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

为了阐明脂多糖对边缘性牙周炎牙周韧带破坏的组织病理学机制，(1)我们首先采用组织培养细胞和免疫组织化学方法研究了牙周韧带成纤维细胞的胶原吞噬和/或胶原溶解活性。我们可以建立大鼠磨牙牙周韧带成纤维细胞系。组织培养板底表面细胞呈多面体、圆形或纺锤形。组织化学和免疫组织化学显示牙周韧带成纤维细胞的特征，碱性磷酸酶阳性，茜素红钙化染色，静脉蛋白和I型胶原免疫染色。他们还发现角蛋白、骨钙素和OX-43（内皮细胞和巨噬细胞的标志物）的免疫组化轻微阳性。我们将这些成纤维细胞在胶原凝胶中进行三维培养，并在电镜下观察培养细胞对胶原的吞噬作用。但是，即使在超微结构水平下，也很难在培养细胞中发现摄入的胶原原纤维。在培养的成纤维细胞中，免疫组化证实胶原蛋白被吞噬的试验也没有成功，因为很难区分摄入的胶原原纤维和细胞内合成的胶原蛋白。另一方面，(2)我们研究了巨噬细胞在外用脂多糖引起的实验性牙周炎中的动态变化，因为巨噬细胞合成胶原酶和/或il -1 β被怀疑是导致牙周韧带破坏的原因。结果显示，脂多糖给药后，炎症性牙周病变的血单核细胞中ED1阳性的渗出组织巨噬细胞增加。这些巨噬细胞产生il -1 β，并被认为参与牙周组织的破坏。lps给药后，病变中Ia抗原阳性的树突状细胞也增加，提示这些巨噬细胞大部分转变为抗原提呈细胞，可能在局部免疫反应的反应性中起重要作用。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