摘要
目的运用单细胞转录组测序分析牙周炎小鼠脑膜的生物学改变,探讨牙周炎小鼠脑膜生物学改变与认知障碍的相关性。方法将30只C57BL/6小鼠按随机数字表法随机分为2组(每组15只),在对照组小鼠上颌双颊侧局部涂抹不含牙龈卟啉单胞菌(Pg)的2%羧甲基纤维素(CMC),在实验组小鼠上颌双颊侧局部涂抹Pg W83和2%CMC的混合物,3次/周,持续16周。观察对照组和实验组小鼠上颌牙槽骨吸收情况、自主活动能力与认知功能的变化、大脑皮层中小胶质细胞和星形胶质细胞激活情况、检测小鼠脑膜和大脑内紧密连接蛋白Occludin mRNA表达情况。然后使用统一流形逼近与投影(UMAP)算法对单细胞转录组各细胞亚群数据进行降维整合处理。对内皮细胞差异基因进行基因本体(GO)和京都基因和基因组数据库(KEGG)富集分析,进一步采用实时荧光定量PCR(RT-qPCR)验证差异基因转录激活因子3(Atf3)、含载脂蛋白L域1(Apold1)的表达情况。结果亚甲蓝染色实验显示,实验组小鼠上颌颊、腭侧釉质牙骨质界到牙槽嵴顶的距离[分别为(185.60±17.60)、(206.90±13.37)μm]均显著大于对照组[分别为(135.33±9.57)、(163.05±14.98)μm](t=5.02,P=0.002;t=4.37,P=0.005)。旷场实验显示实验组小鼠的总路程和平均速度[(971.88±164.57)cm和(3.25±0.55)cm/s]与对照组[(914.24±278.81)cm和(3.05±0.93)cm/s]相比差异均无统计学意义(t=0.65,P=0.525;t=0.65,P=0.520)。新物体识别实验中实验组小鼠相对辨别指数[(48.02±16.92)%]显著低于对照组[(66.27±17.90)%](t=2.40,P=0.027)。Y迷宫实验显示,实验组小鼠的自发交替率[(50.99±14.17)%]显著低于对照组[(63.56±11.88)%](t=2.33,P=0.030)。免疫组化染色结果显示,实验组小鼠脑内小胶质细胞和星形胶质细胞被激活。RT-qPCR结果显示,实验组小鼠脑膜和大脑中紧密连接蛋白Occludin mRNA的表达(分别为0.61±0.10、0.64±0.20)均显著低于对照组(分别为1.02±0.2
ObjectiveTo clarify the potential correlation between biological changes of meninges in periodontitis mice and cognitive impairment by analyzing the biological changes of meninges in periodontitis mice using single-cell RNA sequencing.MethodsThirty C57BL/6 mice were divided into two groups by using random number table method(15 mice in each group).Mice in the control group were locally administered 2%carboxyl methyl cellulose(CMC)without Porphyromonas gingivalis(Pg)on both buccal sides.A mixture of Pg W83 and 2%CMC was applied on both buccal sides in the experimental group mice three times a week,lasting for 16 weeks in total.The absorption of alveolar bone,locomotor activity and cognitive function,the activation of microglia and astrocytes in the cortex were observed and assessed.The mRNA expression levels of Occludin in meninges and brain were detected in two groups.Single-cell RNA sequencing data of meninges were processed by uniform manifold approximation and projection(UMAP).Differential genes expressions of endothelial cells were processed by gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis.In addition,real-time fluorescence quantitative PCR(RT-qPCR)was used to verify the expressions of transcription activating factor 3(Atf3)and apolpoprotein L domain-containing 1(Apold 1).ResultsMethylene blue staining found the distances of buccal and palatal cement-enamel junction-alveolar bone crest in experimental mice[(185.60±17.60),(206.90±13.37)μm]increased significantly compared with the control group[(135.33±9.57),(163.05±14.98)μm](t=5.02,P=0.002;t=4.37,P=0.005).Open field experiment showed the total distance and average speed of mice in the experimental group[(971.88±164.57)cm,(3.25±0.55)cm/s]were not statistically significant compared with the control group[(914.24±278.81)cm,(3.05±0.93)cm/s](t=0.65,P=0.525;t=0.65,P=0.520).The recognition index of the experimental group[(48.02±16.92)%]was lower than the control group[(66.27±17.90)%](t=2.40,P=0.027)by novel object r
作者
江旖婷
徐丽娜
赵旭日
沈慧
邱澈
何智妍
周薇
宋忠臣
Jiang Yiting;Xu Lina;Zhao Xuri;Shen Hui;Qiu Che;He Zhiyan;Zhou Wei;Song Zhongchen(Department of Periodontology,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,China;Laboratory of Oral Microbiota and Systemic Disease,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,China)
出处
《中华口腔医学杂志》
CAS
CSCD
北大核心
2024年第6期595-603,共9页
Chinese Journal of Stomatology
基金
国家自然科学基金(82270974、82071112)
上海市“科技创新行动计划”自然科学基金(22ZR1437500)。
关键词
牙周炎
紫单胞菌
龈
认知障碍
神经炎症
单细胞转录组测序
脑膜
Periodontitis
Porphyromonas gingivalis
Cognitive impairment
Neuroinflammation
Single-cell RNA sequencing
Meninges
