Role Of Wnt/β-catenin Signaling Pathway In Osteogenic Differentiation Of Human Periodontal Ligament Stem Cells In The Microenvironment Of Estrogen

Part Ⅰ.Isolation,culture and identification of human periodontal ligament stem cells IObjective:Isolation,culture and identification of human periodontal ligament stem cells(hPDLSCs),and to provide seed cells for the follow-up experiments.Methods:The periodontal ligament tissues of the middle third of the roots about the premolar teeth were scraped,and the primary periodontal cells werecultured by enzymatic digestion combined with tissue explant method.Cloningand purification of human periodontal ligament stem cells by limited the finite dilution method.Detection of cell proliferation by CCK-8,and the expression of cell surface antigens such as CD 146,Stro-1 and CD45 were detected by flow cytometric analysis.The ability of multi-directional differentiation of the hPDLSCs were identified by alizarin red staining and oil red O staining.Results:1.The cells cultured by the enzyme digestion tissue block and the limited dilution method were adhered cells,spindle or fusiform shaped,To arrange in clusters or swirls;2.The results of flow cytometry showed that the positive expression of CD146 and Stro-1,and negative expression of CD45;3.The osteosynthesis and lipid induction of 21d,alizarin red and oil red O staining were positive,confirming the cell’s multidirectional differentiation ability;4.The results of CCK8 method showed that cells had strong proliferation ability in vitro;Conclusion:The isolated cells were hPDLSCs.Part Ⅱ.Role of Wnt/p-catenin signaling pathway in osteogenic differentiation of human periodontal ligament stem cells in the microenvironment of estrogenObjective:1).To explore the effect of estrogen on Osteogenic differentiation of hPDLSCs;2).To investigate the effect of estrogen on the Wnt/P-catenin signaling pathway in hPDLSCs.3).Using Wnt3a/Xav939 to activate/inhibit the Wnt/β-catenin signaling pathway,to explore the regulation effect of Wnt/β-catenin signaling pathway on the osteogenic differentiation of hPDLSCs.Methods:Selecting the P3 hPDLSCs with good growth status as the research object,Four groups were divided according to different processing factors:Control,10-7M E2,10-7M E2+ 100ng/ml Wnt3a,10-7M E2+ 5×10-3M Xav939.After 7 days of osteogenesis induction,qPCR was used to detect the expression of β-catenin,CyclinDl,ALP,Runx2 and OCN at the gene level;Western blot was used to detect the expression of(3-catenin,GSK3β,P-GSK3β,CyclinD1,Runx2 and OCN at protein level;After 1day,3days,5days,7 days of osteogenesis induction,The activity of alkaline phosphatase(ALP)was detected.Results:1).After 7 days of osteogenesis induction,qPCR showed that the expression of ALP,Runx2 and OCN of hPDLSCs in E2 group was significantly higher than that of Control group(P<0.001);Western Blot showed that the expression of Runx2 and OCN of hPDLSCs in E2 group was significantly higher than that of Control group(P<0.05);After 1day,3 days,5 days,7 days of osteogenesis induction,The ALP activity test showed that:the ALP levels in E2 group were higher than those in Control group(P<0.05),It shows that E2 promotes osteogenesis of hPDLSCs.2).After 7 days of osteogenesis induction,The results of qPCR showed that the gene levels of β-catenin.CyclinD1 were obviously increased,wllich is same with results of Western blot(P<0.001);Western blot was found that the protein levels of P-catenin、P-GSK3β、CyclinDl were increased in E2 group(P<0.05).At the same time,the levels of GSK3β was decreased in E2 group(P<0.05).It indicates that E2 activates the Wnt/β-catenin signal pathway.3).After 7 days of osteogenesis induction,The results of qPCR showed that the gene levels of P-catenin、CyclinD1 were lower than those in group E2(P<0.001);Western blot was found that the protein levels of β-catenin、P-GSK3β、CyclinD1 in E2 + Wnt3a group were higher than E2 group,E2 +Xav 939 group(P<0.05),At the same time,the GSK 3β expression was lower than E2 group,E2 + Xav 939 group(P<0.05).β-catenin、P-GSK3β、CyclinD1 were lower expression in E2 + Xav939 group than in E2 group,while GSK3β was higher than E2 group(P<0.05).This shows that Wnt3a/Xav939 activates/inhibits the Wnt/β-catenin signal path in hPDLSCs.After 1day,3 days,5 days,7 days of osteogenesis induction,The ALP activity test showed that:The ALP expression of E2 + Wnt3 a group was higher than that of E2 group,E2 +Xav939 group(P<0.05),The ALP in E2 group was higher than E2 + Xav939 on 1 day and 3 days(P<0.05),and there was no difference in 5days,7days and E2 + Xav 939(P>0.05).After 7 days of osteogenesis induction,The results of qPCR showed that the gene levels of ALP,Runx2 and OCN in E2 + Wnt3a group were higher than E2 group,E2 + Xav 939 group(P<0.001),and there was no statistical difference between E2 and E2 + Xav 939 groups(P>0.05);Western blot was found that the protein levels of Runx2,OCN in E2 + Wnt3a group were higher than E2 group,E2 + Xav 939 group(P<0.05),and there was no statistical difference between E2 and E2 + Xav 939 groups(P>0.05).The results show that the activation of Wnt/β-catenin signal pathway can stimulate the hPDLSCs osteogenesis differentiation mediated by estrogen,while the inhibition of Wnt/β-catenin signal pathway can not significantly decrease the hPDLSCs osteogenesis differentiation mediated by estrogen.Conclusion:1.Wnt/β-catenin signaling pathway is involved in the regulation of osteogenesis differentiation in hPDLSCs2.Estrogen activates the Wnt/β-catenin signaling pathway in hPDLSCs and stimulates the osteogenesis differentiation differentiation of hPDLSCs3.Wnt/β-catenin signaling pathway is involved in the osteogenesis of hPDLSCs under the action of estrogen.Wnt3a activates the signal pathway of Wnt/β-catenin and further enhance the osteogenesis of hPDLSCs.Xav939 can inhibit the Wnt/β-catenin signaling pathway in estrogen-mediated environments,but does not obvious inhibitory effect on osteogenic differentiation of hPDLSCs...