利用功能基因组学手段开展胰岛素原C肽的受体鉴定及信号通路研究

C-peptide, a byproduct created during insulin maturation, is released simultaneously with insulin. C-peptide is increasingly recognized as an important bioactive peptide, and its defects are involved in the pathogenesis of diabetes complications, especially vascular disease. However, the receptor for C-peptide has not yet been discovered, which has become a most critical question for breakthroughs in the C-peptide field. In previous studies, we have established a stable functional assay system for C-peptide in mouse renal tubular epithelial cells, and set up a platform for cDNA library and genome-wide siRNA library screening based on high-throughput and high-content assay systems. In this project, we plan to identify and characterize C-peptide receptor and its signaling components by three approaches: affinity-amplified pull-down, cDNA library screening and whole-genome siRNA library screening. Then we will utilize tool compounds library to further characterize C-peptide signaling pathways. Based on these explorations, a global picture of C-peptide intracellular signaling pathways would be mapped and further careful studies will be carried out accordingly. The identification of C-peptide receptor and its signaling components is of great significance not only for understanding pathogenesis of diabetes complications, but also for discovery of novel drug targets for the diabetes complications treatment.

C肽是胰岛素成熟过程中被剪切下来的一段多肽，与胰岛素同时释放。近年研究发现C肽自身具有生物学活性，其水平的异常与糖尿病并发症尤其是血管病变的发生发展密切相关。但是，C肽的受体一直未被鉴定，已成为阻碍该领域取得进一步突破的重要问题。前期工作中，我们在小鼠肾小管上皮细胞系中建立了稳定的C肽功能检测体系，并搭建了基于高内涵定量分析的cDNA文库及全基因组siRNA文库高通量筛选系统。我们拟通过亲和沉淀法、cDNA文库过表达法及全基因组siRNA文库筛选法三种方案鉴定C肽受体及参与C肽信号转导的分子；在此基础上，我们将利用工具化合物库进一步开展C肽信号通路的研究。利用这些研究提供的新信息，我们将能从整体水平认识C肽所介导的信号通路，并进一步展开针对性的深入研究。C肽受体和相互作用蛋白的鉴定以及C肽信号通路的研究将使我们更好的认识糖尿病并发症的发病机制，并为糖尿病并发症的防治提供创新性的重要靶点。

C肽是胰岛素成熟过程中被剪切下来的一段多肽，与胰岛素同时释放。近年研究发现C肽自身具有生物学活性，其水平的异常与糖尿病并发症尤其是血管病变的发生发展密切相关。但是，C肽的受体一直未被鉴定，已成为阻碍该领域取得进一步突破的重要问题。本项目计划通过搭建全基因组cDNA过表达文库及全基因组siRNA文库的高通量筛选体系，鉴定C肽受体及其信号通路相关分子。我们利用Western印记法及免疫荧光染色法发现，C肽刺激前后Akt及JNK的磷酸化水平变化比较微弱，还不适合作为高通量筛选的指标，目前我们还在继续探索C肽更加显著的细胞学表型。但是通过本项目的执行，我们顺利完成了全基因组ORF文库整套质粒的制备工作，并建立了成熟稳定的基于自动化液体工作站的全基因组ORF文库过表达筛选体系，以及全基因组siRNA筛选体系。为了建立C肽生物学功能相关的检测体系，我们还建立了适于高通量筛选的免疫荧光染色流程及高内涵成像及图像分析方法，并建立了3D微球体的体外培养及筛选体系。在本项目基金的支持下，我们利用建立的一系列筛选体系，在多种细胞体系中进行了筛选工作，并开展了基于3D微球体的体外药物筛选。这些体系的建立为C肽生物学表型确立后的受体筛选工作奠定了基础。