ABSTRACT
<p><b>OBJECTIVE</b>To study the biological properties of human dental pulp cells (HDPC) by cloning and analysis of genes differentially expressed in HDPC in comparison with human gingival fibroblasts (HGF).</p><p><b>METHODS</b>HDPC and HGF were cultured and identified by immunocytochemistry. HPDC and HGF subtractive cDNA library was established by PCR-based modified subtractive hybridization, genes differentially expressed by HPDC were cloned, sequenced and compared to find homogeneous sequence in GenBank by BLAST.</p><p><b>RESULTS</b>Cloning and sequencing analysis indicate 12 genes differentially expressed were obtained, in which two were unknown genes. Among the 10 known genes, 4 were related to signal transduction, 2 were related to trans-membrane transportation (both cell membrane and nuclear membrane), and 2 were related to RNA splicing mechanisms.</p><p><b>CONCLUSION</b>The biological properties of HPDC are determined by the differential expression of some genes and the growth and differentiation of HPDC are associated to the dynamic protein synthesis and secretion activities of the cell.</p>
Subject(s)
Humans , Cloning, Molecular , Cloning, Organism , Dental Pulp , Fibroblasts , Gene Library , Gingiva , Polymerase Chain ReactionABSTRACT
<p><b>OBJECTIVE</b>To determine whether dentin matrix proteins were expressed by the human odontoblast-like cell line hTERT-hOd-1 in vitro.</p><p><b>METHODS</b>Collagen type I, bone sialoprotein (BSP), dentin matrix protein 1 (DMP1) and the marker for odontoblast, dentin sialophosphoprotein (DSPP) and dentin sialoprotein (DSP) were detected in these cells by immunohistochemistry, RT-PCR and in situ hybridization. During being cultured in mineralizing medium for 5 weeks, the secretion of OC and activity of ALP were measured once a week.</p><p><b>RESULTS</b>DSPP, DMP1, BSP and collagen type I were expressed in hTERT-hOd-1 either at mRNA or protein level. Under the induction of mineralizing medium, the cells showed higher activity of ALP and increased secretion of OC.</p><p><b>CONCLUSION</b>hTERT-hOd-1 expressed dentin extracellular matrix in vitro, which means the cell line has the potential of mineralization.</p>
Subject(s)
Humans , Cell Line , Collagen Type I , Dentin , Extracellular Matrix , Extracellular Matrix Proteins , Immunohistochemistry , In Situ Hybridization , In Vitro Techniques , Odontoblasts , Phosphoproteins , RNA, Messenger , SialoglycoproteinsABSTRACT
<p><b>OBJECTIVE</b>To clone and sequence the upstream of mouse dentin sialophosphoprotein promoter.</p><p><b>METHODS</b>Genomic DNA was obtained from Balb/c mouse blood. The upstream of mouse dentin sialophosphoprotein promoter segments was obtained by PCR. Then the segments were inserted into T-vector. The plasmids were identified by digestion with the restriction enzyme analysis. The positive clone was sequenced and compared with Genebank.</p><p><b>RESULTS</b>The upstream of mouse dentin sialophosphoprotein promoter was divided into three sequences and three different target segments with 997 bp, 1004 bp and 674 bp in length were obtained. After identified, sequenced and compared with Genebank, the sequences of the segments were consistent with those displayed on Genebank by 99%.</p><p><b>CONCLUSION</b>The clone of the upstream of mouse dentin sialophosphoprotein promoter was successful. This work will help to study the regulation of DSPP expression.</p>
Subject(s)
Animals , Mice , Cloning, Molecular , Extracellular Matrix Proteins , Genetics , Mice, Inbred BALB C , Phosphoproteins , Genetics , Promoter Regions, Genetic , Sequence Analysis, DNA , Sialoglycoproteins , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To explicit whether the expression of the mineral-related proteins is regulated by cbfa1 in human dental papilla cells.</p><p><b>METHODS</b>Human dental papilla cells were cultured in vitro and transfected with pcDNA3-cbfa1 recombinant plasmids. After selected with G418 sulfate, a cell clone named PC-3, which could stably express the cbfa1 mRNA and protein, was proved by PCR and western blot. Then the amount of ALP and OC and the expression of OPN, BSP, ON, DMP1, DSP and DSPP were detected by immunohistochemistry, Western blot and PCR methods.</p><p><b>RESULTS</b>We established the human dental papilla cells model PC-3 which could stably express the cbfa1 mRNA and protein. Compared with normal cells, a lot of mineral-related proteins such as ALP, OC, OPN, BSP, ON, DMP1 were upregulated in PC-3 cells.</p><p><b>CONCLUSIONS</b>In human dental papilla cells, cbfa1 can induce the expression of most mineral-related genes and proteins. It may implicate that cbfa1 must play a key role during tooth development and mineralization.</p>