Intercellular Communications in Hepatic Function

描述（由申请人提供）： 该项目的重点是通过控制细胞-细胞相互作用来开发和表征肝组织的体外模型。在体外维持分化的肝细胞功能的困难已经对肝病的基于细胞的疗法以及体外实验（例如药物毒性研究）的进展产生了负面影响。有人提出，一个集成的“仿生”平台，结合关键的肝脏功能（分化的肝细胞，区室化代谢，定向细胞-细胞相互作用，定向流体流动）将作为一个更好的预测平台比现有的模型异源生物代谢和生理实验。初步数据表明，肝细胞与非实质细胞（成纤维细胞）的共培养导致长期分化的功能，尽管“共培养效应”的分子基础和该过程的动力学都没有很好的理解。在拟议的研究中，我们的目标是表征共培养反应的动力学，揭示共培养反应的机制，并将所需的元素纳入模拟肝脏特征的微制造生物反应器阵列中高通量平台。具体目标1将研究同型肝细胞/肝细胞间隙连接通讯和异型（肝细胞/成纤维细胞）接触对分化功能的动态作用。研究人员开发了一种微图案工具，可以控制细胞间的相互作用。电活性微图案化表面将用于从共培养物中动态释放成纤维细胞，并研究对肝功能的影响。“共培养效应”的机制之前使用各种成纤维细胞株的基因表达谱进行了研究。初步结果表明，钙粘蛋白可能发挥作用的异型信号。具体目标2将是研究钙粘蛋白在共培养中的作用，特别是T-钙粘蛋白，一种差异表达超过30倍的候选物。初步结果表明，可以使用受控的氧梯度在体外重建肝脏的区室化功能。具体目标3将是将联合收割机氧梯度和定向流体流与分化的肝细胞（如SA 1和2中所确定的）组合成可用作肝脏的预测模型的小型化生物反应器阵列。将通过检查对充分表征的刺激的反应来评估工程化组织。该项目将导致对细胞-细胞相互作用如何产生协调的器官功能的综合理解，并将建立一个强大的肝功能预测模型，用于药物开发和基础肝脏研究。

DESCRIPTION (provided by applicant): This project focuses on development and characterization of an in vitro model of hepatic tissue by control of cell-cell interactions. The difficulty in sustaining differentiated hepatocyte functions in vitro has negatively impacted progress towards cell-based therapies for liver disease as well as in vitro experimentation (e.g. drug toxicity studies). It is proposed that an integrated 'biomimetic' platform incorporating key hepatic features (differentiated hepatocytes, compartmentalized metabolism, oriented cell-cell interactions, and directional fluid flow) will serve as a better predictive platform than existing models for Xenobiotic metabolism and physiological experimentation. Preliminary data suggest that co-cultivation of hepatocytes with non-parenchymal cells (fibroblasts) results in long-term differentiated functions, though neither the molecular basis for the 'co-culture effect' nor the dynamics of the process are well understood. In the proposed research, we aim to characterize the dynamics of the co-culture response, uncover the mechanisms that underlie the co-culture response, and incorporate the required elements in a microfabricated array of bioreactors that mimics features of the liver in a high-throughput platform. Specific Aim 1 will be to investigate the dynamic role of homotypic hepatocyte/hepatocyte) gap junction communication and heterotypic (hepatocyte/fibroblast) contact on differentiated functions. The investigator has developed a micropatterning tool that enables control of cell-cell interactions. Electroactive micropatterned surfaces will be utilized to dynamically release fibroblasts from co-culture and study the impact on hepatic function. The mechanism of the 'co-culture effect' was investigated previously using gene expression profiling of various fibroblast strains. Preliminary results indicate that cadherins may play a role in heterotypic signaling. Specific Aim 2 will be to investigate the role of cadherins in coculture, in particular T-cadherin, a candidate that was differentially expressed by over 30-fold. Preliminary results indicate that compartmentalized functions of the liver can be recreated in vitro using controlled oxygen gradients. Specific Aim 3 will be to combine oxygen gradients and directional fluid flow with differentiated hepatocytes (as determined in SA1 & 2) into an array of miniaturized bioreactors that can be used as predictive models of the liver. The engineered tissue will be assessed by examining the responses to well-characterized stimuli. This project will lead to an integrated understanding of how cell-cell interactions produce coordinated organ function and will establish a robust predictive model of liver function for pharmaceutical drug development and fundamental hepatic studies.