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

本研究的目的是检测牙龈上皮细胞分泌性白细胞蛋白酶抑制剂（SLPI），并阐明龈沟液（GCF）中白细胞蛋白酶抑制剂（SLPI）与牙周临床参数或龈下菌斑中牙周病细菌存在的可能关系。用免疫细胞化学方法对冰冻切片的牙龈上皮和培养的健康牙龈上皮细胞进行SLPI检测。用放线菌放线菌comitans （A.a）、牙龈卟啉单胞菌（P.g）、Prevotella intermedia （P.i）的IL-1*、β (0-100U/ml)、TNF-*(0- 1000ng /ml)和细胞外囊泡（0- 100ug /ml）孵育4小时后，采用夹心酶联免疫吸附法测定培养的牙龈上皮细胞上清液中的SLPI。牙龈上皮细胞分泌SLPI到培养基中。随着TNF-*浓度的升高，SLPI的分泌明显受到抑制。在P.g . ECV浓度升高的情况下，SLPI的分泌也明显受到抑制。采用DNA探针检测10例成人牙周炎患者龈下菌斑中8种牙周病细菌的存在，并测定龈下菌斑中SLPI水平和中性粒细胞弹性蛋白酶（NE）活性。在32个检查部位，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、连翘芽孢杆菌和密螺旋体存在呈显著负相关，而NE活性在细菌存在时升高。结果表明，具有蛋白水解能力的细菌可降解或灭活蛋白酶抑制剂，表明牙周组织的蛋白水解损伤可能是由活性蛋白酶抑制剂的减少引起的。

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.