| Aim:The loss of teeth is an unavoidable problem for every person’s life.At present,the solution for the absence of teeth usually is: active denture,fixed bridge repair and implant.However,the lack of functional denture,fixed bridge repair need to prepare abutmen ts,dental implants need to accept implant surgery,so the above method is not the most ideal way to replace the teeth repair,therefore,tissue engineering teeth has become the majority of oral research workers to study the goal.Dental development is a complex process involving a large number of molecules and signaling pathways,including both enamel,dentin,cementum and pulp and other different organizations between the coordinated development,including incisors,fangs,before the development of different parts of molars and molars.Therefore,a clear molecular development mechanism in the process of molecular development,as a major problem to solve tissue engineering teeth.Cranial nerve derived epidermal mesenchymal stem cells can be used as carrier cells for studying molecular biological mechanisms during tooth development.Epithelial mesenchymal stem cells are considered to be the source cells of all teeth except enamel.These cells migrate from the cranial nerve crest to the maxillary and mandibula r distortions during embryonic development,interacting with the odontogenic epithelium to form the dental follicles and dental papillae,which are further differentiated into various dental tissues and form dental bone Circumferential membrane,alveolar bone,dentin and pulp.In the bud stage of tooth germ development,the signal of induced formation of teeth from the epithelium to mesenchymal,odontogenic epithelium can induce mesenchymal stem cells to form teeth,which is the classic epithelial-mesenchymal interaction theory.Cranial nerve crest-derived ectomesenchymal stem cells play an important role in this process.However,the precise molecular mechanism of tooth development is still unclear.Therefore,it is important to study the molecular biological mechanism of dental development during the development of tissue engineering teeth.P75 neurotrophic factor receptor(P75NTR)belongs to the tumor necrosis factor superfamily of the low affinity neurotrophic factor receptor.P75 NTR is the first isolated neurotrophic receptor and is highly expressed in neuronal cells during early development and is involved in the proliferation,migration,differentiation,survival and apoptosis of neural crest stem cells during embryogenesis.P75 NTR exhibits different bi ological effects in different cell lines.In our previous studies,it has been found that P75NTR-purified cranial nerve derived epidermal mesenchymal stem cells have neural,adult muscle,cartilage and osteogenesis Such as multi-directional differentiation.To explore whether P75 NTR is involved in the process of tooth development and how to regulate the process of tooth development,and to study its specific molecular biological mechanism,it is the scientific problem that this topic focuses on.Materials and Methods:Part Ⅰ:We first for the two key time of development of tooth germ 12.5 days and 19.5 days,through abdominal surgery,in the same SD pregnant mice to obtain embryonic development of 12.5 days and 19.5 days of cranial nerve crest origin Mesench ymal stem cells(ED12.5EMSCs and ED19.5EMSCs).Stem cell surface markers and P75 NTR were detected by flow cytometry at two different time points.Stem cells have strong ability of multi-directional differentiation.In order to further compare the different iation ability of cells at two different time points,the osteoblast-induced differentiation experiments of two kinds of cells were carried out.Real-time quantitative PCR and western blot Gene ALP,Run X2 were tested.By alkaline phosphatase staining,more intuitive comparison of ED12.5EMSCs and ED19.5EMSCs osteogenic differentiation differences.Part Ⅱ:Clarify the role of P75 NTR in the development of tooth germ.To demonstrate that P75 NTR was involved in tooth germ development and to determine its role i n tooth germ development,we further expressed P75 NTR by silencing P75 NTR and lentiviral transfection techniques using RNA interference techniques,and identified by cell immunofluorescence staining The expression efficiency of P75 NTR in ED19.5EMSCs was detected by real-time quantitative PCR and western blot.The expression of ALP and Runx2 were detected by real-time quantitative PCR and western blot at 0 days,14 days and 21 days after induction.The expression of P75 NTR and osteogenic key genes ALP and Run X2 were detected by real-time PCR and western blot.Alkaline phosphatase staining and alizarin red staining were compared at different time points of osteogenesis induction,ED19.5EMSCs differentiation.Part Ⅲ:In order to clarify the molecular mechanism of P75 NTR involved in tooth germ development.We used P75 NTR + ED12.5EMSCs and P75 NTR + ED19.5EMSCs to prepare Affymetrix expression profiles.Through the GO and Pathway analysis of chip results,it is found that Wnt signal path plays an important role in it.Real-time quantitative PCR and western blot were used to detect the expression of snt,Lrp6,β-catenin,LEF1 and CCND1 in WNT signaling pathway in ED19.5EMSCs.It is further explained that P75 NTR participates in osteogenesis differentiation through Wnt signaling pathway in ED19.5EMSCs.Part Ⅳ:The complex process of tooth development,with a large number of molecules and signal pathways involved,in order