Over-Expression Of GPNMB Enhances Odontoblastic Differentiation Of Human Dental Pulp Stem Cell

Objective:Dental pulp stem cells(DPSCs)maintain the undifferentiated state of stem cells in the pulp tissue.Under certain inducing condition,they can differentiation into odontoblasts.Numerous studies have shown that different inducing factors can be used as regulators to participate in the regulation of DPSCodontogenic differentiation to a certain extent through related signaling pathways.In this study,we used Glycoprotein non-metastatic melanoma protein B(GPNMB)lentiviruses to transfect human dental pulp stem cells(hDPSCs)to observe the effects of GPNMB on odontoblastic differentiation of hDPSCs.Method:1.Healthy impacted teeth or permanent teeth that had to be removed due to orthodontic treatment were collected for the experiement.Enzymatic digestion combined with tissue explant culture was used to extract dental pulp cells from the dental pulp tissue.The cells were purified by single colony selection and limited dilution methods?.By adipogenic,mineralizing or ordontogenic differentiating induction fat droplet formation was detected by oil red O staining for lipid formation,and alizarin red staining was used to detect mineralized/dentate nature of cells forming mineralized nodules.In order to identify the extracted cell as DPSCs and estanblish the experimental cell model,protein immunohistochemistry was used to detect the expression of DSPP and DMP1,and RT-PCR was used to detect the mRNA expression of DSPP and DMP1.Meanwhile,the expression of GPNMB was detected by Western blotting and RT-PCR.2.Dental pulp stem cells more transfected by GPNMB lentivirus.The expression of DSPP,DMP1 and GPNMB were detected by Western blotting.3.The dental pulp stem cells transfected with GPNMB lentivirus were divided into control group and experimental and group,induced by mineralization,Alkaline phosphatase staining was used to compare the activity of alkaline phosphatase.RT-PCR was used to check the expression of DSPP,DMP1,GPNMB mRNA.Results:1.The expanded and cultured cells have the multiple differentiation potential hDPSCs,and the expression of DSPP and DMP1proteins and mRNA were detected during the process of mineralization induction,and the expression levels were enhanced with time.2.The expression of GPNMB protein and mRNA was detected in the process of hDPSCs mineralization induction.The expression increased with the induction time.3.When MOI=50,the transfection of dental pulp stem cells by GPNMB lentivirus was better,and the morphology of hDPSCs did not change significantly after transfection.The expression of GPNMB detected by Western blot was stronger than control group.4.After transfection of GPNMB lentivirus,mineralized was induced.In the experimental group,alkaline phosphatase activity and mRNA expression of DSPP,DMP1 and GPNMB were enhanced compared with the control group.Conclusion:The mRNA expression of DSPP and DMP1 was up-regulated after transfection of dental pulp stem cells by GPNMB lentivirus into mineralized induced.This indicates that GPNMB plays a positive regulatory role in the process of odontoblast differentiation of dental pulp stem cells.