Role Of Toll Like Receptor 9 Signalling Pathway In Odontoblasts

Dental caries is a hight-incidence disease.When the caries reaches dentin, the cariogenic bacteria with their toxic productions will move into the dental pulp cavity through dentinal tubules, result in pulpitis. Dental pulp playsa role of denfense and repair by immune response and the tertiary dentinogenesis.The odontoblast cells could deep into the dentinal tubules, due to long cell processes, which is the first contact with microbial cells of bacteria in the pulpodentinal complex. If so, do odontoblast cells and oral mucosa epithelial cells have the same innate immune function? No studies have reported over the years. TLR9 is the innate immune receptor which specifically identifies the DNA of bacteria containing unmethylated CpG motifs. In many histiocytes it can rapidly activate the innate immune response and trigger the consequent adaptive immune response and it plays an important role in the histiocyte immune defence response. At present, it has made great progress in anti-infective, anti-allergic and anti-cancer therapy research. Our group and foreign articles found that in vitro odontoblast-like cells expressed TLR9, which may mediate odontoblasts innate immune function. But whether odontoblasts express TLR9 in vivo; Or whether TLR9 specifically identify the bacterial DNA? And after TLR9 activation, the situation of intracellular signaling pathways and the expression of downstream gene have never been reported.For the bacterial invasion, the formation of tertiary dentinogenesis is the other main way to play defense and repair capacity except for, immune response. Many studies have confirmed DSPP gene products play an important role in formation and mineralization of dentin, and tertiary dentinogenesis formation. The bacterial infection causes odontoblasts or odontoblast-like cells to form the tertiary dentinogenesis with DSPP gene expression; so does TLR9 signaling pathway activated by bacterial DNA genes in odontoblasts regulate DSPP expression? To our knowledge, no research have been reporte.In summary, this study of TLR9, DSPP gene study by molecular biological methods is to clarify the TLR9 mediated odontoblasts natural immune response and regulation of DSPP gene expression in the intracellular signaling pathways. In the process of caries, the relationship among the bacterial DNA infection, odontoblasts immune response, DSPP gene expression and the formation of tertiary dentinogenesis are important to clarify the cause of pulpitis, dental pulp injury, repair of molecular mechanisms and DSPP gene expression and regulation, which also supplies new ideas and ways to immunize against dental caries and save vital pulp therapy.1 TLR9, DSPP expression in odontoblasts and pulp tissue.Firstly, collect mouse dental pulp tissue, extract RNA, reverse transcription to the tissue block. amplify by PCR and observe the expression of TLR9 mRNA in dental pulp tissue. Secondly extract TRNA, reverse transcription from OLC mouse odontoblasts cell line, amplify by PCR and observe the expression of TLR9 mRNA and DSPP mRNA. It is indicated that TLR9 mRNA has a significant expression in mouse dental pulp tissue, and TLR9 mRNA and DSPP mRNA has a significant expression in OLC cell line upon 1% agarose gel electrophoresis. The molecular weight of a TLR9 mRNA is 198bp, and a DSPP mRNA is 151bp.2 The regulation of CpG DNA on the OLC intracellular TLR9, DSPP gene expression.Cultured OLC cell by the six-orifice plates firstly, stimulated the cell in the time gradient of 0, 3, 6, 9, 12, 24 hours by CpG ODN-A and CpG ODN-B. Then extract TRNA, reverse transcription, amplify by PCR, and use the way of real-time-PCR to observe the changes of TLR9 mRNA and DSPP mRNA expression in OLC cell line. It is indicated that the expressions of TLR9 mRNA and DSPP mRNA has no significant change in CpG ODN-A stimulating group upon result of 1% agarose gelelectrophoresis and real-time-PCR. The expressions of TLR9 mRNA and DSPP mRNA with the stimulation of CpG ODN-B was increased, but then decreased, the highest expression was in the 6 hours group and 3 times to the 0 hour group.3 TLR9 involved in the CpG OND's regulation of the signaling pathways of DSPP gene expression in cells.There were three kinds of cell signaling inhibitors: ERK cell signaling inhibitors (PD98059), p38 cell signaling inhibitors (SB203580) and NF-κB cell signaling inhibitors PDTC inhibited the cell which was stimulated by CpG ODN-B .The concentration of three kinds was 30μmol / L.It is indicated that the ERK pathway group and the NF-κB pathway group has an inhibitory effect on the expression of DSPP mRNA by the way of real-time PCR. There are no significant difference between the p38 pathway group and the blank group.