白细胞介素-22通过调节菌群和E-钙黏蛋白表达保护牙龈上皮屏障的研究  

Study on the protection of gingival epithelial barrier by interleukin-22 through regulating microbiota and E-cadherin expression

作　　者：张驰 张璐 任俊贤 李金雨 谭灵屏 高雳 赵川江[1] Zhang Chi;Zhang Lu;Ren Junxian;Li Jinyu;Tan Lingping;Gao Li;Zhao Chuanjiang(Department of Periodontology,Hospital of Stomatology,Guanghua School of Stomatology,Sun Yat-sen University&Guangdong Provincial Key Laboratory of Stomatology,Guangzhou 510055,China;Department of Stomatology,The Third Affiliated Hospital of Sun Yat-sen University,Guangzhou 510630,China)

机构地区：[1]中山大学附属口腔医院牙周病科·中山大学光华口腔医学院·广东省口腔医学重点实验室,广州510055 [2]中山大学附属第三医院口腔科,广州510630

出　　处：《中华口腔医学杂志》2024年第7期653-662,共10页Chinese Journal of Stomatology

基　　金：国家自然科学基金(81870770,82370958)。

摘　　要：目的研究白细胞介素-22(IL-22)在牙周炎症环境下对牙龈上皮屏障的调控作用及其可能机制。方法构建IL-22敲除小鼠,采用混合菌液口腔灌洗法构建牙周炎模型。收集同窝生野生型牙周炎组及IL-22敲除牙周炎组小鼠(每组7只)口腔菌斑并提取DNA,建立牙周炎相关风险微生物数据库"PD-RiskMicroDB"并结合16S rRNA测序结果,测定两组小鼠口腔菌群变化和微生物功能的关系。通过改良胰酶消化法培养牙龈上皮细胞(GEC),以100μg/L IL-22、感染复数为的100牙龈卟啉单胞菌(Pg)分别或联合刺激GEC,实验分组为:对照组(细胞未加刺激)、IL-22组、Pg组及Pg+IL-22组,处理时间为3和12 h;采用细胞免疫荧光、实时荧光定量PCR(RT-qPCR)及蛋白质印迹法检测各组细胞3 h后上皮屏障蛋白E-钙黏蛋白(E-cadherin)的表达;通过异硫氰酸荧光素-葡聚糖(FITC-D)介导的上皮细胞通透性实验明确各组GEC 3和12 h细胞通透性的变化。RT-qPCR检测同窝生野生型牙周炎组及IL-22敲除牙周炎组小鼠牙龈上皮E-cadherin表达水平。将15只C57BL/6野生型小鼠按随机数字表法随机分为对照组、牙周炎组和牙周炎+IL-22处理组,每组5只。RT-qPCR及免疫组织化学(IHC)染色法检测各组小鼠牙龈上皮E-cadherin的表达水平。结果16S rRNA测序结果显示,与同窝生野生型牙周炎组小鼠相比,IL-22敲除牙周炎组小鼠口腔菌群组成发生改变,其中与牙周组织侵袭相关的菌属丰度显著增高(线性判别分析得分为2.22,P=0.009)。体外细胞实验显示,Pg感染GEC 3 h后,Pg组GEC的细胞连接中断,Pg组E-cadherin荧光强度较对照组减弱,E-cadherin的mRNA(0.69±0.12)和蛋白(0.60±0.12)表达水平均显著低于对照组(分别为1.00±0.00、1.04±0.08)(P=0.043,P=0.003);而Pg+IL-22组的E-cadherin荧光强度较Pg组稍增强,Pg+IL-22组E-cadherin的mRNA(1.16±0.10)和蛋白(0.98±0.07)表达水平均显著高于Pg组(分别为0.69±0.12、0.60±0.12)(P=0.005,P=0.007)Objective To investigate the regulatory effect and mechanism of interleukin-22(IL-22)on the gingival epithelial barrier in the context of periodontal inflammation.Methods IL-22 knockout(IL-22 KO)mice were constructed,and periodontitis mice models were established through oral gavage with polymicrobial inoculation.DNAs were extracted from the oral plaques of IL-22 KO periodontitis mice group(n=7)and their wild-type littermates periodontitis group(n=7)to establish a periodontitis-related oral microbiota database"PD-RiskMicroDB",determining the relationship between changes in oral microbiota and microbial function in two groups using 16S rRNA sequencing results.Gingival epithelial cells(GEC)were cultured by modified trypsinization method,and were stimulated with 100μg/L IL-22,Porphyromonas gingivalis(Pg)(multiplicity of infection:100),separately or together for 3 and 12 hours.The experimental groups were as follows:control group(no stimulation),IL-22 group,Pg group and Pg+IL-22 group.The expression of barrier protein E-cadherin in each group at 3 h was detected by immunofluorescence,real-time fluorescence quantitative PCR(RT-qPCR)and Western blotting.Fluorescein isothiocyanate-dextran-mediated epithelial cell permeability experiment was conducted to clarify the changes in permeability of GEC in each group at 3 and 12 h.The mRNA expressions of E-cadherin in the gingival epithelium of wild-type littermates periodontitis group and IL-22 KO periodontitis group were detected by RT-qPCR.Fifteen C57BL/6 wild-type mice were randomly divided into control group(n=5),periodontitis group(n=5)and periodontitis+IL-22 treatment group(n=5).RT-qPCR and immunohistochemistry(IHC)staining were used to detect the expression level of E-cadherin in the gingival epithelium of each group.Results 16S rRNA sequencing results showed that the composition of oral microbiota changed in IL-22 KO periodontitis group,of which the abundance of bacterial genera related to periodontal tissue invasion was significantly increased(linear discriminant ana

关 键 词：牙周炎 白细胞介素22 菌群调控 牙龈上皮屏障 E-钙黏蛋白 

分 类 号：R781.4[医药卫生—口腔医学]