基于IVF卵泡液差异蛋白-代谢物整体调控网络的肾主生殖理论研究

Our project aims at excavating scientific connotation of “Kidney governing reproduction” theory by analyzing follicular fluid on egg retrieval day of women undergoing in vitro fertilization and embryo transfer (IVF-ET) with Kidney-Yin deficiency, Kidney-Yang deficiency or Kidney-Qi deficiency and intervention of tradition Chinese medicine (TCM). According to the previous seven fund researches, our study attempts to complete the interaction network map and find biomarkers and their related pathway by the correlation analysis of differential protein and metabolites via IPA using the obtained multidimensional massive database of proteomics by using iTRAQ marker on LC-MS platform and metabolomics based on UPLC-Q-TOF/MS, GC-MS and NMR techniques. We try to conduct the quantitative verification of key proteins, targeted metabolites and biomolecules involved in focused pathway and construct the integrated regulatory network platform of differential protein-metabolite of kidney deficiency syndromes by “measure syndrome by TCM” based on LC-MS/MS, Western Blot, RT-Q-PCR and other complementary technologies. Our project provide basis for scientific prediction and diagnosis of kidney deficiency syndrome and exploration of its syndrome essence, and enrich the “kidney governing reproduction” theory via the integrated regulatory network of differential protein-metabolite via biological pathway discovery of “kidney deficiency resulting in infertility” and “reinforcing kidney for promoting reproduction”.

项目以挖掘“肾主生殖”理论的科学内涵为目标，以体外受精-胚胎移植为平台，以肾阴虚、肾阳虚、肾气虚以及补肾中药干预后的女性取卵日卵泡液为研究载体，在前七项基金研究基础上，利用LC-MS平台iTRAQ标记的蛋白质组学及UPLC-Q-TOF/MS、GC-MS、NMR多种代谢组学技术，获取多维海量数据库；借助IPA对差异蛋白和代谢物进行关联整合分析，完成相互作用网络图谱、生物标志物筛选及相关通路绘制。利用LC-MS/MS，Western Blot 和RT-Q-PCR 等多种互补技术，采取“以药测证”，对关键蛋白、目标代谢物及聚焦通路所涉及的生物分子进行定量验证，构建肾虚证的差异蛋白-代谢物互作整体调控网络平台；有助于发现“肾虚致不孕”的实质以及“补肾促生殖”的具体干预机制，为科学预测和诊断肾虚证、探讨其证候本质提供依据，力争在差异蛋白-代谢物互作整体调控网络角度丰富发展“肾主生殖”理论。

本项目以挖掘“肾主生殖”理论的科学内涵为目标，以体外受精-胚胎移植为平台，以肾阴虚、肾阳虚、肾气虚以及补肾中药干预后的女性取卵日卵泡液为研究载体。首先，利用LC-MS平台iTRAQ 标记的蛋白质组学及UPLC-Q-TOF/MS、GC-MS、NMR多种代谢组学技术，获取了多维海量数据库。随后，借助IPA对差异蛋白和代谢物进行关联整合分析，初步筛选出相关差异代谢物及差异蛋白共计116种，完成了Pantothenate and CoA biosynthesis等关键通路的绘制。最后，利用LC-MS/MS，Western Blot 和 RT-Q-PCR 等多种互补技术，采取“以药测证”，对关键蛋白、目标代谢物及聚焦通路所涉及的生物分子进行定量验证，发现补肾中药参与色氨酸代谢、甘氨酸代谢、丝氨酸代谢、苏氨酸代谢、丙酮酸代谢、甘油磷脂代谢、类固醇激素生物合成等多个代谢通路的调节，发现了VNNI、ATRN等补肾中药的多个作用靶点，构建了肾虚证的差异蛋白-代谢物互作整体调控网络平台。本研究进一步阐释了“肾虚致不孕”的实质和“补肾促生殖”的具体干预机制，为科学预测和诊断肾虚证、探讨其证候本质提供了依据，从差异蛋白-代谢物互作整体调控网络角度丰富发展了“肾主生殖”理论。