physical and chemical factors,dental pulp stem cells residing in pulp can be activated and induced to differentiate into odontoblast-like cells,and then secret dentine matrix and eventually form reparative dentine to prevent diseases progression and preserve the activity of pulp.The self-repair potential of dental pulp tissue provides a reliable biological basis for the study of pulp-dentine complex.Therefore,it is of great significance to understand the molecule mechanisms of odontoblast-like cells differentiation into dental pulp stem cells,and to find the key molecules involved in the formation of pulp-dentine complex and the construction of tissue engineered teeth.Stathmin is an important microtublue-associated protein which is related to mineralization of bone.Sonic hedgehog(Shh)as a classical signal pathway regulating tooth development,plays an important role in the differentiation and proliferation of human dental pulp stem cells.Our previous results indicated that the expression of Stathmin in normal dental pulp stem cells is higher than that in carious dental pulp stem cells.To date,the role of Stathmin in regulation of hDPSCs was poor understood and its’ possible molecule mechanisms remained unclear.Thus,the research was to investigate whether Shh signaling pathway is involved in Stathmin’s regulation to the proliferation and osteo-/odontogmic differentiation of hDPSCs.Materials and methodsHuman dental pulp stem cells were enzymatically separated from dental pulp tissues,cultured and passaged.Cell phenotype CD29、CD31、CD90、CD44、CD45 and CD 105 was detected by flow cytometric.HDPSCs multilineage differentiation potential was tested by osteogenic,adipogenic and chondrogenic differentiation.Immunofluorescence was used to reveal the location and expression of Stathmin in human dental pulp stem cells.Previously we designed the Stathmin-targeting sequence of oligonucleotides.The negative control consisted of a scrambled sequence with no homology to any human gene.The optimal conditions for lentivirus transfection was investigated.The knockdown of Stathmin both in mRNA and protein level after lentivirus transfection were confirmed by Realtime PCR and Western blot respectively.CCK8、flow cytometry analysis and Realtime PCR were used to detect the changes of hDPSCs’ proliferation and osteo-/odontogenic differentiation after the silencing of Stathmin.The expression changes of Shh and its downstream signaling molecules were detected by Realtime PCR and Western blot in both Stathmin-shRNA group and Stathmin-Ctrl group.Then,to elucidate the effects of Shh sinaling pathway on Stathmin’s regulation to dental pulp stem cells,we added puromorphamine,an activator of Shh signaling pathway.CCK8,flow cytometry analysis,Realtime PCR and Western blot were used to analyze the changes of proliferation and osteo-/odontogenic differentiation of hDPSCs after Shh signaling pathway was activated.ResultsWe successfully expanded human dental pulp stem cells from human dental pulp.The flow cytometry analysis showed that the hDPSCs expressed the mesenchymal cell surface protein.The positive rate of CD29、CD44、CD90、CD105(MSC-specific cell surface markers)was 99.84%、99.82%、99.72%、100%and the rate of CD31,CD45(hematopoietic surface molecule)was 0.07%、0.23%.The human dental pulp stem cells have osteogenic,adipogenic and chondrogenic potential of multilineage differentiation.Immunofluorescence showed that Stathmin was mainly expressed in the cytomembrane and cytoplasm of hDPSCs.Realtime PCR and Western blot showed that the specific Stathmin shRNA lentivirus was successfully constructed.HDPSCs in both silencing Stathmin lentivirus transfected group and negative control lentivirus transfected group were cultured in mineralized medium for 14 days.Real-time PCR analysis was used to detect the mRNA expression of ALP、BSP、OCN、DSPP.The data showed that the osteo-odontogenic differentiation markers ALP、BSP、OCN、DSPP expressed lower when silencing the Stathmin expression.CCK8 assays and cell cycle analysis showed that the proliferation activity decreased in Stathmin-shRNA group compared with Stathmin-Ctrl group.The expression of Shh and its downstream signaling pathway molecule PTCH1、SMO、GLI1 mRNA and protein were significantly lower in Stathmin-shRNA than those in Stathmin-shRNA group.HDPSCs were treated with Shh signaling pathway activator purmorphamine after transduced with lentiviruses that expressed shRNA-Stathmin.The results of Realtime PCR and Western blot showed that PTCH1,SMO and GLI1 expression were upregulated compared with shRNA-Stathmin control group,confirming that purmorphamine activated Shh signaling pathway in hDPSCs after transduced with Stathmin shRNA lentiviruses.The mRNA expression of ALP,BSP,OCN and DSPP in shRNA-Stathmin+PM group of hDPSCs was significantly higher than that in shRNA-Stathmin group after both cells cultured in mineralized medium for 14 days.CCK8 assays and cell cycle analysis showed that the proliferation rate of shRNA-Stathmin+PM group hDPSCs was higher than that of Stathmin-shRNA group.ConclusionsIn this study,we successfully isolated and cultured human dental pulp stem cells in vitro,which derived from mesenchymal tissue,and they possessed the clonogenic capacity and the potential to undergo odontoblastic,adipogenic and chondrogenic differentiation.Stathmin was expressed in the cytomembrane and cytoplasm of hDPSCs.Stathmin has a positive role in maintaining the proliferation and osteo-/odontogenic differentiation of human dental pulp stem cells.Stathmin may regulate the proliferation and osteo-/odontogenic differentiation of hDPSCs by Shh signaling pathway.