牙周炎症环境中内皮细胞焦亡现象的体内外研究初探  

A preliminary in vivo and in vitro study of endothelial cell pyroptosis in the periodontal inflammatory environment

作　　者：李蕊 王晓雨 叶庆元 王垭铮 张曦戈 葛晓彤 王勤涛 Li Rui;Wang Xiaoyu;Ye Qingyuan;Wang Yazheng;Zhang Xige;Ge Xiaotong;Wang Qintao(Department of Periodontology,School of Stomatology,The Fourth Military Medical University,State Key Laboratory of Oral&Maxillofacial Reconstruction and Regeneration,National Clinical Research Center for Oral Diseases,Shaanxi International Joint Research Center for Oral Diseases,Xi′an 710032,China;Digital Dentistry Center,School of Stomatology,The Fourth Military Medical University,State Key Laboratory of Oral&Maxillofacial Reconstruction and Regeneration,National Clinical Research Center for Oral Diseases,Shaanxi Key Laboratory of Stomatology,Xi'an 710032,China)

机构地区：[1]第四军医大学口腔医院牙周病科、口颌系统重建与再生全国重点实验室、国家口腔疾病临床医学研究中心、陕西省口腔疾病国际联合研究中心,西安710032 [2]第四军医大学口腔医院数字化口腔医学中心,口颌系统重建与再生全国重点实验室国家口腔疾病临床医学研究中心,陕西省口腔医学重点实验室,西安710032

出　　处：《中华口腔医学杂志》2024年第5期486-495,共10页Chinese Journal of Stomatology

基　　金：国家自然科学基金(82170955)。

摘　　要：目的通过体内外模型观察牙周炎局部组织内皮细胞在炎症环境下是否发生焦亡,为探究牙周炎发病机制提供实验依据。方法根据牙周病2018年新分类标准收集无全身疾病、牙周健康者及Ⅲ~Ⅳ期C级牙周炎患者的牙龈组织,免疫组化染色检测牙龈组织中焦亡标志性蛋白消皮素D(GSDMD)的表达水平及分布情况;每组通过结扎3只小鼠上颌第二磨牙2周建立牙周炎模型(结扎组),使用显微CT(micro-CT)检测小鼠牙槽骨吸收情况(以不做结扎处理的小鼠作为对照组);使用免疫荧光染色对健康及炎症小鼠牙龈组织中血管内皮细胞血小板内皮细胞黏附分子(CD31)与GSDMD共定位进行定量分析;体外培养人脐静脉内皮细胞(HUVECs),分别以质量浓度为0.5、1.0、2.5、5.0、10.0 mg/L牙龈卟啉单胞菌(Pg)脂多糖(LPS)联合腺苷三磷酸(ATP)处理HUVECs,同期设置0 mg/L Pg-LPS组为对照组。使用扫描电镜观察HUVECs形态,蛋白质印迹法检测GSDMD的N端结构域(GSDMD-N)蛋白表达,细胞免疫荧光染色检测GSDMD蛋白表达及分布,细胞计数试剂盒(CCK-8)检测HUVECs的增殖能力,碘化丙啶(PI)染色检测HUVECs细胞膜的完整性。结果免疫组化结果显示,牙周炎患者牙龈组织中GSDMD主要分布于血管周围,且表达水平较健康组织显著升高;micro-CT结果显示,结扎组小鼠上颌第二磨牙周围牙槽骨吸收较对照组小鼠显著增多(t=8.88,P<0.001);免疫荧光染色结果显示,结扎组小鼠牙龈组织中血管内皮细胞GSDMD与CD31存在明显的共定位;扫描电镜结果显示,在不同浓度Pg-LPS联合ATP模拟的炎症环境中,HUVECs细胞膜上出现不同大小的孔洞,呈现典型的细胞焦亡形态,其中2.5 mg/L Pg-LPS+ATP组细胞膜上孔洞最多且扩张融合,细胞有裂解死亡的趋势。蛋白质印迹法结果显示,2.5和5.0 mg/L Pg-LPS+ATP组焦亡标志性蛋白GSDMD-N表达显著高于对照组(F=3.86,P<0.01);细胞免疫荧光结果显示,2.5 mg/L Pg-LPS+AObjective To observe whether endothelial cells undergo pyroptosis in the inflammatory periodontal environment by using a model in vivo and in vitro,providing an experimental basis for indepth understanding of the underlying pathogenesis of periodontitis.Methods According to the classification of periodontal diseases of 2018,gingival tissues were collected from periodontally healthy subjects and patients with stageⅢ-Ⅳ,grade C periodontitis,who presented Department of Oral and Maxillofacial Surgery and Department of Periodontology,School of Stomatology,The Fourth Military Medical University from April to May 2022.Immunohistochemical staining was performed to detect the expression level and distribution of gasdermin D(GSDMD),a hallmark protein of cell pyroptosis,in gingival tissues.Periodontitis models were established in each group by ligating the maxillary second molar teeth of three mice for 2 weeks(ligation group).The alveolar bone resorption was determined by micro-CT(mice without ligation treatment were used as the control group),and the colocalization of GSDMD and CD31 were quantitatively analyzed by immunofluorescence staining in gingival tissues of healthy and inflammatory mice.Human umbilical vein endothelial cells(HUVECs)were cultured in vitro and treated with lipopolysaccharide(LPS)of Porphyromonas gingivalis(Pg)combined with adenosine triphosphate(ATP)at various concentrations of 0.5,1.0,2.5,5.0,and 10.0 mg/L,respectively,and the 0 mg/L group was set as the control group at the same time.Scanning electron microscopy was used to observe the morphology of HUVECs.Western blotting was used to detect the expression of gasdermin D-N terminal domains(GSDMD-N)protein and immunofluorescence cell staining was used to detect the expression and distribution of GSDMD.Cell counting kit-8(CCK-8)was used to detect the proliferative ability of HUVECs,and propidium iodide(PI)staining was used to detect the integrity of cell membrane of HUVECs.Results Immunohistochemistry showed that GSDMD in gingival tissues of perio

关 键 词：牙周炎 内皮细胞 细胞焦亡 脂多糖 腺苷三磷酸 

分 类 号：R781.42[医药卫生—口腔医学]