草酸肠道转运及草酸钙结石形成中肠道菌群分泌的关键调控蛋白的宏蛋白质组学筛选和验证

Nephrolithiasis is a common disease, secondary to hypertension. Although calcium oxalate stones make up 80% of the kidney stones, its cause remains unclear, leading to the poor effect of preventive strategies. Previous studies on calcium oxalate stones ignored the vital role of intestinal bacterial flora, and only focused on the direct degradation of oxalic acid by the bacteria. Recently, it’s suggested that bidirectional transport between the gut and intestinal epithelial cells may be a more important factor in regulating the oxalate level in serum. Intestinal bacterial flora had a great impact on the intestinal SLC26 oxalate transporter, however, the key compounds haven’t been known yet. Basing on the previous research, a rat model of calcium oxalate stone will be established by excessive intake of oxalic acid precursor. We will explore the relationships among the intestinal bacterial flora, intestinal SLC26 oxalate transporter and calcium oxalate stones with metagenomics techniques. At first, the differentially expressed proteins of intestinal bacterial flora between stone group and non-stone group will be screened by iTRAQ techniques, followed by a second screening with bioinformatics analysis soft wares. Then, for confirming the differentially expressed proteins, we need to perform a plasmid transfection in vitro and achieve the protein overexpression. Finally, the proteins that are excreted by intestinal bacterial flora and can regulate the SLC26 oxalate transporter will be found out. We believe the results do will help identify the role of intestinal bacterial flora in oxalic acid metabolism and the formation of calcium oxalate kidney stones. It’s also helpful in understanding how the intestinal flora, together with diet and human genes, exerts the synergistic effects on oxalate metabolism. It also will be helpful to find proper intestinal microbiome intervention strategy to reduce nephrolithiasis incidence effectively.

肾结石是仅次于高血压的常见病。80%的肾结石是草酸钙结石，但成因不清，导致预防措施效果不佳。以往研究草酸钙结石忽视了肠道菌群的重要作用，仅关注细菌对草酸的直接降解。最新研究提示草酸在肠腔和肠道细胞间的双向转运也许是调控体内草酸水平的更重要因素。研究发现，肠道菌群会对肠道草酸SLC26转运体产生重要影响，但产生该影响的作用物质暂不清楚。本研究将在前期研究基础上，在大鼠消化道草酸过量摄入模型中，用宏基因组学技术分析肠道菌群，SLC26转运体和草酸钙结石三者间关系。并用iTRAQ技术筛选出结石与非结石组肠道菌群的差异表达蛋白，用生物信息学分析软件二次筛选，体外质粒转染过表达加以验证，找出肠道菌群产生的调控SLC26转运体功能的蛋白质。有助于明确肠道菌群在草酸代谢和草酸钙结石形成中的作用；有助于明确肠道菌群如何与饮食及人体基因协同对草酸代谢发挥影响，有助于制定肠道菌群干预方案，有效降低结石发生率。

肾结石是仅次于高血压的常见病，大部分肾结石的成分是草酸钙结石，但成因不清，因此并无高效的预防措施。本研究跳出以往认识的局限，重视肠道菌群及其代谢产物对草酸平衡的影响，探寻肠道菌群及其代谢产物短链脂肪酸（SCFAs）在该过程中的调控作用和机制，为今后草酸钙结石的防治提供了重要靶点及理论支撑。.首先，我们收集了153例肾草酸钙结石和非结石健康人群粪便标本。通过16S rRNA、宏基因组、气相色谱质谱法等技术手段，检测并分析了肠道菌群的构成及功能差异。我们发现两组人群在肠道菌群的构成和功能上具有显著差异，且产SCFAs的菌群和SCFAs可能在肾草酸钙结石的形成过程中具有重要作用。鉴于目前临床尚无良好的结石发生预测模型，课题组创新性的将其引入了预测肾草酸钙结石发病的模型。我们尝试使用8种不同机器学习方法预测草酸钙结石的发生，发现结合3个特征菌属和5个临床指标，使用随机森林模型的受试者工作特征曲线下面积AUC最高（0.936），该结果具有重要的临床价值。.为了验证SCFAs在肾草酸钙结石形成中的作用，我们建立了肾草酸钙结晶的大鼠模型，通过补充SCFAs发现其可以减少肾草酸钙结晶的形成并影响结晶模型大鼠的肠道菌群的特征及其代谢产物SCFAs的水平。然后，为了探究SCFAs减少肾草酸钙结石形成的作用机制，我们关注各段肠道草酸转运体的表达情况，发现SCFAs可以调控肾草酸钙结晶模型大鼠的肠道草酸转运体SLC26A3/6的表达。最后，为了证明SCFAs是通过影响肠道草酸转运体，进而减少肾草酸钙结晶的形成，课题组使用大鼠腺相关病毒9，构建草酸转运体的过表达和敲低模型，证实了SCFAs通过肠道草酸转运体SLC26A3/6影响肾草酸钙结石的形成。.本研究创新性的提出了肠道菌群及其代谢物产物在肾草酸钙结石形成中的重要作用，为有效预测、预防肾结石提供了理论依据，为预防草酸钙结石形成及复发的制剂研发提供了新思路。