利用干细胞移植建立肝脏和免疫细胞双人源化的小鼠乙肝肝硬化模型

Developing a small animal model that accurately delineates natural history of hepatotropic viruses infection and immunopathophysiology is still a scientific challenge. Recently, we have established a dual humanized mouse model with human bone marrow mesenchymal stem cell (hBMSC) transplantation to model hepatitis B virus (HBV) infection. The implanted hBMSCs rescued fulminant hepatic failure (FHF) in immunodeficient FAH-/-Rag2-/-IL2-/-SCID (FRGS) mice and generated liver and immune dual humanized hBMSC-FRGS mice with robust proliferation of functional human hepatocytes and leukocytes. The sustained HBV infection and human immunoresponse against HBV from hBMSC-derived immune cells (CD45+ and CD4+/CD8+) in the blood, spleen and liver of humanized hBMSC-FRGS mice were observed after 3 weeks of transplantation. The hBMSC-derived hepatocytes reached 60% in humanized FRGS mice after 8 weeks of transplantation. The new humanized mice were more sensitive and efficient for sustained infection of 4 different HBV genotypes than primary hepatocyte-induced humanized mice. Histochemistry and biochemical analysis indicated that the typical characteristics of human chronic hepatitis were observed at 16 weeks after HBV infection. In this study, we aim to developing a dual humanized mouse to model HBV induced liver cirrhosis after optimizing the dose of stem cell transplantation and HBV infection. HBV DNA, cccDNA, HBV specific antigens and antibodies will be performed to clarify the sustained HBV infection. The humanized inflammatory factors, cytokines and immunohistochemistry will be used to observe the immunopathological response and subsequent induced chronic hepatitis and liver cirrhosis. The multifaceted interactions analysis with genomics, proteomics and metabolomics will be used to determine the detailed mechanisms of human HBV infection, acute and chronic liver injury, and subsequent liver cirrhosis. The final results will be helpful to recapitulate a natural diseases progression of HBV infection for the first time, opening opportunities for understanding viral immune pathophysiology and testing antiviral therapies in vivo.

建立肝脏与免疫细胞双人源化的乙肝病毒（HBV）诱导肝硬化动物模型极具挑战意义。课题组已利用人骨髓间充质干细胞移植治疗暴发性肝衰竭技术成功建立肝脏和免疫细胞双人源化的FRGS小鼠，细胞移植3周后，小鼠血液、脾和肝内均可持续分离到人CD45、CD4/CD8等阳性细胞；细胞移植后8周，人肝细胞嵌合率达60%，且可被4种基因型HBV持续感染，感染后16周小鼠出现慢性肝炎的典型生化与病理特征。本研究拟在前期研究基础上进一步优化干细胞移植和HBV病毒感染剂量，建立HBV自然感染的乙肝肝硬化小鼠模型。利用乙肝三系、HBV DNA和cccDNA等标记物明确HBV持续感染，通过炎症细胞因子和免疫组化等观察HBV感染对双人源化小鼠的免疫损伤，利用基因、蛋白和代谢等多组学功能关联分析阐明HBV感染诱导模型小鼠急慢性肝损伤和肝硬化的发生发展机制。为最终阐明乙肝肝硬化发病机制、研发和筛选抗HBV新药等提供保障。

利用干细胞移植建立肝脏和免疫细胞双人源化的小鼠乙肝肝硬化模型极具挑战。本项目在前期利用人骨髓间充质干细胞（hBMSC）肝内移植成功救治暴发性肝衰竭（FHF）猪的研究基础上，构建了肝脏和免疫细胞双人源化小鼠模型，经HBV感染后成功构建了乙肝肝硬化模型，首次模拟了人类HBV自然感染过程，克服了抗乙肝、肝硬化新药研发没有动物模型的国际难题。. 本项目主要研究内容和创新成果如下：. 1. 利用单种hBMSC移植暴发性肝衰竭小鼠构建了肝脏和免疫细胞双人源化小鼠模型，并通过转录组分析揭示植入的干细胞可在14天内快速转分化为功能性肝细胞和多种免疫细胞。. 2. 利用HBV病毒感染双人源化小鼠构建了乙肝肝硬化模型，揭示人源性肝细胞是HBV成功感染的关键，而人源性免疫细胞对HBV的免疫应答反应是HBV持续感染进而形成乙肝肝硬化的分子基础。. 3. 利用项目组前期建立的多中心、前瞻性、开放性乙肝慢加急性肝衰竭（HBV-ACLF）临床大队列，建立HBV-ACLF诊断的中国标准及预后评分系统，揭示HBV病毒激活引起的免疫代谢失衡是HBV-ACLF发生发展的重要分子机制，为肝衰竭临床治疗提供科学依据。. 4. 利用猪血清联合D-gal/LPS急性刺激构建非乙肝肝硬化和肝衰竭大鼠模型，为肝硬化、肝衰竭研究提供更多疾病动物模型。. 上述研究成果已发表SCI论文9篇，其中IF>20的3篇，授权国际发明专利1项，授权中国发明专利4项，申请中国发明专利1项。培养博士生3名、硕士生2名。