Analysis of gene expression concerned with PDL mechanical receptor in biting

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Periodontal ligament (PDL) cells, which consist of osteoblasts, fibloblasts, osetoclasts, and et.al., maintain the attachment of the tooth to alveolar bone. Recently, it has been reported that PDL cells examines function and characterization in vitro. To confirm immune response for periodontal disease casing dental plaque on PDL cells, we investigated the cytokine Mrna expression and production stimulating Prevotella intermedia and Streptococcus mutans. PDL cells were obtained from the middle third of the root surface of healthy human erupted maxillary third molar. The tissue minced and cultured as explants in tissue culture medium [D-MEM with D-Glucose 4500 mg/dl L-Glu NaHCO3 ] containing 10% low-endotoxin fetal calf serum (FCS), 100U penisillin, 25ug/ml streptomysin, amphotericin B as FugizoneTM in 0.85% saline). Cultures were then grown in medium and cloned by limiting dilutions, I.e., the cells obtained from primary cultures. The confluent cultures were used for experimentation bet … More ween the 3th and 4th passages. Bacteria were the kind gift from department of Microbiology, Kyoto Prefectural University of Medicine. Cultures were confluent (1.0× 105cells/well). And stimulated by bacteria (1.0×107/ml) after 24 hours in a 6-well plate. No bacteria were used as control. Total RNA was extracted by ISOGENTM (NIPPON GENE). 1mg of RNA was used for Cdna synthesis with a reverse transcriptase (SuperscriptII RNAse H; GIBCO-BRL) and an oligo (dt) 16 primer. Reaction mixture was used as a template for PCR amplification was run for 35 cycles at 94℃ for 1 min, 60℃ for 1 min, and 72℃ for 1 min. PCR product was analyzed by electrophoresis on a 1.5% agarose gel containing ethidium bromide, and the bands were examined under UV light. The expression of IL-1b, IL-6, IL-8, TNF-α, TGF-α, and TGF-β were detected. This study indicated expression of many inflammatory cytokines. These results suggest that there was a relationship between the cytokine expression and the inflammation of periodontal ligaments on pockets. Furthermore, we may examine immune mechanism which marginal periodontitis affect mechanical stress and Bone Morphogenetic Protein Less