A Study On Tumor Necrosis Factor Receptor-associated Factor 6 Express During The Replacement Of Primary And Permanent Teeth

Tumor necrosis factor receptor-associated factor (TRAF) family of proteins is one group of proteins which transduce the signals in cytoplasm. There are six members of this protein family, their function is led to dramatic progress in the elucidation of the molecular mechanisms of signal transduction emanating from the TNFR superfamily and the Toll/interleukin-1 receptor (Toll/IL-IR) family. They are key molecules in the signals regulating the immune and inflammatory systems, embryogenesis, stress reaction and bone metabolism. Receptor activator of nuclear factor- K B(RANK) is the key molecule in the signals regulating during the procedur of differentiation and maturity of osteoclasts. The signal through the binding of RANK and receptor activator of NF-kB ligand (RANKL) can accelerate the differentiation of osteoclasts and induce the activation of mature osteoclasts. After the binding between RANK and RANKL, TRAF6 can bind cytoplasmic domains of RANKL through TRAF-C domain and oligomerization of the amino-terminal portion of TRAF6 containing the RING and zinc fingersresulted in the activation of downstream kinases such as TAK1, JNK and NF- K B. Through this signal conduction, osteoclasts will maturate. At present, the study of TRAF6 focuses on bone remodeling and pathological mechanism of bone diseases. The replacement of primary and permanent teeth is an important part that belongs to the growth and development of animals' teeth and maxillofacial region. A lot of changes have taken place in the correlated tissues including the primary teeth, alveolar bone, periodontal ligament and the successional permanent teeth germs during this procedure. Until now, there is not any report concerning about the roles of TRAF6 in the replacement of the primary and permanent teeth. So in our study, we observed TRAF6 expressions in deciduous teeth roots, periodontal ligament, alveolar bone and permanent teeth germs under three states of the dogs' teeth (primary dentition roots stability, primary dentition roots resorption, permanent dentition) and osteoclasts in vitro by immunohistochemical technique, in situ hybridization method and cell culture. The main content and results of our researches are below:Part 1 TRAF6 expression during the replacement of dogs' primary and permanent teethThere are two experiments in this part. Immunohistochemical technique and in situ hybridization method were used in this study to observe the TRAF6 expression under three states of the dogs' teeth: primary dentition roots stability, primary dentition roots resorption, permanent dentition.1.1 dogs' primary dentition roots stability stage: The deciduous teeth roots were not absorbed biologically. The permanent teeth germs were developing and dentin and enamel matrix started to form. In the immunohistochemical experiment and in situ hybridization experiment, TRAF6 positive signals wereseen in the multinucleated osteoclasts localized in the absorbed lacunae of the alveolar bone near the germs, as well as ameloblasts, odontoblasts of the permanent teeth germs. These results indicated that osteoblasts expressed TRAF6 involved in the development of dogs' permanent teeth germs during the stages of the primary dentition roots stability.1.2 dogs' primary dentition roots resorption stage: Dogs' deciduous teeth roots were undergoing absorbed biologically and there were a lot of absorbed lacunae on the roots. Dentin and enamel of the permanent teeth germs went on forming and calcifying. Ameloblasts and odontoblasts appeared to tall posts and the nuclears were away from the basement membranes. In the immunohistochemical experiment and in situ hybridization experiment, the TRAF6-positive signals were observed on osteoclasts in alveolar bone, as well as ameloblasts, odontoblasts of the permanent teeth germs and the signal are stronger than primary dentition roots stability stage with a significant defference(P<0.05). These results indicated that osteoblasts, ameloblasts and odontoblasts expressed TRAF6 involved in the development of dogs' permanent teeth germs during the stages of the primary dentition roots resorption.1.3 dogs' permanent teeth dentition stage: Dogs' deciduous teeth shed and permanent teeth erupted. Osteoclasts were low-positive in immunohistochemical experiment and in situ hybridization experiment. The dyeing degree of these positive cells increased significantly (P<0.01). The results indicated the function of osteoblasts is leading in alveolar bone remodeling in this stage.Part 2 TRAF6 expression in osteoclasts culture in vitro In this part of experiment, we used a co-culture system with rat bone marrow cells and osteoblastic cells and added some cytokines such as 1,25(OH)2D3,...