Investigation of mechanism of periodontal destruction in rat experimental periodontitis caused by topical application of lipopolysaccharide

为阐明脂多糖致边缘性牙周炎牙周膜破坏的组织病理学机制，（1）首次采用组织培养细胞，采用组织化学和免疫组织化学方法研究牙周膜成纤维细胞对胶原蛋白的吞噬和/或溶解活性。结论：大鼠磨牙牙周韧带可建立成纤维细胞系。培养板底面细胞呈多角形、圆形或梭形。组织化学和免疫组化显示牙周膜成纤维细胞的特征，如碱性磷酸酶阳性，茜素红染色显示钙化，免疫组化显示波形蛋白和I型胶原。它们还显示角蛋白、骨钙素和OX-43（内皮细胞和巨噬细胞的标记物）的轻微免疫组织化学阳性。我们将这些成纤维细胞在胶原凝胶中进行三维培养 ...更多信息 并试图在电子显微镜观察下证明培养细胞对胶原的吞噬作用。但是，即使在超微结构水平下，也很难在培养的细胞中找到摄入的胶原纤维。在培养的成纤维细胞中以化学方法证明吞噬的胶原蛋白的试验也是不成功的，因为难以区分摄取的胶原蛋白原纤维和细胞中合成的胶原蛋白。另一方面，（2）我们研究了局部应用脂多糖引起的大鼠实验性牙周炎中巨噬细胞的动力学，因为巨噬细胞合成胶原酶和/或IL-1 β被怀疑引起牙周膜的破坏。结果显示，给予LPS后，炎症性牙周病变中来自血液单核细胞的ED 1阳性渗出组织巨噬细胞增加。这些巨噬细胞产生IL-1 β，可能参与牙周组织的破坏。LPS刺激后，Ia抗原阳性树突状细胞也明显增多，提示这些巨噬细胞中有许多已转化为抗原提呈细胞，可能在局部免疫反应中起重要作用。少

To elucidate histopathological mechanism of periodontal ligament destruction in the marginal periodontitis caused by lipopolysaccharide, (1) we have firstly tried to investigate the collagen-phagocytotic and/or collagenolytic activities of periodontal ligament fibroblasts using tissue culture cells with histochical and immunohistochemical methods. We could establish the fibroblastic cell lines from rat molar periodontat ligaments. These cells were polyhedral, round or spindle in shape on the bottom surface of tissue culture plate. Histochemically and immunohistochemically, they showed characteristics of periodontal ligament fibroblasts, such as positive reaction of alkaline phosphatase and alizarin red staining for calcification, immunostaining for vimentin and type I collagen. They also revealed slight immunohistochemical positivities for keratin, osteocalcine and OX-43 (marker for endothelial cell and macrophage). We cultured these fibroblasts three-dimensionally in the collagen gel … More and tried to demonstrate phagocytosis of collagen by the culture cells under the electron microscopic observation. But, it was difficult to find the ingested collagen fibrils in the cultured cells even under the ultrastructural level. Trial to demonstrate the phagicytosed collagen immunohisto-chemically in the cultured fibroblastic cells was also unsuccessful, as it was difficult to differentiate the ingested collagen fibrils from the synthesized collagen in the cells. On the other hand, (2) we studied dynamics of macrophages in the rat experimental periodontitis caused by topical application of lipopolysaccharide, because synthesis of collagenase and/or IL-1beta by macrophage is suspected to cause destruction of periodontal ligament. As a result, it was revealed that ED1 positive exudative tissue macrophages from blood monocytes increased in the inflammatory periodontal lesion after LPS-administration. These macrophages produced IL-1beta and was suggested to participate destruction of periodontal tissue. Ia antigen positive dendritic cells also increased in the lesion after LPS-administration, which suggested that much of these macrophages changed to antigen presenting cells and might have important role for reactivity of local immune response. Less