南方医科大学学报

南方医科大学学报 ›› 2023, Vol. 43 ›› Issue (2): 287-293.doi: 10.12122/j.issn.1673-4254.2023.02.18

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牙龈卟啉单胞菌在体外可破坏脐静脉内皮屏障功能：基于下调ZO-1、occludin和VE-cadherin的表达

曾 娇，李心竹，殷琳莹，陈 婷，侯 晋   

  1. 南方医科大学南方医院口腔科，广东 广州 510515
  • 出版日期:2023-02-20 发布日期:2023-03-16

Porphyromonas gingivalis infection causes umbilical vein endothelial barrier dysfunction in vitro by down-regulating ZO-1, occludin and VE-cadherin expression

ZENG Jiao, LI Xinzhu, YIN Linying, CHEN Ting, HOU Jin   

  1. Department of Stomatology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
  • Online:2023-02-20 Published:2023-03-16

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摘要： 目的 通过体外实验探讨牙龈卟啉单胞菌（P. gingivalis）破坏内皮屏障功能的分子机制。方法 人脐静脉内皮细胞（HUVECs）体外培养形成屏障后，使用感染复数（MOI）100的P. gingivals感染细胞，对照组为未感染P. gingivals的HUVECs；通过跨膜电阻值（TEER），异硫氰酸荧光素（FITC）-右旋糖酐通透性实验和细菌易位实验来评估内皮屏障功能；并使用实时荧光定量聚合酶链式反应（qRT-PCR）和Western blot实验检测P. gingivals对构成内皮屏障结构的主要蛋白紧密连接蛋白1（ZO-1），occludin和VE-cadherin表达的影响。结果 HUVECs在接种培养后第5天，其跨膜电阻值趋于稳定，体外内皮屏障形成。与对照组相比，感染P. gingivals后0.5 h，内皮屏障功能即会发生改变，并且随着感染时间的延长，屏障功能障碍更加显著，主要表现为：跨膜电阻值下降、40 000 /70 000 FITC-Dextran通透性上升以及易位细菌的数量增加；MTT结果显示P. gingivals对HUVECs的增殖能力没有影响（P>0.05）；除此之外，与对照组相比，HUVECs感染P. gingivals后24和48 h后，qRT-PCR和Western blot结果显示被感染的 HUVECs 中 ZO-1，occludin 和 VE-cadherin 的 mRNA 和蛋白水平出现明显的下调表达（P<0.05）。结论 P.gingivalis可以通过下调细胞间连接蛋白ZO-1、occludin和VE-cadherin的表达破坏内皮屏障，导致内皮屏障功能障碍。

关键词: 牙龈卟啉单胞菌；脐静脉内皮细胞；内皮屏障；细胞间连接蛋白；牙周炎

Abstract: Objective To explore the molecular mechanisms of Porphyromonas gingivalis infection-induced umbilical vein endothelial barrier dysfunction in vitro. Methods Human umbilical vein endothelial cells (HUVECs) were cultured in vitro, and after the formation of the endothelial barrier, the cells were infected with P. gingivals at a multiplicity of infection (MOI). The transepithelial electrical resistance (TEER) of the cell barrier was measured, and FITC-dextran trans-endothelial permeability assay and bacterial translocation assay were performed to assess the endothelial barrier function. The expression levels of cell junction proteins including ZO-1, occludin and VE- cadherin in the cells were examined by qRT-PCR and Western blotting. Results In freshly seeded HUVECs, TEER increased until reaching the maximum on Day 5 (94 Ωcm2), suggesting the formation of the endothelial barrier. P. gingivals infection caused an increase of the permeability of the endothelial barrier as early as 0.5 h after bacterial inoculation, and the barrier function further exacerbated with time, as shown by significantly lowered TEER, increased permeability of FITC-dextran (40 000/70 000), and increased translocation of SYTO9-E. coli cross the barrier. MTT assay suggested that P. gingivals infection did not significantly affect the proliferation of HUVECs (P>0.05), but in P. gingivals-infected cells, the expressions of ZO-1, occludin and VE-cadherin increased significantly at 24 and 48 h after bacterial inoculation (P<0.05). Conclusion P. gingivals may disrupt the endothelial barrier function by down-regulating the expressions of the cell junction proteins (ZO-1, occludin, VE-cadherin) and increasing the permeability of the endothelial barrier.

Key words: Porphyromonas gingivalis; umbilical vein endothelial cells; endothelial barrier; cell junction proteins; periodontitis