System Microscopy of Stress Response Pathways in Cholestasis Research
System Microscopy of Stress Response Pathways in Cholestasis Research
复制标题
DOI：
10.1007/978-1-4939-9420-5_13
复制
发表时间：
2019-01-01
期刊：
影响因子：
--
通讯作者：
van de Water, Bob
中科院分区：
文献类型：
--
作者：
Schimming, Johannes P.;ter Braak, Bas;van de Water, Bob
暴露于氧化自由基和细胞因子介导的炎症应激是胆淤积期间肝细胞死亡的确定因素。细胞对抗这些压力源的措施被称为适应性压力反应途径。虽然在疾病的早期阶段，适应性应激途径保护肝细胞，但在长期应激条件下的后期阶段，它们失效了。使用HepG2 BAC-GFP应答报告平台对细胞应激反应途径进行定量成像评估是评估化学物质和基因敲低对适应性应激反应途径激活的影响的有力策略，从而允许系统筛选对胆汁淤积进展的积极或消极影响。该方案允许应用一种高度通用的筛选工具，用于系统评估在胆汁淤积损伤期间具有胆汁淤积倾向的化合物和受影响基因对细胞适应性应激途径激活的影响。该方法涉及高通量活细胞可视化gfp标记的氧化应激反应/Nrf2通路和炎症细胞因子信号的关键蛋白。对单个细胞的时间响应进行定量图像分析，然后进行信息学分析。本章讨论了总体的实用方法。
Exposure to oxidative radical species and cytokine-mediated inflammatory stress are established contributors to hepatocyte cell death during cholestasis. Cellular counter measures against those stressors are called adaptive stress response pathways. While in early stages of the disease adaptive stress pathways protect the hepatocytes, in later stages during prolonged stressed conditions they fail. The quantitative imaging-based assessment of cellular stress response pathways using the HepG2 BAC-GFP response reporter platform is a powerful strategy to evaluate the impact of chemical substances and gene knockdown on activation of adaptive stress response pathways, hence allowing systematic screening for positive or negative influences on cholestasis progression. This protocol allows the application of a highly versatile screening tool for a systematic evaluation of the effect of compounds having cholestasis liability and affected genes during cholestatic injury on cellular adaptive stress pathway activation. The approach involves high-throughput live-cell visualization of GFP-tagged key proteins of the oxidative stress response/Nrf2 pathway and inflammatory cytokine signaling. Quantitative image analysis of temporal responses of individual cells is followed by informatics analysis. The overall practical approaches are discussed in this chapter.