Analysis of gene expression concerned with PDL mechanical receptor in biting

牙周膜(PDL)细胞由成骨细胞、成纤维细胞、破骨细胞等组成，维持牙齿与牙槽骨的附着。最近，有报道称，PDL细胞在体外检测其功能和特性。为了证实牙周病菌斑对PDL细胞的免疫应答，我们研究了刺激中间普氏杆菌和变形链球菌的细胞因子mRNA的表达和产生。PDL细胞取自健康人上颌第三磨牙萌出牙根表面中1/3处。组织块在含有10%低内毒素胎牛血清、100U青霉素钠、25ug/ml链霉菌素、两性霉素B的组织培养液[D-MEM+D-葡萄糖4500 mg/dl L-Glu NaHCO3]中培养。然后，培养物在培养基中生长，并通过限制稀释度进行克隆，即从原代培养获得的细胞。融合后的培养用于…的实验更多是在第三和第四段之间。细菌是京都医科大学微生物学教研室赠送的礼物。培养为融合培养(1.0×105个/孔)。在6孔板中培养24小时后用细菌刺激(1.0×107/ml)。未使用任何细菌作为对照。用ISOGENTM(Nippon基因)提取总RNA。1 mg的RNA用于逆转录酶(SuperscriptII RNase H；GIBCO-BRL)和寡聚(DT)16引物的cDNA合成。以反应混合物为模板，94℃反应1min，60℃反应1min，72℃反应1min，共35个循环。聚合酶链式反应产物在含溴化乙锭的1.5%琼脂糖凝胶上进行电泳分析，并在紫外光下检测条带。检测IL-1b、IL-6、IL-8、肿瘤坏死因子-α、转化生长因子-α、转化生长因子-β的表达。这项研究表明许多炎性细胞因子的表达。这些结果提示，细胞因子的表达与袋上牙周韧带炎症有关。此外，我们还可以研究边缘牙周炎对机械应力和骨形态发生蛋白影响较小的免疫机制。

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