从新疆维哈2型糖尿病人粪便分离的关键菌群致小鼠2型糖尿病的作用及机制

The latest research shows that the gut flora is closely associated with type 2 diabetes mellitus (T2DM). But there is not enough evidence to show a causal relationship between gut flora and T2DM in the world. Our previous study showed that the incidence of T2DM in Xinjiang Kazakhs was far lower than the Uighurs (8.16%), only 1.47%. To explore its causes, we found the characteristics of intestinal flora in normal glucose tolerance (NGT) and T2DM population. Based on Illumina 16S rDNA hypervariable region sequencing technology, the abundance of Desulfovibrionaceae, Veillonellaceae, Aeromonadaceae of feces in Kazakhs with T2DM group were significantly increased, but the content of butyric acid from GC-MS was significantly decreased while butyric acid is beneficial for intestinal health. The abundance of Coriobacteriaceae in Uighurs with T2DM were significantly higher. Compared with the NGT group in Kazakhs, the abundance of Bacillaceae, Fusobacteriaceae were significantly higher in Uighurs NGT group. Therefore, we assume that the differences of intestinal flora of the two ethnics may have a role in the development of T2DM. We intend to isolate、culture and identify these key fecal flora from the Kazakhs and Uighurs . Then they will be tied back to normal, sterile or low bacteria animals to copy animal models of T2DM. We will explore the role and mechanism of T2DM arising from different intestinal flora, to provide more definitive evidence for the "causes and consequences" between intestinal flora and T2DM, and to provide new ideas for early prevention and effective treatment of T2DM..

最新研究表明肠道菌群与2型糖尿病（T2DM）密切相关，但目前还缺少足够证据来说明肠道菌群与T2DM发生的因果关系。本课题组前期研究表明：新疆哈族T2DM发病率低于维族（8.16%），仅为1.47%，对其原因探究，发现两民族正常糖耐量人群（NGT）与T2DM人群差异肠道菌群特征：基于Illumina16S rDNA 高变区测序技术，哈族T2DM组粪便脱硫弧菌、韦荣球菌、气单胞菌丰度显著升高，对肠道有益的丁酸(GC-MS检测)明显下降；维族T2DM组红椿菌菌群丰度显著升高；与哈族NGT组相比，维族NGT组芽孢杆菌，梭杆菌菌群丰度显著升高；故假设维、哈两民族T2DM人群差异肠道菌群可能对T2DM发生发展有一定作用。拟从维哈人群粪便内培养分离鉴定上述差异菌，将其回接到正常、无菌或低菌动物，复制2型糖尿病动物模型，探索差异菌对T2DM的作用及机制，为肠道菌群与T2DM之间的"因果问题"提供明确的佐证

从新疆维、哈两民族人群粪便内对靶标菌进行培养分离鉴定，并分别获得脱硫弧菌、气单胞菌、丹毒丝菌、具核梭杆菌的富集液及韦荣球菌菌液，并将其回接到正常小鼠体内，成功诱导出了以胰岛素抵抗为特征的2型糖尿病小鼠模型，并运用二甲双胍及黄连素进行干预，拟阐明靶标菌对T2DM的发生的机制 。本课题组顺利完成项目计划书内容的同时，还获得了具核梭杆菌富集液，并再次为肠道菌群与T2DM之间的关系增加了实验依据。我们的研究成果如下：1.揭示了从维吾尔族或哈萨克族2型糖尿病患者中获得的靶标菌及其共生菌液可以诱导出正常小鼠的糖脂代谢紊乱--即以胰岛素抵抗为特征的2型糖尿病小鼠模型。.2.量效关系上：共生菌原液就可以诱导胰岛素抵抗模型，稀释液及浓缩液效果都不如共生菌原液。3.高脂饮食比本研究中的靶标菌更容易诱导出正常小鼠的以胰岛素抵抗为特征的2型糖尿病小鼠模型：表现在4周灌胃高脂饮食胰岛素抵抗模型建立，而正常饮食加共生菌液一般在灌胃后8周开始诱导出胰岛素抵抗模型。4.用RT-PCR定量分析小鼠粪便脱硫弧菌共生菌液中脱硫弧菌水平的变化，来衡量定植与否：与正常饮食组小鼠相比，正常饮食+脱硫弧菌组小鼠自第8周至第16周，粪便中脱硫弧菌水平升高，差异具有统计学意义（p<0.01，p<0.01）；与高脂饮食组相比，高脂饮食+脱硫弧菌组小鼠自第4周至第16周，粪便中脱硫弧菌水平升高，证实脱硫弧菌在小鼠肠道内定植成功。这与高脂饮食+脱硫弧菌组灌胃第4周就出现糖脂代谢紊乱，而正常饮食+脱硫弧菌组在第8周出现糖脂代谢紊乱相一致。5.用CM-Dil标记荧光法进行各菌共生菌液定植力检测。此方法已经很稳定，值得推广用于肠菌是否定植的考量。6.正常饮食加共生菌液，高脂饮食，高脂饮食+共生菌液三个组糖脂代谢紊乱的程度逐级升高，提示共生菌液菌能进一步加重高脂诱导的糖脂紊乱 。7.二甲双胍/ 黄连素对高脂饮食+菌组的指标改善较弱于正常饮食+菌组、高脂饮食组，这与我们前期诱导胰岛素抵抗模型时发现的共生菌液能进一步加重高脂诱导的糖脂紊乱相一致，也与-正常饮食加共生菌液，高脂饮食，高脂饮食+共生菌液三个组糖脂代谢紊乱的程度逐级升高相一致，提示二甲双胍/ 黄连素对高脂饮食+共生菌液的糖脂代谢紊乱的治疗存在一定的难度，也进一步证明了共生菌可加重高脂诱导的糖脂代谢紊乱，其机制值得进一步深入探讨。