Modelling bile formation and flow in vitro to understand normal healthy liver function and how damage-associated molecular changes predispose towards

胆汁是胆盐（BS）、磷脂酰胆碱（PC）、胆固醇、盐和废物的复杂混合物，在胆小管中形成。胆汁流动受损（胆汁淤积）会导致肝损伤，患者会出现急性或慢性疾病。驱动胆汁从肝细胞流出的关键转运蛋白，BS输出泵（BSEP）和PC floppase（ABCB 4），已在KJL和RJT实验室中鉴定和表征（Groen et al Gastro 2011; Byrne et al.，Gastro 2002）。这些转运蛋白的突变导致急性进行性家族性胆汁淤积，在没有肝移植的情况下，这种急性进行性家族性胆汁淤积在儿童时期是致命的。较轻形式的转运蛋白功能不全（或抑制）更为普遍，易发生一系列肝病，包括妊娠期胆汁淤积症、胆石病、胆道胆管炎、硬化性胆管炎和肝细胞癌（Nicolaou et al J Path 2014）。尚未描述与继发性病理易感性相关的分子变化。进展缓慢是因为体内胆管难以进入，无法取样，也因为体外培养的肝细胞迅速丧失了制造胆汁的能力（其中维持低水平胆汁形成，例如在3D培养物中，它被分泌到球体内部的封闭隔室中，从而阻止精确的动力学测量）。我们将开发一种新的transwell灌注系统，用于肝细胞培养，维持胆汁形成和流动（图1）这将使我们能够诱导胆汁淤积并确定驱动疾病进展的根本变化。肝细胞（iHEP）将使用TR实验室中开发的技术衍生自iPSC（Rashid等人，JCI 2010，Nature 2011）。在分化之前将纯合ABCB 4-S320 F突变引入iPSC中。ABCB 4-S320 F不寻常地引起一系列肝脏病理（Andress等人，Hepatol 2014）。胆汁淤积将根据需要通过妊娠激素和/或胆汁淤积药物抑制野生型（WT）或ABCB 4-S320 F来诱导。在测量基因表达和细胞因子释放到顶室和底室的变化之前，将表征胆汁复杂性和流量。

Bile, a complex mixture of bile salts (BS), phosphatidylcholine (PC), cholesterol, salts and waste products, is formed in the biliary canaliculi. Impaired bile flow (cholestasis) causes liver damage and patients present with acute or chronic disease. The key transporters driving bile flow from the hepatocyte, the BS export pump (BSEP) and the PC floppase (ABCB4), have been identified and characterised in the KJL and RJT labs (Groen et al Gastro 2011; Byrne et al., Gastro 2002). Null mutations in these transporters cause acute forms of progressive familial cholestasis that are fatal in childhood in the absence of liver transplant. Milder forms of transporter insufficiency (or inhibition), which are much more prevalent, predispose to development of a spectrum of liver disease including gestational cholestasis, gallstone disease, biliary cholangitis, sclerosing cholangitis and hepatocellular cancer (Nicolaou et al J Path 2014). The molecular changes associated with predisposition to secondary pathologies have not been described. Progress is slow because the biliary canaliculi in vivo is inaccessible precluding sampling and also because hepatocytes cultured in vitro rapidly lose the ability to make bile (where low level bile formation is maintained, e.g. in 3D cultures, it is secreted into closed compartments internal to the spheroid preventing accurate kinetic measurements).We will develop a novel transwell perfusion system for hepatocyte culture that maintains bile formation and flow (Fig 1) and this will allow us to induce cholestasis and define the fundamental changes that drive disease progression. Hepatocytes (iHEPs) will be derived from iPSCs using technology developed in the TR lab (Rashid et al., JCI 2010, Nature 2011). Homozygous ABCB4-S320F mutations will be introduced in the iPSCs prior to differentiation. ABCB4-S320F, unusually, causes a spectrum of liver pathologies (Andress et al., Hepatol 2014). Cholestasis will be induced by inhibition of wild type (WT) or ABCB4-S320F by gestational hormones and/or cholestatic drugs as necessary. Bile complexity and flow will be characterised before measurement of changes in gene expression and cytokine release into the apical and basal chambers.