Kiili M, Cox S W, Chen H Y, Wahlgren J, Maisi P, Eley B M, Salo T, Sorsa T
Faculty of Veterinary Medicine, University of Helsinki, Helsinki, Finland.
J Clin Periodontol. 2002 Mar;29(3):224-32. doi: 10.1034/j.1600-051x.2002.290308.x.
To determine the cellular and molecular forms of MMP-8 (collagenase-2) and MMP-13 (collagenase-3) associated with chronic adult periodontitis by examining the species present in gingival crevicular fluid (GCF) and enzyme distribution in gingival tissue.
30-s GCF samples were collected directly from the periodontal pockets of 12 untreated patients using filter paper strips. After elution into buffer, the samples were examined by Western immunoblotting with polyclonal antibodies for MMP-8 and MMP-13 and quantification by scanning image analysis. Individual band intensities were expressed as a percentage of total sample absorbance and mean patient values were calculated. Gingival tissue from 6 patients was fixed in formalin and embedded in paraffin wax. MMP-8 and MMP-13 were localised using the same antibodies and an avidin-biotin-peroxidase detecting system. Double staining was performed with a contrasting substrate reaction.
The majority of MMP-8 staining in pre-treatment GCF was present in 80, 75 and 60 kD bands corresponding to prepro-, pro- and active forms of PMN-type enzyme. 43 and 38 kD bands evidently represented active, fibroblast-type MMP-8. Immunoreactivities at >100 kD and < or =30 kD were probably enzyme-inhibitor complex and degraded fragments, respectively. MMP-13 was seen mainly as 60 kD proenzyme with some 40 kD active enzyme and a small proportion of >100 kD complex. The percentages of MMP-8 PMN-type enzyme and MMP-13 proenzyme bands correlated significantly with gingival and bleeding indices (p<0.05). Immunohistochemistry demonstrated MMP-8 in PMNs, sulcular epithelial and also plasma cells in inflamed gingival connective tissue. MMP-13 immunoreactivity was detected in the sulcular epithelium and in macrophage-like cells.
Multiple species and elevated levels of both MMP-8 and MMP-13 from many rather than single cellular sources in the diseased periodontium are identified in untreated periodontitis GCF and active forms contribute to GCF collagenase activity.
通过检测龈沟液（GCF）中存在的种类以及牙龈组织中的酶分布，确定与成人慢性牙周炎相关的基质金属蛋白酶-8（胶原酶-2）和基质金属蛋白酶-13（胶原酶-3）的细胞和分子形式。
使用滤纸直接从12例未经治疗的患者的牙周袋中采集30秒的GCF样本。洗脱到缓冲液中后，用针对基质金属蛋白酶-8和基质金属蛋白酶-13的多克隆抗体进行蛋白质免疫印迹分析，并通过扫描图像分析进行定量。各条带强度以总样本吸光度的百分比表示，并计算患者的平均值。将6例患者的牙龈组织用福尔马林固定并石蜡包埋。使用相同的抗体和抗生物素蛋白-生物素-过氧化物酶检测系统对基质金属蛋白酶-8和基质金属蛋白酶-13进行定位。用对比底物反应进行双重染色。
治疗前GCF中大部分基质金属蛋白酶-8染色出现在80、75和60 kD条带中，分别对应于PMN型酶的前体、前酶和活性形式。43和38 kD条带明显代表活性成纤维细胞型基质金属蛋白酶-8。大于100 kD和小于或等于30 kD的免疫反应性可能分别是酶-抑制剂复合物和降解片段。基质金属蛋白酶-13主要表现为60 kD的酶原，伴有一些40 kD的活性酶和一小部分大于100 kD的复合物。基质金属蛋白酶-8的PMN型酶和基质金属蛋白酶-13的酶原条带百分比与牙龈和出血指数显著相关（p<0.05）。免疫组织化学显示，在PMN、龈沟上皮以及炎症牙龈结缔组织中的浆细胞中存在基质金属蛋白酶-8。在龈沟上皮和巨噬细胞样细胞中检测到基质金属蛋白酶-13免疫反应性。
在未经治疗的牙周炎GCF中，从患病牙周组织的多种而非单一细胞来源中鉴定出多种形式且水平升高的基质金属蛋白酶-8和基质金属蛋白酶-13，其活性形式对GCF胶原酶活性有贡献。
