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Abstract
The molecular mechanisms control the function of PDL(periodontal ligament) cells and/or fibroblasts remain unclear. PDLs17, PDL-specific gene, had previousely identified the cDNA for a novel protein from cultured PDL fibroblasts using subtraction hybridization between gingival fibroblasts and PDL fibroblasts. The purpose of this study was to determine the regulation by growth factors and cytokines on PDLs17 gene expression in cultured human periodontal ligament cells and observe the immunohistochemical localization of PDLs17 protein in various tissues of mouse.
Primary PDL fibroblasts isolated by scraping the root of the extracted human mandibular third molars. The cells were incubated with various concentration of human recombinant IL-1β, PDGF-BB and TGF β for 48h and 2 weeks. At each time point total RNA was extracted and the levels of transcription were assessed by reverse transcription-polymerase chain reaction (RT-PCR assay). Polyclonal antiserum raised against PDLs17 peptides, CLSVSYNRSYQINE and SEAVHETDLHDGC, were made, and stained the tooth, periodontium, developing bone, bone marrow and mid-palatal suture of the mouse.
The results were as follows.
1. PDLs17 mRNA levels were increased in response to PDGF (10ng/ml) and TGF β(20ng/ml) after treatment of the IL-1β, PDGF-BB and TGFβ for 48 h.
2. PDLs17 was up-regulated only by TGFβ(20 ng/ml) after treatment of the IL-1β, PDGF-BB and TGF β for 2 weeks and unchanged by the other stimulants.
3. PDLs17 was a novel protein coding the 142 amino acid peptides in the ORF and the nucleotide sequences of the obtained cDNA from RT-PCR was exactly same as the nucleotides of the database.
4. Immunohistochemical analysis showed that PDLs17 is preferentially expressed in the PDL, differentiating osteoblast-like cells and stromal cells of the bone marrow in the adult mouse.
5. The expression of PDLs17 protein was barely detectable in gingival fibroblasts, hematopoetic cells of the bone marrow and mature osteocytes of the alveolar bone.
These results suggest that PDLs17 might upregulated by PDGF-BB or TGFβ and acts at the initial stage of differentiation when the undifferentiated mesenchymal cells in the bone marrow and PDL differentiate into multiple cell types. However, more research needs to be performed to gain a better understanding of the exact function of PDLs17 during the differentiation of bone marrow mesenchymal and PDL cells.
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