| Part I.Establishment of the diabetes mellitus rat model anddetection the level of DNA methylation in periodontal ligamentObjective: To establish the diabetes mellitus(DM)rat model and check out the level of DNA methylation in periodontal ligament.Methods: 8-week-old male Sprague-Dawley rats were randomly divided into 2 groups: streptozotocin(STZ)-induced diabetes mellitus group and vehicle citrate buffer-induced group.After successfully established the DM rat model,we first collected mandibular bones from 18-week-old diabetic rats and detected the bone mass using microCT analysis.Next,to assess the DNA methylation status of rat periodontal ligaments,we measured 5-mC by immunohistochemistry at postnatal week 18.Results:MicroCT analysis demonstrated a marked reduction in bone mass as well as trabecular bone volume in alveolar bones of diabetic rats compared with control littermates.Notably,analysis of histological sections revealed that 5-mC expression was obviously higher in diabetic rats compared with normal littermates.Conclusion: These data were consistent with clinical observations and suggested that cells exposed tohigh glucose(HG)may result in a reduction in bone mass and density by increasing the levels of DNA methylation in periodontal ligament tissues.Part II.Characterization of human periodontal ligament stem cells Objective: Cultivation and identification of human periodontal ligament stem cells(hPDLSCs).Methods: Healthy premolars were extracted from patients(n= 12,14–18 years of age)with written consent signed by parents during orthodontic treatment.Periodontal ligaments,collected from the middle third of the root,were cultured in alpha-Modified Eagle's Medium.Isolated h PDLSCs were identified by pluripotent examination,immunofluorescence and flow cytometric analysis,in which hematopoietic and other precursor contamination could be excluded.Results: After being separately cultured in osteogenic or adipogenic medium for 21 days,mineralized nodules were stained with alizarin red solution and oil droplets were stained with oil red O solution.hPDLSCs which were cultured in neurogenic media for 2 hours formed axon-like structures.Immunocytochemical analysis showed that isolated hPDLSCs were negative for CK14 and positive for vimentin.The results from flow cytometric analysis showed stromal origin of these isolated cells.Conclusion: These results revealed the stromal origin of these isolated cells with mesenchymal stem cell characteristics and the absence of hematopoietic and other precursor contamination.Part III.Effect of DNA methyltransferase inhibitor on theosteogenic potential of hPDLSCs under HG conditionObjective: To explore the effects of DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine(5-aza-dC)on the osteogenic potential of hPDLSCs under HG condition.Methods: cells were exposed to either5.5 mM(Control,normal glucose)or 30 mM glucose(high glucose: HG)for 4 days,and the relative mRNA expression of DNMT1,DNMT3 a and DNMT3 b was analyzed using quantitative RT-PCR.Moreover,global DNA methylation analysis was at applied at 3 and 7.ALP activity at day3 and 5,alizarin red staining and quantitative calcium measurement at day 14 in HG condition?5-aza-dC?HG and 5-aza-dC and control in mineralization media was respectively investigated.The expression of osteoblastic markers(ALP?OCN/OCN?OPN/OPN?OSX/OSX)in the mentioned four groups were measured by real-time RT-PCR and Western blot.Results: Treatment with HG resulted in a significant increase in expression of DNMTs.Both in the control group and the HG group,treatment of cells with 1 ?M 5-aza-dC led to a reduction in methylation.Cells cultured in the presence of 1 ?M 5-aza-dC exhibited robust ALP activity compared with the control group at day 3,and was further evaluated at day 7.Similarly,alizarin red staining showed that treatment of cells with 5-aza-dC resulted in substantially higher levels of stainingthan in the control group,whereas HG conditions resulted in reduced mineralization nodule formation by hPDLSCs.Consistent with the above results,treatment with 5-aza-dC led to higher expression levels of these osteogenic-related genes than in controls.Furthermore,cells exposed to HG showed a significant decrease in expression compared with other groups,and 5-aza-dC administration resulted in recovered expression levels under HG conditions.In addition,Western blot analysis confirmed the expression changes of OPN,ALP and OSX at the protein level.Conclusion: HG increases the DNA methylation level in hPDLSCs.5-aza-dC rescues the osteogenic differentiation capacity of h PDLSCs under HG conditions.Part IV.The canonical Wnt signaling pathway is involved in5-aza-dC-induced osteogenic differentiation of hPDLSCs under normal and HG conditions.Objective: To explore the canonical Wnt signaling pathway involvement in 5-aza-dC-induced osteogenic differentiation of h PDLSCs under normal and HG conditions.Methods: hPDLSCs were incubated with osteogenic medium for 3 or 7 days under normal or HG conditions,the protein levels of non-phospho(active)?-catenin(Ser33/37/Thr41),p-GSK-3?(Ser9)and Lef1 were detected by Western blot analysis.We then used rhDKK1 to block canonical Wnt signaling pathway activity andconveyed Western blot analysis to detect the expression levels of osteogenic differentiation related proteins.Results: hPDLSCs exposed to30 mM glucose showed reduced activity of the canonical Wnt signaling pathway compared with control groups at day 3 and 7.Furthermore,the expression of these Wnt related proteins was increased significantly by5-aza-dC administration compared with control cells.in the presence of rhDKK1,5-aza-dC induced expression levels of osteogenic differentiation-related proteins were decreased.This was also observed in the presence of HG.Conclusion: Together these results indicate that5-aza-dC promotes osteogenic differentiation of hPDLSCs via the canonical Wnt signaling pathway. |
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