Detection and Change of Secretory Leukocyte Protease Inhibitor Produced by Gingival Epithelial Cells in Periodontal Disease Progression.

本研究项目的目的是检测牙龈上皮细胞中的分泌性白细胞蛋白酶抑制剂（SLPI），并阐明龈沟液（GCF）中的SLPI与牙周临床参数或龈下菌斑中牙周病细菌的存在之间可能的关系。在冰冻切片牙龈上皮和来自健康牙龈的培养牙龈上皮细胞上通过免疫细胞化学方法检测SLPI。与来自伴放线放线杆菌 (A.a)、牙龈卟啉单胞菌 (P.g) 和中间普雷沃氏菌 (P.i) 的 IL-1*、β (0-100U/ml)、TNF-*(0-1,000ng/ml) 和细胞外囊泡 (ECV) (0-100mug/ml) 一起孵育 4 小时，SLPI使用夹心酶联免疫吸附测定法测量培养的牙龈上皮细胞的上清液。在非刺激下牙龈上皮细胞将SLPI分泌到培养基中。随着TNF-*浓度的增加，SLPI的分泌受到显着抑制。在P.g ECV浓度增加的情况下，SLPI的分泌也显着受到抑制。测量了10名成年牙周炎患者的SLPI水平和GCF中的中性粒细胞弹性蛋白酶（NE）活性，并通过DNA探针分析检测了龈下菌斑样本中是否存在8种牙周病细菌。在 32 个检查部位中，SLPI 水平与 GCF 体积和牙龈炎症的存在呈正相关。 SLPI水平与P.g、福赛拟杆菌和密螺旋体的存在呈显着负相关，而在细菌存在下，NE活性增加。结果表明，具有产生蛋白水解酶能力的细菌降解或灭活了蛋白酶抑制剂，提示牙周组织的蛋白水解损伤可能是由于活性蛋白酶抑制剂的减少造成的。

The purpose of the present research project was to detect the secretory leukocyte protease inhibitor (SLPI) in gingival epithelial cells and to clarify the possible relationship between SLPI in gingival crevicular fluid (GCF) and the periodontal clinical parameters or the presence of periodontopathic bacteria in subgingival plaque.SLPI was immunocytochemically detected on frozen sectioned gingival epithelium and cultured gingival epithelial cells from healthy gingiva.After four hours incubation with IL-1*, beta (0-100U/ml), TNF-*(0-1,000ng/ml), and extracellular vesicles (ECV) (0-100mug/ml) from Actinobacillus actinomycetemcomitans (A.a), Porphiromonas gingivalis (P.g), and Prevotella intermedia (P.i), SLPI in supernatant of cultered gingival epithelial cells was measured using sandwich enzyme linked immunosorbent assay. Gingival epithelial cells secreted SLPI into culture medium under non stumuli. The secretion of SLPI was significantly suppressed under the increasing concentration of TNF-*. And the secretion of SLPI was also significantly suppressed under the increasing concentration of P.g ECV.SLPI levels and neutrophil elastase (NE) activity in GCF were measured and subgingival plaque samples were tested for the presence of eight periodontopathic bacteria by DNA probe analysis in ten adult periodontitis patients. In thirty two examined sites, the SLPI levels were positively associated with the GCF volume and the presence of gingival inflammation. Statiscally significant negative correlations were found between SLPI levels and the presence of P.g, Bacreroides forsythus and Treponema denticola, whereas, the NE activity increased in the presence of the bacteria.The results demonstrated that the bacteria with the capacity to produce proteolytic enzymes degraeded or inactivated the protease inhibitor, suggesting that the proteolytic damage of periodontal tissue might be caused by the decrease of active protease inhibitor.