The Role Of GSK3β In Tooth Mineralization Anomalies Induced By TNAP Deficiency

Hypophosphatasia(HPP) is a systemic genetic disease induced by the mutation of ALPL gene. Due to the decrease of ALPL gene encoded tissue nonspecific alkaline phosphatase(TNAP), abnormal metabolism of bone and tooth mineralization will be caused. Recent studies have found that ALPL encoded TNAP protein not only plays an important role in the mineralization process, but also may lead to the abnormal development of bone and tooth by affecting the biological behavior of stem cells. As an important basis for the maintenance of normal tissue development, mesenchymal stem cells play an important role in the tissue renewal and repair process. But the cell biological behavior of mesenchymal stem cells with DNA defects will determine the clinical manifestations of patients with genetic diseases.The research group found in the early stage that bone marrow mesenchymal stem cells(BMMSC) derived from HPP children have abnormal differentiation ability. At the same time, the decreased expression of Active-β-catenin suggested that Wnt/β-catenin signaling pathway may be involved in the functional change of stem cells under low ALP activity. However, the functional change of dental pulp stem cells and periodontal ligament stem cells which are closely related to the tooth development and mineralization has not been reported under the abnormality of ALPL gene.This experiment mainly includes the following parts: First, with the ALPL gene knock-out mice Akp2+/- as the model, the influence of partial knockout of ALPL gene on tooth hard tissues was observed. Second, through the lentiviral transfection method, the normal people derived ALPL gene of hPDLSC and h DPSC were knocked out to observe the influence of the ALPL gene knockout on stem cell functions and detect the changes of GSK3β and the related signaling pathways. Third, the influence of GSK3 phosphorylation inhibitor LiCl on the functions of hPDLSC and h DPSC after the knockout of ALPL gene as well as the recovery effect on the abnormality Akp2+/- mouse tooth and alveolar bone were observed to verify the function of GSK3β in the abnormal mineralization of tooth caused by the abnormal function of TNAP.Part I The effect of ALPL Knockout on Dental Hard Tissues in MiceObjectiveTo observe the effect of ALPL knockout on teeth and alveolar bones of mice.Method1. Use Micro-CT to scan the unilateral jawbones of wild-type and Akp2+/- mice and observe the changes in teeth and alveolar bones of Akp2+/- mice; Select the crown dentin of first molars for quantitative analysis of mineralization density at the tissue level.2. Observe the dentin mineralization of wild-type and Akp2+/- mice with scanning electron microscope.Results1. Micro-CT scan results showed that Akp2+/- mice had obvious defects in jawbones and alveolar bones and enlarged dental pulp cavity compared with wild-type mice;2. Scanning electron microcopy images showed that Akp2+/- mice had lower dentin mineralization and visible collagen fiber exposure compared with wild-type mice.ConclusionCompared with wild-type mice, Akp2+/- mice have some level of alveolar bone defects and incomplete dentin mineralization.Part II The Effect of ALPL Knockout on hPDLSC and h DPSC FunctionsObjectiveTo verify the effect of ALPL mutation on hPDLSC and hDPSC Functions.Method1. Culture hPDLSC and h DPSC from normal tissues in vitro and knock out ALPL genes in hPDLSC and h DPSC through lentivirus transfection; then use Western-Blot and alizarin red staining methods to detect the changes in osteogenic capacity of hPDLSC and h DPSC after ALPL knockout;2. Measure the expression of P-GSK3β in hPDLSC and h DPSC after ALPL knockout with Western-Blot.Results1. Western-Blot results showed that after ALPL knockout, both the expression of ALP protein in hPDLSC and h DPSC and other osteoblasts-related protein expression dropped; and DSP expression in h DPSC also reduced.2. Alizarin red staining results showed a decrease in osteogenic capacity of hPDLSC and h DPSC after ALPL knockout.3. Western-Blot results showed a reduced expression of P-GSK3β in hPDLSC and hDPSC after ALPL knockout.Conclusion1. Lentivirus transfection method can effectively knock out ALPL genes in hPDLSC and h DPSC, and the osteogenic capacity of hPDLSC and h DPSC is restrained after ALPL knockout;2. After ALPL knockout, the expression of P-GSK3β in hPDLSC and h DPSC is reduced to suggest that Wnt signaling pathway is inhibited.Part III The Effect of GSK3β Inhibitor on the Functions of hPDLSC and h DPSC after ALPL KnockoutObjectiveTo observe LiCl’s effect on the osteogenic capacity of hPDLSC and h DPSC after ALPL knockout and verify the function of GSK3β in the functional abnormality of stem cells caused by ALPL mutation.MethodApply osteogenic induction to hPDLSC and h DPSC after ALPL knockout and add GSK3β inhibitor LiCl in the process; after osteogenic induction for 21 days, observe the changes in osteogenic capacity of hPDLSC and h DPSC with alizarin red staining method.ResultAlizarin red staining results showed that the addition of LiCl in the process of osteogenic induction could greatly improve the osteogenic capacity of hPDLSC and h DPSC.ConclusionInhibiting GSK3β to activate Wnt signaling pathway could improve the osteogenic capacity of hPDLSC and h DPSC after ALPL knockout, and it is verified that ALPL mutation affects the osteogenic capacity of stem cells by GSK3β.Part IV The Function of GSK3β Inhibitor in Abnormality of Dental Hard Tissues in Akp2+/- MiceObjectiveTo observe the function of LiCl in abnormality of dental hard tissues in Akp2+/- mice.MethodInject LiCl(100mg/Kg) into Akp2+/- mice, compare them to normal Akp2+/- mice, and observe the changes in alveolar bone defects and dentin mineralization with Micro-CT scan and scanning electron microscope.Results1. Micro-CT scan results showed an obvious improvement in alveolar bone defects of the experimental group(mice with LiCl injection) compared with the control group;2. Scanning electron microscope images showed a great increase in dentin mineralization of the experimental group.ConclusionAn injection of GSK3β inhibitor LiCl could greatly improve the abnormalities in teeth and alveolar bones of Akp2+/- mice; it is proved that ALPL mutation may affect the function of tooth-derived mesenchymal stem cells through signaling pathway like GSK3β and eventually lead to mineralization abnormalities in teeth and periodontium development.