蛋白质组芯片与iTRAQ技术联用研究穿心莲内酯抗炎靶点及作用机制

Andrographolide is widely used as anti-inflammatory monomer in clinic. The anti-inflammatory mechanism of andrographolide has been explored many years , and a lot of useful data have been obtained. However, the specific anti-inflammatory targets and signaling pathways of andrographolide are still unclear. At present, the direct screening of andrographolide protein targets is mainly based on affinity purification, which is easy to lose low abundant proteins, and interference exists between proteins in complex system. Proteomic chip as one of the mainstream drug target screening technologies can effectively screen low abundance protein targets from cells. ITRAQ technology can find signal pathways involved in drug action with high throughput. The applicant has been engaged in protein chip research for a long time. The applicant has been engaged in protein chip and proteomics research for a long time, and has successfully screened several molecular targets of human diseases. At the same time, the preliminary experiments of this subject have proved that the combination of proteomic chip and iTRAQ technology is technically feasible. In conclusion, this subject propose to screen andrographolide specific anti-inflammatory targets by proteomic chip and selected the correct specific targets by its anti-inflammatory response pathway from iTRAQ technology, and then verify the results to explore new anti-inflammatory targets and mechanisms of Andro.

穿心莲内酯（Andro）是临床上使用广泛的抗炎中药单体，学界对穿心莲内酯的抗炎机制进行多次探索，取得大量有益数据，但该药特异性抗炎靶点和信号通路研究仍不完善。目前Andro靶点筛选主要基于亲和纯化方法，该方法易造成蛋白损失和相互干扰。蛋白质组芯片作为主流药物靶点筛选技术，能有效筛选低丰度蛋白靶点；iTRAQ技术是成熟的差异蛋白分析方法，可以系统筛选药物作用信号通路。将蛋白质组芯片与iTRAQ技术联用既能提高蛋白质组芯片筛选靶点的特异性，又能明确药物作用的信号通路。本课题申请人长期从事蛋白质组学研究，成功筛选到多个人类疾病分子靶点，同时前期实验证明蛋白质组芯片和iTRAQ技术联合用于Andro抗炎靶点和信号通路研究具有技术可行性。综上，本课题提出运用蛋白质组芯片对穿心莲内酯特异性抗炎靶点进行筛选，借助iTRAQ技术得到的信号通路对抗炎靶点进行遴选并验证，探索穿心莲内酯抗炎特异性新靶点和机制。

穿心莲内酯（Andro）是临床上使用广泛的抗炎中药单体，学界对穿心莲内酯的抗炎机制进行多次探索，取得大量有益数据，但该药特异性抗炎靶点和信号通路研究仍不完善。目前Andro靶点筛选主要基于亲和纯化方法，该方法易造成蛋白损失和相互干扰。蛋白组芯片作为主流药物靶点筛选技术，能有效筛选低丰度蛋白靶点；定量蛋白质组学是成熟的差异蛋白分析方法，可以系统筛选药物作用信号通路。将蛋白质组芯片与定量蛋白质技术联用既能提高蛋白质组芯片筛选靶点的特异性，又能明确药物作用的信号通路。..本项目的主要研究计划包括：.第一：穿心莲内酯的生物素标记，药效评价, 穿心莲内酯的人蛋白质组芯片的靶点筛选。预期建立穿心莲内酯炎症研究细胞模型、药效评价数据、候选靶点。.第二：定量蛋白质组学分析穿心莲内酯的抗炎信号通路并实验验证，通过抗炎信号通路对候选靶点进行遴选，通过分子生物技术对靶点和信号通路进行二次验证。预期实现穿心莲内酯抗炎靶点与抗炎通路的实验验证、功能验证，提出穿心莲内酯新的抗炎靶点和机制。.第三，预期发表SCI文章2篇。..根据项目的科研计划，本基金严格按照计划开展科研任务，研究内容上，完成了计划设定的研究内容：1..穿心莲内酯生物素标记和细胞模型建立、药效评价；2..生物芯片和组学联用筛选穿心莲内酯抗炎靶点，针对PDCD2L靶点进行了抗炎功能和机制研究、验证；3. 拓展开发了中药分子靶点筛选的新技术新方法：本项目负责人以第一和通讯作者共发表标注基金号SCI论文4篇，中文核心1篇，超额完成成果论文考核指标。. .总结而言，按照原定计划，本项目完成了任务书规定的研究内容，发表论文等指标超额完成课题计划，提出、建立的中药靶点生物芯片联用蛋白质组学筛选新策略，并在多个中药靶点筛选工作获得应用，所积累的技术体系形成了对外服务，为解读中药作用机制贡献力量。