to verify that P75 NTR is indeed involved in ED19.5EMSCs osteogenic differentiation process,rather than differentiation process of a concomitant phenomenon.We used lentivirus to transfect P75 NTR and used plasmid to express the expression of sost.Real-time PCR and western blot were used to detect Lrp6,β-catenin,LEF1 and CCND1.And through alizarin red staining,more intuitive observation of different methods of treatment after ED19.5EMSCs mineralized nodules formation.Results:Part Ⅰ:A Comparative Study on the Biological Behavior of ED12.5EMSCs and ED19.5EMSCs1.CD45-but CD29 +,CD44 +,CD90 +,CD105 +,CD146 + were detected by stem cell molecular marker detection.2.The expression of P75 NTR in ED19.5EMSCs was significantly higher than that in ED12.5EMSCs.3.Real-time PCR and western blot showed that the expression level of ALP and Run X2 were significantly higher in ED19.5EMSCs than that in ED12.5EMSCs after 14 days of osteogenic induction.4.The results of alkaline phosphatase staining showed that the number of mineralized nodules of ED19.5EMSCs was more than that of ED12.5EMSCs after 14 days of osteogenic induction.Part Ⅱ:The effect of P75 NTR on osteogenic differentiation of ED19.5EMSCs.1.The expression level of P75 NTR in ED19.5EMSCs decreased after P75 NTR was silenced,and the expression level of P75 NTR in ED19.5EMSCs increased after P75 NTR.2.Cell immunofluorescence staining showed that silence and overexpression were achieved with good efficiency.3.Real-time quantitative PCR and western blot showed that the expression of ALP and Run X2 decreased after P75 NTR,while the expression of ALP and RunX 2 increased after P75 NTR was overexpressed.There was a significant difference between the experimental group and the control group after 14 days of osteogenesis induction.4.Alkaline phosphatase staining and alizarin red staining showed that the number of mineralized nodules between the experimental group and the control group was significantly different after 14 days of osteogenic induction.After 21 days of osteogenesis induction,alizarin There were significant differences in the number of mineralized nod ules between the red staining group and the control group.Part Ⅲ:Study on the Effect of P75 NTR on Wnt Pathway in ED19.5EMSCs.1.The Affymetrix expression profile results show that the Wnt classical pathway plays an important role in ED19.5EMSCs.2.Real-time quantitative PCR results showed that the expression of sost,Lrp6,β-catenin and CCND1 were decreased after P75 NTR,and the expression levels of sost,Lrp6,β-catenin,LEF1 and CCND1 were increased after overexpression of P75 NTR.3.Western blot analysis showed that the expression of P75 NTR,β-catenin and Lrp6 decreased and the expression of Sper was increased after P75 NTR was increased.The expression of P75 NTR,β-catenin and Lrp6 increased and the expression of sost decreased.Part Ⅳ:Molecular Mechanism of P75 NTR on the Osteogenic Differentiation Ability of ED19.5EMSCs by Wnt Signaling Pathway.1.The expression of Lrp6,β-catenin,LEF1 and CCND1 was the highest in overexpression of P75 NTR.The expression level of Lrp6,β-catenin,LEF1 and CCND1 was the lowest after overexpression of sost The expression levels of Lrp6,β-catenin,LEF1 and CCND1 were higher than those of overexpression in the sost group,but lower than that in the P75 NTR group.2.Western blot analysis showed that the expression level of P75 NTR,Lrp6 and β-catenin was the highest when P75 NTR was overexpressed.The expression level of P75 NTR,Lrp6 and β-catenin was the lowest and sost expression was the highest P75 NTR and sost,the expression level of P75 NTR,Lrp6 and β-catenin was higher than that of overexpression of sost group,but lower than that of overexpression of P75 NTR group.3.The results showed that the number of mineralized nodules in overexpression sost group was the least,and the number of overexpressed P75 NTR group was the highest.At the same time,the number of mineralized nodules was lower than that of P75 NTR group,But higher than the oost expression of sost group,while it was higher than the blank control group,indicating that ED19.5EMSCs in the process of osteogenesis differenti ation sost inhibitory effect is lower than the P75 NTR promotion.Conclusion:1.The results showed that ED19.5EMSCs had stronger osteogenic differentiation potential in the same SD pregnant mice,which obtained different embryonic development sites at different embryonic development time points.2.This study found that P75 NTR in ED19.5EMSCs for osteogenic differentiation ability to promote the role.3.Wnt pathway plays an important role in ED19.5EMSCs.P75 NTR can positively regulate the activity of Wnt signaling pathway.4.P75 NTR activates the Wnt pathway by inhibiting the activity of sost,and further promotes osteogenic differentiation in ED19.5EMSCs.In summary,our results suggest that the Wnt pathway plays a leading role in ED19.5EMSCs.P75 NTR can negatively regulate sost,activate Wnt signaling pathway activity,and promote osteogenic differentiation.This study not only deepens the understanding of the bone-to-brain differentiation of cranial nerve derived epidermal mesenchymal stem cells during tooth development,but also suggests that P75 NTR can alter the Wnt signaling pathway under cranial neural crest-derived ectomesenchymal stem cells And to provide a new theoretical and experimental basis for the study of tissue engineered teeth. |
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