A novel inhibitor in bone formation from periodontopathic bacteria

为了研究中间普雷沃氏菌超声提取液的抑菌效果。用不同的细菌提取物培养放线菌、牙龈卟啉单胞菌等口腔相关细菌，以及影响骨形成的大肠杆菌、MC3T3-E_1克隆细胞，保留成骨活性。与对照相比，在1 ~ 1000 ng ml^<-1>的浓度范围内，添加中间普雷沃氏菌和放线菌的超声提取物可使碱性磷酸酶活性呈剂量依赖性降低。相比之下，其他口腔细菌（包括牙龈卟啉单胞菌、紫噬细胞菌、milleri链球菌、sanguis链球菌和大肠杆菌）的超声提取物即使存在100 ng ml^<-1>蛋白，也没有降低碱性磷酸酶的活性。在细胞培养物中加入经过加热和胰蛋白酶处理的中间普氏菌和放线菌提取物也能抑制细胞中碱性磷酸酶的活性，这表明抑制因子不是蛋白质性的。多粘菌素B对中间普雷沃菌和放线菌提取物的细胞碱性磷酸酶活性无明显影响，说明多粘菌素B对细胞碱性磷酸酶活性的抑制作用不是脂多糖。在Sephadex G-200柱洗脱的290 kDa以上的分子质量中观察到两种提取物的抑制作用。经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳，部分纯化得到了普雷沃氏菌中间介质的抑制物质，并显示出分子量为170 ~ 190 kDa的宽频带。这些结果表明，中间普雷沃氏菌和放线菌可能在抑制骨形成和促进骨吸收方面发挥重要作用。

In order to study the effects of sonicated extracts from Prevotella intermedia. Actinobacillus actinomycetemcomitans, Porphyomonas gingivalis, and other oral-related bacteria, as well as Escherichia coli on bone formation, clone MC3T3-E_1 cells, which have retained osteoblastic activity, were cultured with various bacterial extracts.The addition of the sonicated extracts from Prevotella intermeedia and Actinobacillus actinomycetemcomitans decreased the alkaline phosphatase activity in a dosedependent fashion over the concentration range of 1-1000 ng ml^<-1> compared with the control. By contrast, the sonicated extracts from other oral bacteria including Prophyromonas gingivalis, Capnocytophaga ochracea, Streptococcus milleri and Streptococcus sanguis, and Escherichia coli did not decrease the alkaline phosphatase activity even in the presence of 100 ng ml^<-1> protein.The addition of prevotella intermedia and Actinobacillus actinomycetemcomitans extracts that had been treated with heat and trypsin to the cell cultures also inhibited alkaline phosphatase activity in the cells, suggesting that inhibitory factors are not proteinaceous. Polymyxin B did not change the alkaline phosphatase activity in the cells treated with the extracts from Prevotella intermedia and Actinobacillus actinomycetemcomitans, suggesting that the inhibitory activity of the extracts is not lipopolysaccharide.The inhibitory effect of both extracts was observed in the molecular mass over 290 kDa eluted from Sephadex G-200 column. The inhibitory substances of Prevotella intermedia were partially purified and showed broad band with estemated molecular weight of 170-190 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.These results indicate that Prevotella intermedia and Actinobacillus actinomycetemcomitans may play an important role in inhibiting bone formation as well as in stimulating bone resorption.