Invesgation Of Estrogen On Osteogenic Differentiation Of Human Periodontal Ligament Stem Cells Rugulating Through Wnt Signaling Pathway

Part Ⅰ:Culture,purification and identification Human periodontal ligament stem cells in vitro.Objective:The molecular biology technique was used to culture,purify and identify human periodontal ligament stem cells（hPDLSCs）in vitro,and provide the research object for the second part of the experiment.Methods:The periodontal ligament tissue in the middle of the root of fresh premolar was scraped and digested by type I collagenase,and Primary hPDLSCs were isolated and purified by limiting dilution assay.Flow cytometry（FCM）was used to detect the expression of surface antigens related to mesenchymal stem cell.Alizarin red S staining after osteogenic induction and oil red O staining after adipogenic induction to detect the multidirectional differentiation potential of the cells.Detection of cell proliferation by CCK-8 method.Results:1.The primary cells digested by type I collagenase and the cells isolated and purified by limiting dilution assay showed a long fusiform shape similar to that of fibroblasts,which grew like a vortex around the tissue mass.2.The results of flow cytometry showed that the expression of Stro-1,CD146,CD90,CD29 was positive and the expression of CD31,CD45 was negative,which suggested that the cultured cells are derived from mesenchymal cells and are not contaminated by endothelial cell lines.3.The formation of irregular calcium nodules was found 21 days after osteogenesis induction under microscope,and a large number of round red staining lipid droplets were formed in the cells after 21 days of lipid induction under microscope,which proved that the cultured cells had the potential of osteogenic differentiation and adipogenesis differentiation.4.The CCK-8 method showed that cell grew in"S"shape,which had strong proliferative ability in vitro.Conclusion:Periodontal ligament stem cells cultured by enzyme digestion tissue block method and limited dilution cloning method had the potential of osteogenic differentiation and adipogenesis differentiation and MSCs characteristics,and had strong proliferative activity.Part Ⅱ:Invesgation of estrogen on osteogenic differentiation of human periodontal ligament stem cells rugulating through Wnt signaling pathwayObjective:1.Investigation of the change of noncanonical Wnt/Ca²⁺signaling pathway in estrogen-mediated osteogenic differentiation of hPDLSCs.2.To research the regulation of estrogen on osteogenic differentiation of hPDLSCs through non-canonical Wnt/Ca²⁺signaling pathway and canonical Wnt signaling pathway.3.To investigate the interplay between the noncanonical Wnt/Ca²⁺signal pathway and the canonical Wnt signal pathway within estrogen mediated in the process of the osteogenic differentiation of hPDLSCs.Methods:The research object is the third generation of hPDLSCs,and they were split into 6 groups:Blank Group,Control group,Estrogen Group（estrogen intervention）,L690,330 Group(estrogen intervention after inhibiting noncanonical Wnt/Ca²⁺pathway),XAV939 group（estrogen intervention after inhibiting canonical Wnt pathway）,L690,330+XAV939 group(estrogen intervention after inhibiting noncanonical Wnt/Ca²⁺pathway and canonical Wnt pathway).They were each given cultured of Osteogenic induction.Using fluo-4 fluorescence probe detected the intracellular Ca²⁺concentration.The mRNA expression level of CaMKII、NLK that is noncanonical Wnt/Ca²⁺signal pathway marker gene,the expression ofβ-catenin that is canonical Wnt signal pathway marker gene and RUNX2,OCN that is osteoblast-associated gene were detected by Real-time PCR（RT-PCR）.Alkaline Phosphatase（ALP）staining and ALP activity were used to detect the expression of ALP at morphologic and molecular levels,Meanwhile,Alizarin red staining observe the develepment of calcium nodules.Results:1.After 7 days of hPDLSCs induction and culture,the results of Fluo-4 fluorescence probe indicated that the concentration of Ca²⁺increased after osteogenic induction of hPDLSCs（P<0.01）,and the concentration of Ca²⁺increased further after the addition of estrogen（P<0.01）.After 7 and 14 days of hPDLSCs osteogenesis induction,the RT-PCR test results indicated that the mRNA expression of CaMKII and NLK in Control group was significantly increased than that in Blank group（P<0.001）,and the mRNA expression level of CaMKII,NLK in E2 group was significantly higher than that in Control group（P<0.05）,and the difference was more significant with the increase of induction time（P<0.01）.2.After 7 days of hPDLSCs osteogenesis induction,the results of ALP staining showed that there was no significant difference between L690,330 group and XAV939 group compared with E2 group,while L690,330+XAV939 group was lighter than E2 group.the ALP activity test showed that the expression of ALP in L690,330 group and XAV939 group was lower than that in E2 group,but there was no significant difference between L690,330 group and XAV939group compared with E2 group（P>0.05）,but the expression level of ALP in L690,330+XAV939 group was significantly lower than that in E2group（P<0.001）.After 7 and 14 days of hPDLSCs osteogenesis induction,the RT-PCR test results indicated that the mRNA expression of RUNX2,OCN in L690,330 group and XAV939 group was lower than that in E2 group,but there was no significant difference between L690,330 group and XAV939 group compared with E2 group（P>0.05）,but the mRNA expression level of RUNX2,OCN in L690,330+XAV939group was down-regulated that in E2 group（P<0.001）.The formation of calcium nodules in L690,330 group and XAV939 group was lower than that in E2 group,but there was no significant difference Compared with E2 group（P>0.05）,the calcium nodule formation in L690,330+XAV939group was significantly lower than that in E2 group（P<0.001）.3.After 7days of hPDLSCs induction and culture,the results of Fluo-4fluorescence probe indicated that the intracellular concentration of Ca²⁺in L690,330 group was down-regulated that in E2 group（P<0.05）,the intracellular concentration of Ca²⁺was increased than that in E2group（P<0.05）,and the intracellular Ca²⁺concentration in L690,330+XAV939 group was lower than that in E2 group（P<0.01）.After 7 and14 days of hPDLSCs osteogenesis induction,the RT-PCR test results indicated that the mRNA expression level of CaMKII,NLK in L690,330group was significantly lower than that in E2 group（P<0.001）,but the mRNA expression level ofβ-catenin was significantly up-regulated compared with E2 group（P<0.001）.The mRNA expression level of CaMKII,NLK in XAV939 group was up-regulated than that in E2group（P<0.01）,but the mRNA expression level ofβ-catenin in XAV939group was significantly lower than that in E2 group（P<0.001）.The mRNA expression level of CaMKII,NLK,β-catenin in L690,330+XAV939 group was significantly lower than that in E2 group（P<0.001）.Conclusion:1.The noncanonical Wnt/Ca²⁺signaling pathway is involved in the regulation of osteogenic differentiation of hPDLSCs mediated by estrogen.2.Estrogen promotes osteogenic differentiation of hPDLSCs through the non-canonical Wnt/Ca²⁺signaling pathway and the canonical Wnt signaling pathway.3.The noncanonical Wnt/Ca²⁺signaling pathway and the canonical Wnt signaling pathway play an interactive role in estrogen-mediated osteogenic differentiation of hPDLSCs.