具核梭杆菌来源的外膜囊泡对口腔上皮细胞闭合蛋白Claudin-4表达的影响  

Effects of Fusobacterium nucleus derived outer membrane vesicles on claudin-4 expression in oral epithelial cells

作　　者：杨成龙 杨蒙 王宇峰[2] 宋晨成 杜观环[2] 唐国瑶[2] YANG Cheng-long;YANG Meng;WANG Yu-feng;SONG Chen-cheng;DU Guan-huan;TANG Guo-yao(School of Stomatology,Weifang Medical University.Weifang 261021,Shandong Province;Department of Oral Medicine,Shanghai Ninth People's Hospital,Shanghai Jiao Tong University School of Medicine,College of Stomatology,Shanghai Jiao Tong University,National Center for Stomatology,National Clinical Research Center for Oral Diseases,Shanghai Key Laboratory of Stomatology,Shanghai Research Institute of Stomatology.Shanghai 200011;Department of Laboratory Medicine,Shanghai Ninth People's Hospital,Shanghai Jiao Tong University School of Medicine.Shanghai 200011,China)

机构地区：[1]潍坊医学院口腔医学院,山东潍坊261021 [2]上海交通大学医学院附属第九人民医院口腔黏膜病科,上海交通大学口腔医学院,国家口腔医学中心,国家口腔疾病临床医学研究中心,上海市口腔医学重点实验室,上海市口腔医学研究所,上海200011 [3]上海交通大学医学院附属第九人民医院检验科,上海200011

出　　处：《上海口腔医学》2023年第2期126-131,共6页Shanghai Journal of Stomatology

基　　金：国家自然科学基金(82101008)。

摘　　要：目的:探讨具核梭杆菌(Fusobacterium nucleatum,F.n)分泌的外膜囊泡(outer membrane vesicles,OMVs)对人口腔角质形成细胞(human oral keratinocytes,HOK)闭合蛋白Claudin-4表达及其对口腔上皮屏障功能的影响。方法:厌氧条件下培养具核梭杆菌,透析法提取OMVs。采用Nanosight及透射电镜(transmission electron microscopy,TEM)对提取的OMVs进行表征分析。OMVs以不同质量浓度(0~100μg/mL)刺激HOK 12 h,并以100μg/mL OMVs刺激HOK 6、12 h,采用实时转录PCR和蛋白质印迹法分析闭合蛋白Claudin-4在基因和蛋白水平的表达;倒置荧光显微镜观察HOK与OMVs共定位以及Claudin-4蛋白的定位分布。利用Transwell上室体外构建人口腔上皮屏障,采用跨膜电阻测量仪(EVOM2)检测屏障跨上皮电阻(transepithelial electrical resistance,TER),以异硫氰酸荧光素-右旋糖酐(FD-4)渗透率评估屏障通透性。采用GraphPad Prism 8.0软件包对数据进行统计学分析。结果:与对照组相比,OMVs刺激组HOK中Claudin-4在蛋白水平和基因水平表达均显著降低(P<0.05)。免疫荧光显示,细胞间Claudin-4的荧光连续性表达被破坏。与对照组相比,OMVs刺激可降低口腔上皮屏障的TER值(P<0.05),提高FD-4的透过率(P<0.05)。结论:具核梭杆菌来源的OMVs能通过抑制闭合蛋白Claudin-4表达,损伤口腔黏膜上皮的屏障功能。PURPOSE:To investigate the effect of outer membrane vesicles(OMVs)secreted by Fusobacterium nucleatum(F.n)on Claudin-4 of human oral keratinocytes(HOK)and oral epithelial barrier function.METHODS:Fusobacterium nucleatum was cultured under anaerobic conditions.The OMVs were extracted by dialysis and characterized by nanosight and transmission electron microscopy(TEM).HOK were stimulated with OMVs at different mass concentrations(0-100μg/mL)for 12 h,and stimulated with 100μg/mL OMVs for 6 h and 12 h respectively.The expression of Claudin-4 at gene and protein level was analyzed by RT-qPCR and Western blotting.Inverted fluorescence microscope was used to observe co-localization of HOK and OMVs and localization and distribution of Claudin-4 protein.Human oral epithelial barrier was constructed by Transwell apical chamber.Transepithelial electrical resistance(TER)of barrier was measured with a transmembrane resistance measuring instrument(EVOM2),and the permeability of the barrier was evaluated by transmittance of fluorescein isothiocyanate-dextran(FD-4).Statistical analysis was performed with GraphPad Prism 8.0 software package.RESULTS:Compared with the control group,the expression of Claudin-4 at protein and gene level in the HOK of OMVs stimulated group was significantly reduced(P<0.05),and immunofluorescence showed that the continuity of Claudin-4 fluorescence among cells was destroyed.OMVs stimulation decreased TER value of oral epithelial barrier(P<0.05)and increased the transmittance of FD-4(P<0.05).CONCLUSIONS:OMVs derived from Fusobacterium nucleatum may damage oral mucosal epithelial barrier function through inhibiting the expression of Claudin-4.

关 键 词：口腔扁平苔藓 上皮屏障功能 具核梭杆菌 外膜囊泡 CLAUDIN-4 

分 类 号：R781.5[医药卫生—口腔医学]