Zhou Jing, Windsor L Jack
Department of Oral Biology, Indiana University School of Dentistry, Indianapolis, IN 46202, USA.
J Periodontal Res. 2006 Feb;41(1):47-54. doi: 10.1111/j.1600-0765.2005.00835.x.
Studies have shown that Porphyromonas gingivalis and host matrix metalloproteinases (MMPs) play important roles in the tissue destruction associated with periodontal disease. It is still unclear which MMPs or their inhibitors are regulated by P. gingivalis at the transcriptional and/or at the protein levels. Therefore, this study was conducted to determine what effects P. gingivalis supernatant has on the collagen degrading ability of human gingival fibroblasts (HGFs) and how it regulates the activation, mRNA expression, and inhibition of MMPs.
Culture supernatant from P. gingivalis ATCC 33277 was added to HGFs cultured in six-well plates coated with Type I collagen. At certain time intervals, the cell conditioned media was collected for zymography and/or western blot analyses to determine the MMP and tissue inhibitor of MMPs (TIMP) protein levels. The cells were then removed and the collagen cleavage visualized by Coomassie blue staining. The mRNA expression of multiple MMPs and TIMPs by the treated and untreated HGFs was determined by reverse transcription-polymerase chain reaction.
The collagen in the six-well plates was degraded more rapidly by the HGFs treated with 10% v/v P. gingivalis supernatant. More active MMP-1, MMP-2, MMP-3, and MMP-14 were detected in the conditioned media from the HGFs treated with the P. gingivalis supernatant. TIMP-1, but not TIMP-2, was decreased in the presence of the P. gingivalis supernatant. MMP-1 mRNA expression by the treated HGFs increased more than two-fold over the untreated HGFs. MMP-3 mRNA was unchanged, MMP-2 mRNA had a slight increase, MMP-14 mRNA decreased, and MMP-15 increased. MMP-12 mRNA was induced in the P. gingivalis treated HGFs. TIMP-1 and TIMP-2 mRNA had a slight increase with P. gingivalis treatment.
Porphyromonas gingivalis increased the collagen degrading ability of HGFs, in part, by increasing MMP activation and by lowering the TIMP-1 protein level, as well as by affecting the mRNA expression of multiple MMPs and TIMPs.
研究表明牙龈卟啉单胞菌和宿主基质金属蛋白酶（MMPs）在与牙周病相关的组织破坏中起重要作用。目前仍不清楚哪些MMPs或其抑制剂在转录水平和/或蛋白质水平上受牙龈卟啉单胞菌调控。因此，本研究旨在确定牙龈卟啉单胞菌上清液对人牙龈成纤维细胞（HGFs）胶原降解能力有何影响，以及它如何调节MMPs的激活、mRNA表达和抑制。
将牙龈卟啉单胞菌ATCC 33277的培养上清液添加到接种于包被有I型胶原的六孔板中的HGFs中。在特定时间间隔，收集细胞条件培养基用于酶谱分析和/或蛋白质印迹分析，以确定MMP和基质金属蛋白酶组织抑制剂（TIMP）的蛋白质水平。然后去除细胞，通过考马斯亮蓝染色观察胶原裂解情况。通过逆转录-聚合酶链反应测定经处理和未经处理的HGFs中多种MMPs和TIMPs的mRNA表达。
用10% v/v牙龈卟啉单胞菌上清液处理的HGFs能更快地降解六孔板中的胶原。在用牙龈卟啉单胞菌上清液处理的HGFs的条件培养基中检测到更具活性的MMP-1、MMP-2、MMP-3和MMP-14。在牙龈卟啉单胞菌上清液存在的情况下，TIMP-1水平降低，而TIMP-2水平未降低。经处理的HGFs中MMP-1 mRNA表达比未经处理的HGFs增加了两倍多。MMP-3 mRNA未变化，MMP-2 mRNA略有增加，MMP-14 mRNA减少，MMP-15增加。牙龈卟啉单胞菌处理的HGFs中诱导了MMP-12 mRNA表达。牙龈卟啉单胞菌处理后，TIMP-1和TIMP-2 mRNA略有增加。
牙龈卟啉单胞菌通过增加MMP激活、降低TIMP-1蛋白质水平以及影响多种MMPs和TIMPs的mRNA表达，部分提高了HGFs的胶原降解能力。
