In Vitro Liver Models to Investigate the Progression of Liver Fibrosis

摘要 肝纤维化，由慢性肝损伤和细胞外积聚引起 基质(ECM)蛋白是几种慢性肝病的特征。肝脏的动态变化 微环境(LME)被广泛认为是肝纤维化进展和发展的关键参与者 治疗反应。LME成分，包括实质(肝细胞)和非肝细胞之间的相互作用 实质细胞(肝窦内皮细胞、肝星状细胞、库普弗细胞)，信号 分子(配体-胶原、层粘连蛋白)，以及来自细胞外基质的机械信号，都与 肝纤维化的进展。星状细胞活化是肝纤维化的标志；然而， 肝细胞和LSECs的功能尚未被广泛了解。此外，通过这些机制， LME成分调节肝功能，各种信号级联反应知之甚少，因此限制了 制定最佳的肝病诊断和治疗方案(例如，酒精性肝病， 非酒精性脂肪性肝病、非酒精性脂肪性肝炎以及乙型和丙型肝炎)。因此，有一个 迫切需要开发模拟动态LME组分的体外模型并有效地研究其 在肝纤维化中的作用。为了研究LME对细胞信号的直接影响，使用体外肝脏是必要的 模拟肝纤维化的基本复杂性和动态性的模型，并实现更大的 翻译效度。本应用程序的目标是使用多学科方法开发三个 独立的体外肝脏模型，研究不同的LME成分(肝细胞-LSEC相互作用， ECM硬度、配体类型和密度)调节肝细胞和LSEC功能，这些LME起什么作用 各种成分在肝纤维化的进展中起着重要作用。拟议研究的具体目的是：1) 研究肝细胞-低分子肝素相互作用(S)对肝功能的影响，2)研究 刚性改变肝细胞功能，以及3)决定配体类型/密度在调节肝细胞中的作用 功能。这项工作将为利用体外肝脏模型 准确描述正常和疾病状态下的肝功能和代谢，尤其是如何 LME调节肝功能的发育和维持。重要的是，这种模式是创新的，因为它 将按时间顺序模拟纤维化阶段，类似于临床情况，并拥有一个 比动物模型更具可控性和系统性。这一项目预计将产生渐进的影响 关于肝纤维化及相关领域的研究，因为保留LME的肝脏模型的可用性将 促进了解LME活动背后的分子机制，这些机制对 维持肝脏生物学。

ABSTRACT Liver fibrosis, which results from chronic liver damage in conjunction with the accumulation of extracellular matrix (ECM) proteins, is characteristic of several chronic liver diseases. Dynamic changes to the liver microenvironment (LME) are widely recognized as a critical participant in liver fibrosis progression and therapeutic responses. LME components, including interactions between parenchymal (hepatocytes) and non- parenchymal cells (liver sinusoidal endothelial cells [LSECs], hepatic stellate cells, kupffer cells), signaling molecules (ligands-collagen, laminin), and mechanical cues from ECM, have been implicated in the progression of liver fibrosis. Stellate cells activation is the hallmark of liver fibrosis; however, the effect on hepatocytes and LSECs function has not been extensively understood. Also, the mechanisms by which the LME components regulate liver function and various signaling cascades are poorly understood, thus limiting the development of optimal diagnosis and treatment regimes for liver diseases (e.g., alcoholic liver disease, nonalcoholic fatty liver disease, non-alcoholic steatohepatitis, and hepatitis B and C). Therefore, there is a critical need to develop in vitro models that simulate the dynamic LME components and effectively study their role in liver fibrosis. To study the direct effects of LME on cell signaling, it is imperative to use in vitro liver models to simulate the fundamental complexity and dynamism of liver fibrosis and to achieve greater translational validity. The goal of this application is to use a multidisciplinary approach to develop three independent in vitro liver models to study how different LME components (hepatocytes-LSEC interactions, ECM stiffness, ligand type and density) regulate hepatocyte and LSEC function and what role these LME components play in the progression of liver fibrosis. The specific aims of the proposed study are to: 1) investigate the effect of hepatocytes-LSEC interaction(s) on hepatic function, 2) investigate how variation in stiffness alters hepatic cell function, and 3) determine the role of ligand type/density in regulating liver cell function. This work will provide a significant advancement in the ability to utilize in vitro liver models to accurately describe the liver function and metabolism in normal versus diseased states, and especially how the LME regulate the development and maintenance of liver function. Importantly, this model is innovative as it will chronologically emulate the fibrosis stage, is similar to clinical conditions, and boasts an environment that is more controlled and systematic than animal models. This project is expected to have a progressive impact on the study of liver fibrosis and related fields because the availability of a liver model that retains LME will facilitate understanding of the molecular mechanisms that underlie LME activities in mechanisms critical for the maintenance of liver biology.