基于单细胞RNA测序的体内胆管上皮细胞向肝细胞转化机制研究

In recent years, lineage tracing experiments demonstrated that hepatocytes regeneration is accomplished by hepatocytes themselves, no new hepatocytes have been found to be generated from other cells. A recent report described that under the precondition of inhibiting the proliferation of hepatocytes, hepatocytes injuries can induce bile duct epithelial cells (BECs) generate new hepatocytes. We modified traditional liver regeneration models to simulating pathologic changes of chronic human liver diseases, under such conditions non-parenchymal-cells derived hepatocytes replenished a large fraction of the regenerated liver parenchyma in severe injuries (up to 55.7% of regenerated parenchymal cells). Importantly, we identified that BEC function as a source for hepatocyte regeneration using a BEC-specific tracing model. Biliary epithelial cells undergoing BEC-to-hepatocyte reprogramming via an Hnf4α+CK19+ bi-phenotypic state, which possesses part of transcriptional and morphological characters of both BECs and hepatocytes. In order to clarify the process and mechanism of BEC-to-hepatocyte reprogramming, we plan to identify sub-populations of BEC pools based on transcriptome profile revealed by single-cell RNA sequencing, and to find out specific markers for the BEC sub-populations that underwent converting into hepatocytes. We will identify their conversion process, and to screen specific genes and signal pathways relating to the conversion between sub-populations. These genes and pathways might be involved in regulating the lineage conversion and could be therapeutic targets. Organoid platform will be adopted to verify the lineage conversion process, and to find out effective regulatory genes or pathways. Based on these, an organoid platform for producing mouse and human bi-phenotypic cells and hepatocytes would be established. In conclusion, the project will further reveal the process of liver regeneration, and will identify molecular targets for the promoting-liver-regeneration-therapies. The platform for generating bi-phenotypic cells and hepatocytes in vitro can be used for drug research, personalized drug screening and autologous cell transplantation therapy, which has direct application value.

近几年细胞谱系追踪试验发现体内肝细胞再生完全由肝细胞自身完成，其它细胞不产生新的肝细胞。只有同时抑制肝细胞增殖并引入肝损伤，才能诱导胆管上皮细胞产生肝细胞。本课题组通过建立更符合人体肝病病理表型的两种小鼠肝再生模型，发现造模后多至55.7%的肝细胞来源于其它细胞，进一步的谱系追踪试验鉴定出胆管上皮细胞转化产生了肝细胞。且发现该转化经过“双相细胞”阶段（同时具有胆管上皮细胞和肝细胞部分表达属性和形态特征）。本项目组拟采用单细胞RNA测序技术鉴定向肝细胞转化的胆管细胞亚群和转化途径，筛选该群细胞的标记物和调控转化的靶点。再利用类器官体平台验证转化过程和调控靶点。并据此建立产生鼠源和人源双相细胞及肝细胞的类器官体转化平台。该项目的实施可望进一步揭示肝再生过程，为开发促进肝脏自身再生的新疗法找到靶点。体外产生双相细胞及肝细胞的平台，可用于药物研发、个性化药物筛选及自身细胞移植治疗，有直接应用价值。

项目组构建了特异标记胆管上皮细胞的模式动物，改进构建了模拟人体慢性肝病的肝损伤模型，能够诱导胆管细胞向肝细胞转化。采用单细胞RNA测序技术检测胆管上皮细胞向肝细胞转化的过程，鉴定了向肝细胞转化的双相细胞表达特征以及可能的调控分子。.通过特异标记胆管上皮细胞的模式动物，分离原代肝脏胆管上皮细胞，构建胆管细胞类器官并大量扩增。模拟体内胆管细胞向肝细胞转化过程，构建了胆管细胞向双相细胞及肝细胞转化的类器官技术平台，并持续优化该技术平台，稳定获取双相细胞及肝细胞。.通过优化构建的肝损伤模型，结合特异标记肝细胞的模式动物，发现肝细胞在慢性肝损伤过程发生去分化并获得克隆性增殖及双相分化的能力。模拟体内过程，我们在体外诱导肝细胞的去分化并形成类器官。.发现构建特异敲除肝细胞TGF受体的模式动物，发现抑制TGF通路可抑制肝损伤过程肝细胞的炎症反应与上皮细胞间质转化（EMT），促进肝细胞功能。相应结果投稿于Journal of Digestive Diseases，已修回。.项目负责人分别在2021中华医师协会消化病年会、2019中华医学会消化病分会年（三等奖）、2019第十届全国疑难与重症肝病会议（三等奖）、2019感染及肝病卓越研究论坛、2022第五届东方药理论坛等学术会议报告及获奖。.项目负责人组成员在该项目工作基础上，2020年获得国家自然科学基金面上项目，入选长征医院金字塔人次工程（A类），该项目的实施培养博士后2名，博士1名。