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型白细胞蛋白酶抑制剂（SLPI）的表达，并探讨其与龈沟液（GCF）中SLPI的关系采用免疫细胞化学法检测牙龈上皮细胞和正常牙龈上皮细胞的SLPI，并观察龈下菌斑中SLPI的表达情况，以及牙周临床参数和龈下菌斑中牙周致病菌的存在。用IL-1*、β（0- 100 U/ml）、TNF-*（0- 1000 ng/ml）和来自伴放线放线杆菌（Actinobacillusactinomycetemcomitans，A. a）、牙龈卟啉单胞菌（Porphiromonasgingivalis，P.g）和中间普雷沃氏菌（Prevotellintermedia，P.i）的细胞外囊泡（extracellularvesicle，ECV）（0- 100 μ g/ml）孵育4小时，用夹心酶联免疫吸附测定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到无菌培养液中。随着TNF-* 浓度的增加，SLPI的分泌受到明显抑制。检测10例牙周炎患者龈沟液中SLPI含量和中性粒细胞弹性蛋白酶（NE）活性，并对龈下菌斑进行DNA探针检测。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、Bacreroides dentithus和Treponema denticola呈显著负相关，而NE活性在细菌存在时增加，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减少所致。

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.