Promoter Analysis Of Human Dentin Sialophosphoprotein(DSPP) Gene And The System Establishment For Evaluating Odontogenic Differentiation

The presence of dentin sialophosphoprotein (DSPP) marks the differentiation of cells towards odontoblast. Traditional detection method of DSPP expression include RT-PCR, immunohistochemistry, Western-Blot, etc., all of which are time-consuming and onerous and incapable of positioning. A detection system for Dspp expression using Dspp promoter and report genes had been established within the mice. However, a system using human DSPP promoter was never reported. Therefore, this study attempts to analyze the structure of DSPP promoter, based on which we construct hDSPP promoter-LacZ report system, realizing rapid detection of differentiation status of cells towards odontoblast.Based on the inducible expression property of DSPP, this study firstly establish human cell line that express DSPP, by inducing human dental mesenchymal stem cells to differentiate towards odontoblast cells. After culture, real time-PCR analysis showed the DSPP expression of human dental mesenchymal stem cells was increased, suggesting Dexamethasone could induce differentiation of dental stem cells to odontoblast-like cells. Secondly,online (TESS and Genomatix Software Suite) analysis was carried out on DSPP putative promoter region including basic element and cis-element, the result of which was used to design primers for cloning different sequences (-4000—+54,-2500—+54,-1447—+54,-1027—+54)of DSPP putative promoter and construct pGL3-LUC-DSPP promoter reporting recombination vector that encompass those sequences. The cotransfection of CMV-Renilla and pGL3-LUC-DSPP promoter into human dental mesenchymal stem cells was realized by lipofection. After48-72h of Dexamethasone induced culture, dual luciferase report presented that the activity of-2500—+54in hDSPP putative promoter region ranks at the top,-1447—+54comes next,suggesting considerable activity of the cloned promoter. At last, we used the sequence-2500—+54to construct hDSPP promoter-LacZ lentivirus reporting vector, and lentivirus infected human dental mesenchymal stem cells, followed by subsequent culturing and X-Gal staining. Comparison of cells with recombination lentivirus and control lentivirus, some cells with phDSPP (-2500-+54)-LacZ lentivirus was found blue, indicating the feasibility of using phDSPP (-2500-+54)-LacZ to detect DSPP expression in vitro.The analysis of hDSPP promoter and establishment of its reporting vector facilitate rapid detection differentiation of seed cells towards odontoblast-like cells and researches on growth and transcription factors during tooth development.