| Objective:Teeth are essential organs for mastication,speech,and aesthetics.Tooth loss not only affects people’s daily life and leads to the decline of people’s quality of life,but also is an essential problem for people’s health around the world.Tooth regeneration is the fundamental solution to this problem.This project intends to study the possible molecular mechanism of Shh signaling pathway in regulating the development of second dentition in mice,and provides a theoretical reference for further tooth regeneration therapy.Methods:1.ICR pregnant mice between E13.0 and E17.0 were orally administered a single dose of vismodegib(150mg/Kg)to inhibit Hh signaling pathway and we observed the possible effects of Hh signaling pathway on dentition development and other craniofacial development in different embryonic stages of mice;2.A stable model of lingual supernumerary teeth in the first mandibular molar of ICR mice was established by administering vismodegib(150mg/Kg)to E15.5 pregnant mice.After successfully establishing the model,HE staining was used to identify the origin of supernumerary teeth.To evaluated the effect of vismodegib on cell proliferation,apoptosis,migration,epithelial-mesenchymal transition,and angiogenesis,the molar section between two groups was analyzed by immunohistochemical staining for PHH3/Ki67,Cleaved caspase 3,E-cadherin,CK14/vimentin,and CD34.Shh signaling pathway molecule Shh/Ptc1/Glil protein and mRNA expression were also testified by IHC and RT-qPCR to evaluate the effect of vismodegib which can inhibit the expression of Shh signaling pathway during tooth development,and the source of lingual supernumerary teeth of the first mandibular molar and its possible mechanism of formation were further explored.3.The model of lingual supernumerary teeth of the first mandibular molar of mouse in different periods was established by administering vimodegib(150mg/Kg)to pregnant mice at E10.5 and E15.5.And Sox-2 and Sox-9 immunohistochemical staining were used to study the relationship between the supernumerary teeth induced in two periods and stem cells with positive expression of Sox-2.On this basis,RNA-seq was used to detect the relevant differential genes that inhibit the formation of lingual supernumerary teeth induced by Shh signaling pathway in mice.Screening related differential genes by bioinformatics,verifying the expression of differential genes related to odontogenesis by RT-qPCR and looking for possible differential genes that can inhibit the formation of lingual supernumerary teeth induced by Shh signaling pathway in mice;4.After the mouse second dentition model was established by administering vismodegib to E15.5 pregnant mice.In order to explore the possible signal pathway of inhibiting Shh signaling pathway to induce the formation of second dentition in mice,the expression of key molecules in signal pathways of Wnt,Bmp and Fgf were detected by immunohistochemical staining and RT-qPCR was used to detect the expression changes of related differential genes 24 hours after administration.Results:1.Vismodegib,an inhibitor of Hh signaling pathway,can induce fetal mice to form multiple incisors,cleft palate,discontinuous palate rugae,supernumerary first molars,short fingers,and other developmental deformities during the craniofacial development of mouse embryos,which is intensive related with the time of embryo development.The incidence of supernumerary teeth induced by vismodegib in mice during the middle and late stages(E13.0-E17.0)of tooth development was different,and the key time of the highest incidence for inducing supernumerary incisors and supernumerary molars were E14.5 and E15.5.2.In the model of lingual supernumerary teeth in the first mandibular molar of mouse established by giving pregnant mouse vismodegib at E15.5,expression changes of molecules related to Shh signaling pathway in the experimental group and the control group were detected at E16.5 by IHC and RT-qPCR.It was found that the expression of signal molecules Ptcl and Glil downstream of Shh signal pathway in experimental group was significantly lower than that in control group,and the difference was statistically significant(P<0.001).The HE staining of E16.5,E17.5,E18.5 and P1.5 were analyzed,it was found that vismodegib promoted the thickening of the rudimentary successional dental lamina(RSDL)on the lingual side of the first molar of fetal rats in the experimental group.And the RSDL further developed to tooth germ-like tissue structures such as bud stage and cap stage,while the control group kept the original structure of residual successional dental lamina all the time.IHC detection of E16.5 showed that the positive expression of cell proliferation index PHH3,Ki67 and cell migration index E-cadherin were significantly up-regulated in the RSDL region of the experimental group,but there was no significant difference in the expression of apoptosis index cleave caspase 3,epithelial mesenchymal index CK14,vimentin and angiogenesis index CD34 between the experimental group and the control group.3.The expression of Sox-2 and Sox-9 in the lingual supernumerary teeth model induced at E10.5 and E15.5 were detected by IHC.It was found that Sox-2 positive cells were expressed in the buccal side of supernumerary teeth induced at E10.5 and extended to the lingual epithelium of the first molar,while Sox-2 in supernumerary teeth induced at E15.5 was located at the tip of the lingual RSDL of the first molar.The results suggested that the supernumerary teeth induced at E10.5 and E15.5 were all related to Sox-2 positive expression stem cells.On this basis,29 differentially expressed genes that were jointly involved in the formation of multiple teeth in the two periods were screened out by means of RNA-seq and bioinformatics.The results of mRNA expression of differential genes related to Wnt,Fgf and Bmp signaling pathways associated with odontogenesis detected by RT-qPCR analysis were basically consistent with the sequencing results.It was found that inhibition of Shh signaling pathway by vismodegib may affect the development of tooth morphology and number by up-regulating the expression of Inhba,Fgf20 and down-regulating the expression of Hhip,Ptc1,Ptc2,Foxf2,Wnt7b,Sostdc1,Foxi3.4.In the model of administration of vismodegib to pregnant mice at E15.5 to induce the development of second dentition by inhibiting the Shh signaling pathway,the expression of Sostdc1 at E16.5 in the experimental group was significantly lower than that in the control group,and the difference was statistically significant(P<0.05).The expression of Lefl and β-catenin in Wnt signaling pathway was detected by IHC.It was found that Lef1 expression in RSDL of E16.5 experimental group was significantly up-regulated and β-catenin changed from cell membrane type to nuclear type.Micro-CT was used to measure the size of M1,it was found that the buccal-lingual diameter and the proximaldistal-medial diameter of M1 in the experimental group were smaller than those in the control group,and the difference was statistically significant(n=6,P<0.001).Further IHC detection showed that the expression of Fgf20,Bmp4,Inhba and related downstream molecules p-ERK1/2 and Runx2 in the lingual side of the first molar in the experimental group were significantly up-regulated compared with those in the control group.Conclusion:Studies have found that the regulation of Hh signaling pathway in craniofacial development is time-dependent.During the development of mouse second dentition,the key time points for vismodegib to inhibit the formation of supernumerary incisors and supernumerary molars induced by Shh signaling pathway are E14.5 and E15.5.Morphological studies have found that supernumerary molars are the degenerated second dentition of mice,which can be further developed by activating the proliferation and migration of RSDL cells.The formation may be related to the activation of Wnt signaling pathway expression in RSDL which was activated by down-regulation of Sostdc1 expression and upregulation of Inhba and FGF20 expression caused by the inhibition of Shh signaling pathway.In summary,Shh signaling pathway may inhibit the formation of second dentition by inhibiting the development of residual continuous dental plaque in the early stage of second dentition development in mice. |
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