基于代谢组学蛋白质组学多组学技术的锌缺乏生物标志物筛选和代谢通路分析研究

Zinc deficiency is a global public-health problem, the same as in China. Standing untreated zinc deficiency can cause series severe consequences, including growth retardation, diarrhea, dermatitis and impaired immunity. Current methods for assessing Zn status are limited to measuring plasma or serum Zn within populations suspected of deficiency. Despite the high prevalence of Zn deficiency in the human population there are no methods currently available for sensitively and specifically assessing Zn status among individuals. Although we have learned a great deal about zinc physiological and biochemical function, a comprehensive new research between zinc deficiency and body metabolism was necessary: how to effect the physiological status or lead to pathological status, how to participate in the catalysis, structure and regulating function of the overall body and weather there were other adverse effects of zinc on the body. .Therefore, this study was to utilize a multi-omics method of proteomics and metabonomics using liquid chromatography and mass spectrometry to identify biomarkers (proteins, metabolites) that were sensitive to changes in Zn deficiency in zinc deficiency animals. Then, the reliable biomarkers of zinc deficiency in human were identified and verified by zinc nutrition intervention and zinc defiency human. The zinc nutrition states were assessed by the established method of assessing body's zinc nutrition state and zinc supplements were guided. On the other hand, we investigated the effect of the metabolic profiling of zinc deficiency by pathway analysis based on the multi-omincs. The combination of the genes/protein/metabolites, multi-omics method, pathway analysis software and metabolic database based on their topologies was used to recall disease-relevant subpathways and find novel subpathways, and elucidate the possible molecular mechanisms of pathological alterations and speculate other adverse effects of zinc on the body.

锌缺乏是世界性的公共卫生问题，在我国同样普遍存在。严重的锌缺乏可导致机体生长迟缓、腹泻和免疫低下等。虽然血清或血浆锌水平可用于人群锌缺乏的评估，但是其敏感性和特异性差，不适用于个体锌缺乏的评价。另外，尽管对锌复杂的生物化学功能和生理作用有较详细的了解，但是对于锌缺乏如何影响机体生理状态或导致疾病，如何参与机体的整体催化、结构和调节，对机体还有哪些未知不利影响，仍缺乏整体系统深入的研究。因此，本研究首先利用代谢组学和蛋白质组学多组学联合技术筛选锌缺乏大鼠的差异代谢产物；然后通过多组合联合技术在锌缺乏人群验证动物实验结果，确定人群特异的锌缺乏生物标志物并建立评价机体锌缺乏的方法，准确评价机体锌营养状态，指导合理补锌。同时，通过多组合、差异蛋白/代谢产物/基因、代谢通路软件和代谢数据库等联合的代谢通路分析完善已有的和发现新的锌缺乏相关代谢通路，尝试解析和完善锌缺乏对机体改变的代谢途径和机制。

在本课题中，我们成功通过低锌膳食建立锌缺乏大鼠模型，完成了血液、尿液和粪便的代谢组学实验，血液、肝脏、心脏和海马体的蛋白质组学实验，粪便的肠道菌群多样性分析和宏基因组分析，多种组织的离子组学研究，筛选出大量可靠的锌缺乏生物标志物，140个差异代谢产物，1900多个差异蛋白质，12个金属离子和10几个差异差异肠道菌群。基于选出可靠的锌缺乏生物标志物，通过多组学联合分析发现许多新的锌缺乏潜在危害，例如外源性物质的代谢与转运受损、心脏疾病、癌症、衰老、肠道菌群紊乱等；丰富对已知危害的认识，例如生长发育迟缓、免疫功能受损、血糖升高、骨密度降低等。.其中谷胱甘肽代谢通路同时被多个组学验证，证实锌缺乏可影响机体的谷胱甘肽代谢通路，经定量检测分析和Western Blot验证，证实代谢组学和蛋白质组学结果，筛选出5个生物标志物。评估锌缺乏易感人群小学生和物业保洁人员的锌营养状态，并对锌缺乏物业保洁人员进行随机的补锌干预实验，验证筛选的5个生物标志物，初步证实谷胱甘肽转移酶欧米伽1是可靠的锌缺乏生物标志物，与血清锌和膳食锌相关，具有较高的灵敏度和特异度。.利用ICP MS 检测了锌缺乏大鼠血液、尿液、粪便、肝脏中16种矿物质和微量元素含量分析，发现锌缺乏可导致体内12种微量元素发生变化。.通过重复的动物实验，阐明了锌缺导致骨密度降低的具体机制：锌缺乏通过钙调蛋白拮抗钙，影响机体的钙代谢，尤其是钙的分布，为了维持血钙水平，增加PTH水平动员骨钙导致骨密度降低。.通过重复的动物实验、水迷宫实验和蛋白质组学实验，发现轻度锌缺乏对刚断乳大鼠大脑发育和功能基本没有影响，但需要进一步验证。.通过心脏超声、病例切片、蛋白质组学和差异蛋白质的PRM验证，发现锌缺乏可显著影响大鼠心脏功能，例如松弛素代谢通路。.我们的锌缺乏多组学研究证实锌缺乏可影响机体的蛋白质、代谢产物、肠道菌群和金属离子，发现许多新的生物标志物和潜在危害，增加和完善我们对锌缺乏对机体危害的认识。