牙周炎环境下人牙龈成纤维细胞线粒体稳态的失衡  

Mitochondrial homeostasis imbalance in HGFs in periodontitis

作　　者：黄珺玲 王津津 王勤涛 HUANG Junling;WANG Jinjin;WANG Qintao(State Key Laboratory of Oral&Maxillofacial Reconstruction and Regeneration&National Clinical Research Center for Oral Diseases&Shaanxi Key Laboratory of Stomatology&Department of Periodontology,School of Stomatology,the Fourth Military Medical University,Xi'an 710032,China)

机构地区：[1]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、第四军医大学口腔医院牙周病科,陕西西安710032

出　　处：《口腔疾病防治》2024年第12期916-924,共9页Journal of Prevention and Treatment for Stomatological Diseases

基　　金：国家自然科学基金项目(82101064,82170955)。

摘　　要：目的探讨牙周炎环境下人牙龈成纤维细胞(human gingival fibroblasts,HGFs)线粒体稳态的失衡变化,为牙周炎发病机制的探索提供基础。方法本研究已获得单位伦理委员会的批准。收集2023年6月1日至2023年12月31日于第四军医大学口腔医院牙周病科进行牙周手术治疗患者的牙周组织,所有受试者均在术前签署知情同意书。分别在冠延长术和牙周翻瓣术时收集牙龈组织健康受试者[健康对照组(control组)]和牙周炎受试者[牙周炎组(CP组)]的牙龈结缔组织各6例;原代培养牙周健康受试者来源的HGFs,并将其分为对照组(NC组)和Pg.LPS组,NC组用不含药物的培养基培养24 h,Pg.LPS刺激组用含有5μg/mL牙龈卟啉单胞菌脂多糖(Porphyromonas gingivalis lipopolysaccharide,Pg.LPS)的培养基培养24 h,利用透射电镜观察上述各组牙龈结缔组织和HGFs中线粒体的数量、形态和结构,并对其数量、周长及表面积进行定量分析;利用Mi⁃toSOXTMRed染液、TMRM染液和ATP检测试剂盒测定比较不同刺激条件下HGFs中线粒体活性氧(reactive oxy⁃gen species,ROS)、线粒体膜电位和线粒体三磷酸腺苷(adenosine triphosphate,ATP)的生成水平。结果透射电镜结果提示,牙周炎组牙龈结缔组织HGFs及Pg.LPS刺激组HGFs中线粒体的形态异常,线粒体嵴断裂甚至消失;炎症刺激下线粒体数量显著减少,线粒体表面积和周长显著增加(P<0.05)。此外,经Pg.LPS刺激后,HGFs中线粒体ROS水平显著高于对照组(P<0.05),而线粒体膜电位及ATP生成水平显著低于对照组(P<0.05)。结论牙周炎环境下,HGFs线粒体数量、形态结构及功能均发生显著改变,HGFs线粒体稳态失衡与牙周炎发生发展密切相关。Objective To investigate the changes of mitochondrial homeostasis of human gingival fibroblasts(HGFs)in periodontitis,and to provide a basis for exploring the mechanism of periodontitis.Methods This study has been re⁃viewed and approved by the Ethics Committee.Gingival tissue was collected from patients who underwent periodontal surgery at the Department of Periodontology,School of Stomatology,the Fourth Military Medical University,from June 1,2023 to December 31,2023.All of the subjects signed informed consent forms prior to surgery.Gingival connective tis⁃sues were collected from patients with chronic periodontitis(CP group)and healthy individuals(control group)undergo⁃ing flap surgery and crown lengthening surgery,respectively.There were 6 cases in each group.The primary HGFs obtained from healthy periodontal subjects were cultured and divided into the control group(cultured in complete medium for 24 h)and the Pg.LPS group(cultured in medium with 5μg/mL Pg.LPS for 24 h).The number,morphology,and structure of mitochondria in gingival connective tissue and HGFs were observed by transmission electron microscopy.The number,circumference,and surface area of mitochondria were quantitatively analyzed.MitoSOXTMRed,TMRM,and an ATP kit were used to determine the production levels of mitochondrial reactive oxygen species,mitochondrial membrane potential,and ATP in each group of HGFs.Results Transmis⁃sion electron microscopy showed that the morphology and structure of mitochondria were abnormal in the gingival connective tissues of the periodontitis group and HGFs,which were stimulated by Pg.LPS.The mitochondrial ridges were broken or were not visible in these groups.The number of mitochondria decreased significantly,and the surface area and circumference of the mitochondria in⁃creased significantly(P<0.05).In addition,after stimulation by Pg.LPS,the reactive oxygen species level in HGFs was significant⁃ly higher than that in the control group(P<0.05).The mitochondrial membrane potential and the level of ATP

关 键 词：线粒体稳态 牙周炎 牙龈结缔组织 冠延长术 牙周翻瓣术 牙龈成纤维细胞 牙龈卟啉单胞菌 脂多糖 线粒体活性氧 线粒体三磷酸腺苷 

分 类 号：R78[医药卫生—口腔医学]