Role Of TRAF6 In Regulating The Function Of Odontoblast Cells

Odontoblast is a special type of cell in pulp-dentin complex, and responsible for dentin formation. Tumor necrosis factor receptor-associated factor 6 (TRAF6), a member of TRAF superfamily, is a crucial signaling molecule regulating bone metabolism, development of embryo and plays a key role in LPS/IL-1, CD40, RANK signal transduction pathway and etc.. As we known, dentin is a sort of highly mineralized tissue, shares many similar things with bone during development. Thus, the aim of this study was to investigate whether TRAF6 was involved in dentin formation and if so, the role of TRAF6 in regulating the function of odontoblast cells. The present study consists of three parts:Part One: Expression of TRAF6 in developing tooth germ and odontoblast-like cell line MDPC-23TRAF6 was examined in rat developing tooth germ by immunohistochemistry and in odontoblast-like cell line, MDPC-23 by Semi-quantitive RT-RCR and immunofluoescent staining. The results showed that TRAF6 strongly expressed in preodontoblasts and odontoblasts in rat tooth germ with a spatiotemporal expression pattern from early bell stage topostnatal.7^th day. TRAF6 also expressed in MDPC-23 at both mRNA and protein levels. These suggested that TRAF6 may play a role in regulating the fuction of odontoblast cells.Part Two: effect of TRAF6 on odontoblast cell proliferation and extracellular matrix protein expression by specific RNA interferenceTwo recombinant plasmids pSUPER-TRAF6siRNA were constructed and then transient transfected into MDPC-23 cells. The effectiveness of the plasmids was detected by inhibitory rate of TRAF6 through RT-PCR and Western blot approaches. After that, pSUPER and pcDNA3.1+ vectors were remolded into a new pVIC vector, which met the expectations of both RNA interference (RNAi) and stable transfection, followed by pVIC-TRAF6siRNA construction and transfection into MDPC-23 cells via the Lipofectamine? 2000 method.G418-resistant colonies were selected by adding G418 (300μg/ml) to the medium for several weeks. Viable MDPC-23/pVIC-TRAF6siRNA colonies were further screened by RT-PCR and Western blot. Finally, one colony, whose TRAF6 expression was interfered around 80%, was decided and used for further investigation.MTT assay and flow cytometry (FCM) were applied then to examine the cell proliferatation and extracellular matrix protein expression. The results demonstrated that TRAF6 potently promoted cell proliferatation and increased ALP and OC, but downregulated OPN, BSP and DSP expression in the MDPC-23/pVIC-TRAF6 siRNA clony. These implicated that TRAF6 may involved in the process of dentin mineralizationPart Three: effect of TRAF6 on the response of odontoblast cellto LPS stimulus...