学龄前儿童“口腔菌群年龄”的遗传基础和过程机制

Over the entire life span, human health and nutrition are closely linked to their co-inhabiting and co-evolving oral microbiota. In our past published work, we proposed a novel, microbiome-based concept of “Oral Microbial Ages” (OralMA) in healthy children of 4~6 year old that can model the chronological ages of the hosts; moreover, the OralMA would be retarded or delayed, as compared to the chronological age of host upon the onset of oral caries. However the genetic nature and mechanisms of OralMA remain poorly understood. To tackle these problems, this study proposed the following specific aims. (i) The plaque and saliva microbiota will be longitudinally sampled and multi-dimensionally dissected, with particularly high temporal resolution, in a cohort of age-controled children. The OralMA will be identified and validated based on correlation analysis between the cohort-wide, time-series metagenomics datasets and host chronologic age. (ii) A mechanistic model of the oral microbiota (and in particular the OralMA-associated bacterial species) that links the 16S sequences, genomes and function will be established, via DNA-probe-based Fluorescence In Situ Hybridization (FISH) and the single-cell phenotyping-genotyping technologies (consisting of single-cell Raman imaging, Raman-activated cell sorting and the coupled single-cell sequencing). (iii) OralMA will be validated in another cohort population and its value in prediction of caries onset be tested. These efforts will eluciate the genetic nature and mechanisms of the Oral Microbial Age, and might provide new strategies and new tools for preventive intervention of oral diseases in children.

口腔菌群与人类终生相伴,且与人体健康关系密切。我们前期针对4~6岁儿童，首次提出和定义了“口腔菌群年龄(Oral Microbial Age；OralMA)”，并发现其可定量表征宿主的生理年龄，且在龋病发生时会产生明显滞后的现象。但这些观察的遗传基础和过程机制尚不清楚。为了阐明这一问题，我们将首先，针对学龄前儿童开展口腔菌群纵向追踪研究，通过高频率、多位点的宏基因组和宿主年龄的关联分析，精确甄选OralMA的核心物种并鉴别其基因型；其次，依托与序列特异DNA荧光探针耦合的单细胞分析技术(单细胞拉曼成像、分选和与之耦合的测序)，从细胞个体水平建立OralMA的物种、基因组、个体功能及与菌群功能间的研究模型；最终，在健康和龋病人群中验证OralMA，并评估其用于监测宿主口腔健康状态的应用潜力。这些努力将阐明OralMA的遗传基础和过程机制，从而为腔疾病的预防开拓新思路和新方法。

口腔菌群与人类终生相伴,且与人体健康关系密切。我们前期针对4~6岁儿童，首次提出和定义了“口腔菌群年龄(Oral Microbial Age；OralMA)”，并发现其可定量表征宿主的生理年龄，且在龋病发生时会产生明显滞后的现象。但这些观察的遗传基础和过程机制尚不清楚。为了阐明这一问题，我们针对4岁儿童在单牙位尺度开展了2150个口腔菌群历时13个月的纵向追踪研究。首先，通过高频率（3个时间间隔）、多位点的宏基因组和宿主年龄的关联分析，精确甄选OralMA的核心物种并鉴别其基因型；其次，依托 单细胞拉曼技术，从细胞个体水平建立口腔菌群的物种、基因组、个体功能及与菌群功能间的研究模型；最终，在健康和龋病人群中验证OralMA，并评估了其用于监测宿主口腔健康状态的应用潜力。本研究重要发现是：（1）在健康儿童中，OralMA微生物群落从前（切牙）到后（磨牙）沿生态梯度变化；（2）龋齿儿童中的OralMA的空间异质性会显著下降，表明龋齿诱导/依赖的口腔微生物群年龄在空间上存在重组现象,且龋病延迟了口腔微生物群发育，在所有空间牙位表现上相对一致；（3）精确甄选出OralMA的核心物种并鉴别其基因型和功能，并基于每个独立牙位的口腔结构特点，建立了特异性OralMA龋病诊断预警模型。OralMA可以93％的准确度诊断龋齿并以84％的准确率预警龋病的发生。此外，本研究优化现有宏基因组和单细胞拉曼平台：（1）我们发现酶和珠子联用的DNA提取方法可获得最为精准的口腔菌群结果，而V3-V4区和V4-V5区扩增可提供最好的重复性结果，本部分工作为建立标准化宏基因组测序平台提供了理论依据；（2）初步建立了唾液中未知微生物的单细胞分离和核酸扩增方法，获得高质量的扩增基因组DNA，用于文库构建和测序，为本项目中“复杂群落中活体细胞的原位表型和功能分析”提供了技术方法。这些努力将阐明OralMA的遗传基础和过程机制，从而为腔疾病的预防开拓新思路和新方法。