血清16S rDNA水平与糖尿病的关联性研究

In the previous studies, significant associations were observed between the markers of enterocyte damage and diabetes mellitus (DM). Moreover, the gut bacteria translocation caused by enterocyte damage was proved as a critical factor involved in DM development in animal experiments. Serum 16S rDNA, mainly produced by bacteria in peripheral blood, is widely used as an indicator for bacteria translocation in previous studies. However, the population evidences for the associations between serum 16S rDNA and DM remain insufficient so far. A lack of quantitative measurement method with higher sensitivity and better specificity for the serum 16S rDNA detection is an important reason that stands in the way of the further research. In order to analyze the relationships between serum 16S rDNA and DM in population in the present study, firstly, we will develop the new real-time PCR for the serum 16S rDNA quantitative measurement. Secondly, we will conduct the nested case-control study based on the large cohort study established before (including 18550 subjects with detailed baseline information and serum samples). Thirdly, the DM patients and controls will be selected in the follow-up study according to the exclusion and inclusion criteria. The 16S rDNA levels in baseline serum samples will be quantified in all of selected subjects. Lastly, we will analyze the associations between the serum 16S rDNA and DM, and the effects of the interaction between 16S rDNA and traditional DM risk factors on the DM development. Our study will provide the new strategies for the research on the pathogeny and prevention of diabetes.

既往研究报道肠黏膜上皮细胞损伤的标志物与糖尿病密切相关，而且在动物实验中发现肠黏膜上皮细胞损伤引起的肠道细菌易位是参与糖尿病发病的重要机制。血清16S rDNA主要是肠道细菌易位至外周血后降解产生。但是，迄今为止血清16S rDNA与糖尿病的关联性尚无可靠的人群证据。缺乏敏感性高、特异性好的定量检测方法是阻碍研究的重要原因之一。我们拟通过建立基于Real-time PCR的血清16S rDNA定量检测方法，结合已有的大队列研究人群基础（已具有18550例对象的基线资料和血清标本），采用巢式病例对照研究方法，针对新发生的糖尿病病例和相应的对照，进行基线16S rDNA的检测，分析并明确血清16S rDNA与糖尿病的关联性、16S rDNA与其他传统危险因素的交互作用对糖尿病发病的影响。本研究将为糖尿病的病因研究和防控研究提供新的思路。

糖尿病在我国是一个严重的公共卫生问题。本研究采用巢式病例对照研究方法，通过高通量测序技术检测、分析血清16S rDNA 的水平，探讨血清16S rDNA与糖尿病之间的相关性，结果显示：1）糖尿病组中BMI、WC、SBP、DBP、ALT、TC和TG水平显著高于对照组，而HDL水平显著低于对照组（P<0.05）；2）高通量测序优质序列共9244618条，其中糖尿病组优质序列3000391条，对照组优质序列6244227条；3）本次测序共聚类到453个OTU，注释到17个门、29个纲、51个目、123个科和284个属，Alpha多样性结果显示，糖尿病组和对照组的Chao指数、Shannon指数和Simpson指数均无统计学差异，群落组成情况显示，在门、纲、目、科和属水平上，糖尿病组和对照组人群优势菌的组成均无显著差异；4）糖尿病组和对照组在门、纲和目水平上主要细菌的相对丰度无统计学差异。但在科水平上，糖尿病组中Burkholderiales_incertae_sedis菌科的相对丰度低于对照组（P=0.024）；在属水平上，糖尿病组中Aquabacterium菌属的相对丰度低于对照组（P=0.024）；5）糖尿病组中Actinotalea菌属、Pseudoclavibacter菌属和Sediminibacterium菌属的检出率高于对照组，而Pseudonocardia菌属、Bacteroides菌属、Afipia菌属和Xanthomonas菌属的检出率均低于对照组（P<0.05）；6）在属水平上，共筛选出7种菌属与糖尿病之间存在相关性，其中Actinotalea菌属、Pseudoclavibacter菌属、Sediminibacterium菌属、Ensifer菌属和Alishewanella菌属与糖尿病之间呈正相关，而Pseudonocardia菌属和Xanthomonas菌属呈负相关；7）Xanthomonas菌属预测糖尿病的ROC曲线下面积最大0.627；将各相关性菌属联合曲线下面积0.731。采用传统危险因素预测糖尿病的曲线下面积0.759，采用传统危险因素+相关性菌属ROC曲线下面积0.818。提示血清16S rDNA的组成与糖尿病的发病显著相关，联合传统危险因素和相关性菌属能够提高糖尿病的预测能力，研究结果将为糖尿病的病因研究和和防控研究提供新的思路。

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