Department of Periodontics, College of Dentistry, University of Baghdad, Baghdad, Iraq.
Biomaterials, School of Dentistry, Institute of Clinical Sciences, College of Medical and Dental Sciences, The University of Birmingham, Birmingham, UK.
J Periodontal Res. 2018 Aug;53(4):565-574. doi: 10.1111/jre.12546. Epub 2018 Apr 27.
Epithelial-mesenchymal transition (EMT) is a process by which epithelial cells acquire a mesenchymal-like phenotype and this may be induced by exposure to gram-negative bacteria. It has been proposed that EMT is responsible for compromising epithelial barrier function in the pathogenesis of several diseases. However, the possible role of EMT in the pathogenesis of periodontitis has not previously been investigated. The aim of this study therefore was to investigate whether gram-negative, anaerobic periodontal pathogens could trigger EMT in primary oral keratinocytes in vitro.
Primary oral keratinocytes were harvested from labial mandibular mucosa of Wistar Han rats. Cells were exposed to heat-killed Fusobacterium nucleatum and Porphyromonas gingivalis (100 bacteria/epithelial cell) and to 20 μg/mL of Escherichia coli lipopolysaccharide over an 8-day period. Exposure to bacteria did not significantly change epithelial cell number or vitality in comparison with unstimulated controls at the majority of time-points examined. Expression of EMT marker genes was determined by semiquantitative RT-PCR at 1, 5, and 8 days following stimulation. The expression of EMT markers was also assessed by immunofluorescence (E-cadherin and vimentin) and using immunocytochemistry to determine Snail activation. The loss of epithelial monolayer coherence, in response to bacterial challenge, was determined by measuring trans-epithelial electrical resistance. The induction of a migratory phenotype was investigated using scratch-wound and transwell migration assays.
Exposure of primary epithelial cell cultures to periodontal pathogens was associated with a significant decrease in transcription (3-fold) of E-cadherin and the upregulation of N-cadherin, vimentin, Snail, matrix metalloproteinase-2 (3-5 fold) and toll-like receptor 4. Bacterial stimulation (for 8 days) also resulted in an increased percentage of vimentin-positive cells (an increase of 20% after stimulation with P. gingivalis and an increase of 30% after stimulation with F. nucleatum, compared with controls). Furthermore, periodontal pathogens significantly increased the activation of Snail (60%) and cultures exhibited a decrease in electrical impedance (P < .001) in comparison with unexposed controls. The migratory ability of the cells increased significantly in response to bacterial stimulation, as shown by both the number of migrated cells and scratch-wound closure rates.
Prolonged exposure of primary rat oral keratinocyte cultures to periodontal pathogens generated EMT-like features, which introduces the possibility that this process may be involved in loss of epithelial integrity during periodontitis.
上皮-间充质转化（EMT）是上皮细胞获得间充质样表型的过程，这种转化可能是由革兰氏阴性细菌暴露引起的。有研究提出，EMT 是导致多种疾病中上皮屏障功能受损的原因。然而，EMT 在牙周炎发病机制中的可能作用尚未得到研究。因此，本研究旨在探讨革兰氏阴性、厌氧牙周病原体是否可在体外诱导原代口腔角质形成细胞发生 EMT。
从 Wistar Han 大鼠下唇颏部黏膜中分离原代口腔角质形成细胞。将细胞与热灭活的具核梭杆菌和牙龈卟啉单胞菌（100 个细菌/上皮细胞）以及 20μg/mL 的大肠杆菌脂多糖孵育 8 天。与未刺激对照组相比，在大多数检测时间点，细菌暴露并未显著改变上皮细胞数量或活力。刺激后 1、5 和 8 天，通过半定量 RT-PCR 检测 EMT 标记基因的表达。通过免疫荧光（E-钙黏蛋白和波形蛋白）和免疫细胞化学检测 Snail 激活来评估 EMT 标记物的表达。通过测量跨上皮电阻来确定细菌攻击后上皮单层协调性的丧失。通过划痕和 Transwell 迁移实验研究迁移表型的诱导。
牙周病原体暴露于原代上皮细胞培养物中与 E-钙黏蛋白转录显著降低（约 3 倍）以及 N-钙黏蛋白、波形蛋白、Snail、基质金属蛋白酶-2（~3-5 倍）和 Toll 样受体 4 的上调相关。细菌刺激（8 天）还导致波形蛋白阳性细胞的百分比增加（与对照组相比，牙龈卟啉单胞菌刺激后增加 20%，具核梭杆菌刺激后增加 30%）。此外，牙周病原体显著增加了 Snail 的激活（60%），并且与未暴露的对照组相比，培养物的电阻抗降低（P<.001）。细菌刺激显著增加了细胞的迁移能力，这通过迁移细胞的数量和划痕愈合率都可以看出。
原代大鼠口腔角质形成细胞培养物长时间暴露于牙周病原体可产生 EMT 样特征，这提示该过程可能与牙周炎中上皮完整性丧失有关。
