线虫脂质代谢高通量高内涵RNAi筛选系统的研究

Lipid droplet is the main place of lipid storage in vivo, and it is directly related to the prevention of the human metabolic diseases and the development of biomass energy. There are lipid droplets from prokaryotic bacteria to higher animals. Abnormal lipid metabolism can cause atherosclerosis, diabetes and other metabolic diseases. C. elegans has become an important model organism for studying lipid metabolism because its diverse means of operation and highly homologous to higher animal genes. The use of C. elegans genome-wide RNAi screening to determine the important genes that regulate fat metabolism, is of greate significance for in-depth understanding of the molecular mechanisms of fat regulation, and development of drug in treatment of lipid metabolism related diseases. Equipment named Worm-Sort is currently used for C. elegans high-throughput RNAi screening, this device is capable of high-throughput screening C. elegans of different fluorescence intensity, but cannot distinguish difference of lipid droplet size and lipid droplet number, Because of poor sensitivity of this device, it is difficult to reach the resolution of lipid droplet level. The project intends to develop a high throughput high content RNAi screening system which can distinguish lipid droplet size and number in C. elegans, to find out the key genes that affect lipid metabolism. This system based on a microscope system, and resort to automatic control and pattern recognition technology.

脂滴是生物体内脂质存储的主要场所，它与对人类代谢疾病的防治和对生物能源的开发直接相关，从原核生物细菌到高等动物人都有脂滴的存在。脂代谢的异常会导致动脉粥样硬化和糖尿病等多种代谢类疾病。基于与高等动物基因的高度同源性以及操作手段的多样性，线虫已成为研究脂代谢的重要模式生物。 利用线虫进行全基因组RNAi筛选，确定调节脂肪代谢的重要基因，对深入理解脂肪调控的分子机制，开发治疗脂类代谢疾病药物具有重要意义。目前常用来做高通量RNAi筛选的设备是Worm Sort，该设备能高通量筛选出荧光强度不同的线虫，但筛选的分辨率较低，不能区分脂滴大小及数量的变化，难以达到脂滴量级的高通量筛选。本项目拟以线虫作为模式动物进行脂质代谢相关研究，并结合显微镜系统、自动控制、模式识别等多种技术研究方法，开发出能够对线虫中脂滴大小及数量变化等进行高通量高内涵RNAi筛选的系统，同时筛选出影响脂质代谢的关键基因。

本项目以线虫作为模式动物进行脂质代谢相关研究，并结合显微镜系统、自动控制、模式识别等多种技术和研究方法，开发出能够对线虫中脂滴大小及数量变化等进行高通量高内涵RNAi筛选的系统，并基于这套系统筛选出影响脂质代谢的关键基因。项目按照原定研究计划顺利开展，我们成功搭建了高通量高内涵自动化筛选系统，并在该系统上开展了基因筛选、药物筛选等方面的研究，包括线虫全基因组RNAi筛选确定CAB-1为特异性调节致密核心囊泡分泌的因子的研究、在小分子化合物库中筛选Glut4上膜激动剂和CRAC通道抑制剂的研究等，研究成果以SCI论文等形式产出。在此基础上，为进一步提高筛选效率，我们还发明了一种将流式技术和微流控技术结合在一起的高通量分选装置，并申请了国家发明专利。在软件和算法研究方面，我们开发了由应用层、连接层和硬件层构成的控制软件系统，并且在自动对焦算法和目标识别算法等方面进行了深入的研究，取得了较好的研究成果，发表了SCI论文，该论文被选为期刊封面文章。目前，所研制的系统运行稳定，今后还将继续在基因筛选、药物筛选等方面发挥重要的作用。