【作者】 陶一帆； 凌均棨； 高燕；

【Author】 Yifan Tao;Junqi Ling;Yan Gao.;Operative Dentistry and Endodontics,Affiliated Stomatological hospital,Guanghua School of Stomatology,Sun Yat-Sen University,Guangzhou,China.Guangdong Province Key Laboratory of Stomatology;

【机构】 中山大学光华口腔医学院·附属口腔医院·牙体牙髓病科广东省口腔医学重点实验室；

【摘要】 目的:牙髓根尖周病是由多细菌参与的慢性感染性疾病,根管治疗虽是目前最有效的治疗手段但仍有2%～14%的失败率,既往研究均将感染根管内菌群作为整体而忽略了个体间的功能差异,即"功能异质性"。因此,针对感染根管内菌群开发新型单细胞研究体系,在单细胞尺度监测个体功能,有望在最深层面揭示菌群功能,为牙髓根尖周病的"精准"治疗提供理论依据和实验基础。基于单细胞拉曼技术能够原位,非侵害性,快速并且高通量地检测微生物群落内各个细菌细胞化学表征的特点,本研究提出首先利用重水同位素拉曼标记技术,基于抗菌药物作用下细菌对重水的代谢差异,实现对细菌耐药性的快速判断并且提出菌体细胞耐药性的定量指标;同时,结合单细胞拉曼分选和基因组技术在基因水平上对上述方法进行验证和机制研究,为口腔单细胞拉曼研究体系进一步临床应用提供更精准、高效的方法和工具。方法:选取前期研究基础中己发现的能够代表龋病患者菌群结构和功能变化的关键微生物逐一为研究对象。(1)活体单细胞的表型鉴定。分别采集口腔常见致龋菌不同生长时期的拉曼图谱。(2)活体单细胞的基因型鉴定。将对应的致龋菌依据拉曼光谱进行单细胞分选,通过单细胞DNA扩增获取物种基因组信息。(3)建立完善的致龋微生物拉曼表型-基因型比对数据库。结果:本研究结果表明单细胞拉曼技术可作为口腔微生物分类的标准和依据。结合基因组测序验证手段,单细胞拉曼技术可作为口腔群落微生物的直接、快捷和原位判别手段。进一步采用重水拉曼技术在单细胞水平表征不同口腔抑菌药物的作用效果,证实拉曼光谱中的重水峰是细胞生理代谢活性的生物学指示标志。另外,该技术在快速鉴别筛选耐药性和易感菌株方面可直接跳过细菌培养阶段,0.5 h内即可得到可靠结果。结论:本课题组推测重水拉曼技术能够作为一种普适性的、无损且定量的口腔药物评价手段,理论上该技术可以扩展到所有微生物细胞和所有对细胞代谢水平产生影响的药物,因而具有重要的临床和科学价值。更多还原

【Abstract】 Objectives:Pulpal and periapical disease,a polymicrobial infection,is mediated not by a single pathogen,but by a microbial community.Post-treatment disease has been reported to occurr in 2%-14%of root-filled teeth,The "functional heterogeneity" between individual members were neglected in investigations of endodontic microbiota by former studies.Therefore develop a new single-cell research system for endodontic microbiota and interrogation of the fuctional activity of both microbial community and its individual members,at the resolution of "a single-cell",will be able to unravel the "deepest" functional heterogeneity in the endodontic microbiota.Methods:First we set up the D₂ O-Raman evaluation system in cariogenic oral pathogen Streptococcus mutans UA159 and further test its ability in serving as a universal method for rapid,general applicability and quantitative assessment of antimicrobial effects.Results:By using D₂ O-Raman as model system,we proposed "Minimum Inhibitory Concentration based on Metabolic Activity"（MIC-MA） to evaluate the metabolism-inhibiting efficacy of drugs.In addition,Heterogeneity Index of MIC-MA（MIC-MA-HI） was proposed to quantitatively assesses the among-cell heterogeneity of metabolic activity after drug regimens.Conclusion:we developed a novel Raman stable isotope probing（Raman-SIP） with reverse labelling to study metabolic interactions in a two-species community,this study demonstrates that Raman-SIP with D₂ O labelling is an externally label-free,semi-quantitative,sensitive and reliable strategic approach for probing cellular metabolism,metabolic activity and interactions in microbial communities at the single cell level.更多还原