| Background:As the public pays more and more attention to oral problems,scholars also pay more attention to the repair of tooth loss.At present,it is generally believed that tissue engineering teeth are the best for the restoration of missing teeth.However,the process of tooth development is very complex,finding the appropriate seed cells is essential.Extracranial neural crest-derived mesenchymal stem cells are considered to be the progenitor cells of maxillofacial hard tissue development.Mesenchymal stem cells are formed after the migration of embryos from cranial neural crest-derived cells to the first gills at the early stage of development,then differentiate into other tissues in maxillofacial region.p75 neurotrophic factor receptor(p75NTR)is a low affinity receptor for nerve growth factor.And it is one of members of the tumor necrosis factor superfamily.It plays a vital role in the nervous system.In recent years,more and more studies have also proved it can regulate the development of jaw and positively enhance the mineralization and differentiation ability of mesenchymal stem cells.Our research group found that p75 NTR may further regulate tooth development by affecting the transcriptional activity of homologous box transcription factors.Then,we found that Mage-D1 may be an important synergistic factor downstream of p75 NTR,so the hypothesis of p75NTR-Mage-D1-Dlx/Msx signaling pathway is put forward.Purpose:This study aims to confirm the binding of Mage-D1 with activated p75 NTR,and the roles in mineralization of ectomesenchymal stem cells(EMSCs).Methods:Part I:EMSCs of E19.5d SD rats were obtained by tissue block adherence method,and cultured in vitro.And the cells were identified by flow cytometry.The proliferation ability of the mesenchymal stem cells compared after 100ng/m L NGF stimulation and lentivirus interference with Mage-D1 by CCK-8.Part Ⅱ:The optimal concentration of Mage-D1 binding to p75 NTR was screened by immunofluorescence.Based on this condition,the proximity ligation assay was used to detect the real-time binding of Mage-D1 and p75 NTR stimulated by 100ng/m L NGF in mesenchymal stem cells.And compare the binding strength after 100ng/m L NGF stimulation and lentivirus interference with Mage-D1.ARS and ALP staining were used to compare the mineralization differences caused by different binding intensities.RT-PCR to detect m RNA levels of ALP,Runx2,OCN,BSP,OPN,Msx1 and Dlx1,Western blot to detect the protein expression levels of ALP,Runx2,BSP,Col1,Msx1 and Dlx1.Results:Part I: E19.5d EMSCs were successfully obtained and cultured.The cells obtained by flow cytometry were CD44(98.0%),CD90(98.2%),CD29(97.7%),CD146(97.9%),CD105(97.8%),CD45(0.3%).There were no significant differences in cell proliferation ability after 100ng/m L NGF stimulation and lentivirus interference Mage-D1 by CCK-8.Part Ⅱ: The optimal concentration for immunofluorescence of Mage-D1 and p75 NTR is Mage-D1 1:50 and p75 NTR 1:100;the proximity ligation assay detects,the binding of Mage-D1 and p75 NTR increased gradually with the addition of NGF time,and most of them were located in the cytosol and very few in the nucleus.The binding intensity after100ng/m L NGF stimulation was the strongest,after lentivirus interference Mage-D1 was the weakest.The results of ARS and ALP staining showed that the mineralization staining of 100ng/m L NGF stimulated group was the strongest and the lentivirus Mage-D1 was the weakest.RT-PCR detected the m RNA levels of ALP,Runx2,OCN,BSP,OPN,Msx1 and Dlx1 showed that the group by 100ng/m L NGF has the strongest expression,and the lentivirus interfered with Mage-D1 were the weakest.The Western blot results was consistent with RT-PCR.Conclusion:In this study,the binding of p75 NTR to Mage-D1 increased with time after 100ng/ml NGF activation,while the binding of Mage-D1 to p75 NTR decreased when Mage-D1 was interfered by lentivirus.It was also found that the osteogenic differentiation ability of EMSCs changed with the binding strength of Mage-D1 to p75 NTR. |
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