Research of bacteria related to periapical diseases activate MMP and inactivate TIMP

基质金属蛋白酶（MMPs）在细胞外基质（ECM）的稳态中起关键作用。基质金属蛋白酶及其抑制剂--金属蛋白酶组织抑制剂（tissue inhibitors of metalloproteinases，TIMPs）的产生失衡在组织破坏中起着重要作用。据报道，MMP-1和MMP-9被与根尖周病的产生相关的细菌激活。然而，关于MMP-2前体的激活和TIMP失活的报道很少。1）MMP-2活性的测定将含有PL细胞或HT 1080细胞和来自牙龈卟啉单胞菌（P.g.）采用明胶酶谱法进行分析。当用SBE处理PL细胞或HT 1080细胞时，在明胶酶谱上观察到对应于活性MMP-2的可见条带。2）TIMP-1和TIMP-2蛋白的测量TIMP-1的量通过Kodama等的EIA方法测量。SBE处理的PL细胞。用提取物处理的HT 1080细胞培养物获得的上清液中TIMP-1和TIMP-2的量在P.g. SBE浓度依赖性方式。3）TIMP-1活性的测定纯化的TIMP-1溶液与SBE反应后，通过明胶酶谱法评价保留在溶液中的TIMP-1对MMP-1或MMP-2的抑制活性。P.G. SEE可显著降低TIMP-1对MMP-2的抑制活性。

Matrix metalloproteinases (MMPs) play a critical role in the homeostasis of the extracellular matrix (ECM). The imbalance between production of MMPs and that of their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), plays an important role in tissue destruction. It has been reported that MMP-1 and MMP-9 are activated by the bacteria associated with the production of periapical diseases. However few reports regarding the activation of pro-MMP-2 and inactivation of TIMP have been published. The purpose of the present study was to evaluate the activation of pro-MMP-2 and inactivation of TIMP in cultured cells.l )Measurement of MMP-2 activityThe supernatant obtained from medium containing PL cells or HT1080 cells and a sonicated bacterial extract (SEE) from P. gingivalis (P.g.) was analyzed by the gelatin zymography method. When PL cells or HT1080 cells were treated with the SBE, visible bands that corresponded to active MMP-2 were observed on the gelatin zymograms.2) Measurement of TIMP-1 and TIMP-2 proteinsThe amount of TIMP-1 was measured by the EIA method of Kodama et al. TIMP-1 was not detected in the supernatant obtained from the medium of cultures of P.g. SBE-treated PL cells. The amount of TIMP-1 and TIMP-2 in the supernatant obtained from HT1080 cell cultures treated with the extract decreased in a P.g. SBE concentration-dependent manner.3) Measurement of TIMP-1 activityAfter a solution of purified TIMP-1 had been reacted with SBE, TIMP-1 retained in the solution was evaluated by gelatin zymography in terms of its inhibitory activity toward MMP-1 or MMP-2. P.g. SEE remarkably decreased the inhibitory activity of TIMP-1 toward MMP-2.