A High Throughput Imaging Assay for Hepatic Lipid Droplet Formation

描述（由申请方提供）：非酒精性脂肪性肝病（NAFLD）在美国成年人群中普遍存在（总体发生率约为20%），并且通常与肥胖和糖尿病相关（约60%的糖尿病患者发生）。NAFLD表现为肝细胞内脂滴的数量和大小增加，如果不治疗，可进展为炎性疾病脂肪性肝炎和肝功能障碍。该提案响应分子库筛选中心网络（MLSCN）的PAR-06-545，提出了一种能够高通量/高含量筛选化学库以检测改变肝脂滴形成的试剂的测定法。该试验采用AML-12细胞（鼠肝细胞）或HuH 7细胞（来源于人肝细胞）。将以高通量形式铺板的细胞暴露于油酸，其诱导脂滴形成，固定并染色脂滴，并利用机器人荧光显微镜工作站成像。利用自动图像分析软件来量化脂滴的数量和大小。我们的测定具有非常高的Z'值的再现性（Z' > 0.5的油酸的影响），并且是廉价和快速执行。使用三酸甘油酯C（长链酰基-CoA合成酶（ACSL）的抑制剂）阻断油酸诱导的脂滴形成（对于该效应，Z' > 0.5）来证明测定功效。因此，该测定将提供一种极好的方法来鉴定可开发用于治疗NAFLD疾病或用作化学探针以鉴定肝甘油三酯合成和脂滴形成途径中的蛋白质的新试剂。

DESCRIPTION (provided by applicant): Non-alcoholic fatty liver disease (NAFLD) is prevalent (overall occurrence of ~20%) in the US adult population and is very commonly associated with obesity and diabetes (occurs in ~ 60% of diabetic patients). NAFLD presents as increased number and size of lipid droplets within the hepatocytes, and, if untreated, can progress to the inflammatory disease steatohepatitis and liver dysfunction. This proposal, in response to PAR-06-545 for the Molecular Libraries Screening Centers Network (MLSCN), proposes an assay that will enable high- throughput/high-content screening of chemical libraries to detect agents that alter hepatic lipid droplet formation. The assay employs AML-12 cells (murine hepatocyte) or HuH7 cells (derived from human hepatocytes). Cells plated in a high-throughput format, are exposed to oleic acid, which induces lipid droplet formation, fixed and stained for lipid droplets, and imaged utilizing a robotic fluorescence microscopy workstation. Automated image analysis software is utilized to quantify the number and size of lipid droplets. Our assay has an exceptionally high Z' value for reproducibility (Z' > 0.5 for the effect of oleic acid) and is inexpensive and rapid to perform. Assay efficacy was demonstrated using triacsin C, an inhibitor of long-chain acyl-CoA synthetase (ACSL), to block oleic acid-induced lipid droplet formation (Z' > 0.5 for this effect). Thus, the assay will provide an excellent method to identify new agents that could be developed for treatment of NAFLD disease or used as chemical probes to identify proteins in the hepatic triglyceride ester synthesis and lipid droplet formation pathways.