A High Throughput Imaging Assay for Hepatic Lipid Droplet Formation

描述（由申请人提供）：非酒精性脂肪性肝病（NAFLD）在美国成年人中很普遍（总发生率约为20%），并且通常与肥胖和糖尿病（约60%的糖尿病患者发生）相关。NAFLD表现为肝细胞内脂滴数量和大小增加，如果不治疗，可发展为炎症性疾病脂肪性肝炎和肝功能障碍。作为对分子文库筛选中心网络（MLSCN） PAR-06-545的回应，本提案提出了一种检测方法，可以实现高通量/高含量的化学文库筛选，以检测改变肝脂滴形成的药物。该试验使用AML-12细胞（小鼠肝细胞）或HuH7细胞（来源于人肝细胞）。细胞以高通量的形式被镀，暴露在油酸中，油酸诱导脂滴形成，固定和染色脂滴，并利用机器人荧光显微镜工作站成像。使用自动图像分析软件来量化脂滴的数量和大小。我们的测定方法具有极高的重现性Z值（油酸的影响为Z′>.5），而且成本低廉，操作迅速。实验结果表明，长链酰基辅酶a合成酶（ACSL）抑制剂三聚丙氨酸C （triacsin C）可以阻断油酸诱导的脂滴形成（Z' > 0.5）。因此，该检测将提供一种极好的方法来鉴定新的药物，这些药物可以用于NAFLD疾病的治疗，也可以用作化学探针来鉴定肝脏甘油三酯酯合成和脂滴形成途径中的蛋白质。

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.