具有肝脏再殖能力的人肝细胞体外培养体系的建立

The liver is a vital organ controlling numerous metabolic functions. A remarkable feature of the liver is its outstanding regenerative capability after injury. However, in fatal liver diseases, the liver loses regeneration capability and liver transplantation is only curative therapy. Due to organ shortage, transplantation of primary hepatocytes has been evaluated both in clinics and animal models as a promising alterative to whole-organ transplantation. However, the cellular source of human hepatocyte is limited because of the shortage of organs and protocols to expand human hepatocyte in vitro. Recently, the cell plasticity has recognized to plays an important role in liver regeneration. Both human and mouse hepatocytes can be reprogramed to proliferative biliary-like progenitor cells in liver injury model. Based on the idea of cell plasticity of human hepatocytes, we have developed a new medium to reprogram human hepatocytes to liver progenitor-like cells. In this project, we would like to optimize the culture medium and characterize the proliferation and hepatic maturation ability of human hepatocyte derived liver progenitor cells. Moreover, we will transplant these cells to a mouse model of fatal metabolic liver disease and prove their liver repopulation ability in vivo. Success of this project will hold great promise for regeneration medicine.

肝脏是体内至关重要的器官，调控着大量的物质代谢。同时其也是体内具有强大再生能力的器官之一。但在晚期肝病患者中，肝脏失去再生能力。肝脏移植成为唯一有效的治疗手段。由于肝脏供体的缺乏，肝细胞移植被认为可能成为整肝移植的替代疗法并在临床和动物模型中验证了其有效性。然而，因为缺少肝脏供体和扩增肝细胞的技术，人肝细胞的来源同样匮乏，限制了肝细胞移植的应用发展。肝细胞的可塑性在肝脏再生中的重要性正被逐步认识。人和鼠肝细胞都可以体内重编程为可增殖的胆管样肝前体细胞。基于肝细胞可塑性理论，我们近期建立了一种将人肝细胞重编程为肝前体细胞的培养体系。本项目将系统地优化该培养体系，并鉴定重编程获得的肝前体细胞的扩增能力和体外再分化为成熟肝细胞的能力，最终在Fah-/-Rag2-/-IL2rg-/-小鼠模型中证明其体内肝脏再殖能力。本项目的成功将为肝脏再生医学提供新的移植细胞来源。

人原代肝细胞移植在治疗肝脏疾病方面具有广阔的应用前景。然而，由于肝脏供体短缺以及肝细胞体外培养困难，使这种治疗方式严重受到限制。在肝脏慢性损伤的过程中，体内成熟的肝细胞会被重编程为一种同时表达肝细胞基因和肝前体细胞基因的可增殖的肝细胞。这就提示在体外可以利用体内的这种重编程策略获得可增殖的人肝细胞。我们建立了一种新的培养体系可以让人原代肝细胞体外扩增10000倍。这些体外扩增的细胞是既具有肝细胞特征又表达肝前体细胞基因的双表型肝细胞。更重要的是，这些扩增出来的细胞具有与原代肝细胞相似的体内整合的能力。而且这些可增殖肝细胞在移植整合后或者体外肝向诱导体系中都会进一步分化为成熟的肝细胞。这项研究提供了一种体外显著扩增人原代肝细胞的培养方法，为体外肝脏疾病的模型构建以及为肝脏病人的肝细胞移植治疗奠定了坚实的基础。