RESUMO
Odontogenesis consists of a series of consecutive tooth morphogenesis stages, in which apoptosis is involved to eliminate the unnecessary cells. Autophagy, a lysosome or endosome-mediated self-degradation process, is indicated to participate in embryogenesis and tissue morphogenesis associated with apoptosis. This study hypothesized that autophagy may be involved and associated with apoptosis in odontogenesis. The transcripts of autophagy-related genes (Atg5, Atg7, and Atg12) were positively detected in tooth germs at embryonic day (E) 14.5 and postnatal day (P) 5.5 by quantitative real-time PCR. The protein expression of Atg5-Atg12 conjugate and lipidation of LC3 (microtubule-associated protein 1 light chain 3, autophagic marker) were revealed in the developing tooth germs by western blot. Meanwhile, LC3 was immunolocalized in the enamel organ and dental papilla at embryonic stages (E13.5-E18.5), especially stage E14.5 cervical loop and the PEK that facing the mesenchyme. At postnatal stages (P1.5-P15.5), besides the dental epithelium cells, LC3 was detected in the differentiating and differentiated odontoblasts, dental follicle cells, and Hertwig's epithelium root sheath cells. Moreover, double-immunofluorescence analysis revealed the partial colocalization of LC3 and TUNEL signal in the E14.5 PEK that facing the mesenchyme, the E16.5 stratum intermedium and outer enamel epithelium, the P5.5 stratum intermedium and stellate reticulum. Nevertheless, LC3 was also found in non-apoptotic cells. Furthermore, the transmission electron microscopic images revealed the presence of autophagy, as well as the partial colocalization of autophagic vacuoles and apoptotic nuclei during tooth development. Our findings imply the developmental appearance of autophagy and its partial colocalization with apoptosis during odontogenesis.
Assuntos
Autofagia , Dente Molar/embriologia , Odontogênese , Germe de Dente , Animais , Apoptose , Autofagia/genética , Proteína 12 Relacionada à Autofagia , Proteína 5 Relacionada à Autofagia , Proteína 7 Relacionada à Autofagia , Western Blotting , Regulação da Expressão Gênica no Desenvolvimento , Idade Gestacional , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Endogâmicos ICR , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Dente Molar/metabolismo , Dente Molar/ultraestrutura , Proteínas/genética , Proteínas/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Germe de Dente/metabolismo , Germe de Dente/ultraestruturaRESUMO
The present study was designed to investigate the direct role of Shh molecule on cytodifferentiation and cusp formation. Affi-gel blue beads soaked in exogenous Shh-N, Shh antibody or BSA control protein were implanted between the epithelium and mesenchyme of isolated molar germs at the cap stage. The recombinants were grafted for culture under the kidney capsules respectively. In compared to the control, additional Shh-N protein could not enhance the ameloblasts and odontoblasts differentiation of the explanted tooth germs. While, application of Shh antibody retarded these events. After 4 weeks of subrenal culture, the teeth dissected from the explants treated with Shh-N were multicuspid. Most of the teeth harvested from the Shh antibody group were small and single irregularly shaped cusp was visible. The main cusp height in this group was reduced. The results indicated Shh signaling pathway is critical for odontoblast and ameloblast differentiation and patterns cusp formation.
Assuntos
Diferenciação Celular , Proteínas Hedgehog/metabolismo , Dente Molar/citologia , Dente Molar/embriologia , Transdução de Sinais , Animais , Células Cultivadas , Dente Canino/citologia , Esmalte Dentário/citologia , Esmalte Dentário/metabolismo , Dentina/citologia , Dentina/metabolismo , Regulação da Expressão Gênica , Camundongos , Odontoblastos/citologia , Odontoblastos/metabolismo , Receptores Patched , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
INTRODUCTION: Dentin sialoprotein (DSP) is a dentin extracellular matrix protein, a unique marker of dentinogenesis and plays a vital role in odontoblast differentiation and dentin mineralization. Recently, studies have shown that DSP induces differentiation and mineralization of periodontal ligament stem cells and dental papilla mesenchymal cells in vitro and rescues dentin deficiency and increases enamel mineralization in animal models. THE HYPOTHESIS: DSP as a nature therapeutic agent stimulates dental tissue repair by inducing endogenous dental pulp mesenchymal stem/progenitor cells into odontoblast-like cells to synthesize and to secrete dentin extracellular matrix forming new tertiary dentin as well as to regenerate a functional dentin-pulp complex. As DSP is a nature protein, and clinical procedure for DSP therapy is easy and simple, application of DSP may provide a new avenue for dentists with additional option for the treatment of substantially damaged vital teeth. EVALUATION OF THE HYPOTHESIS: Dental caries is the most common dental disease. Deep caries and pulp exposure have been treated by various restorative materials with limited success. One promising approach is dental pulp stem/progenitor-based therapies to regenerate dentin-pulp complex and restore its functions by DSP induction in vivo.