基于人骨髓间充质干细胞移植的大鼠乙肝病毒感染模型研究

Hepatitis B virus (HBV) infection is one of the most prevalent infectious diseases associated with various liver diseases, including acute, chronic and fulminant hepatitis, hepatic cirrhosis and end-stage hepatic failure. Although the availability of HBV vaccine and the development of antiviral treatments, there are still more than 350 million people worldwide chronically infected with HBV. To clarify the precise mechanisms of HBV infection, viral life cycle, persistence of infection, and associated carcinogenesis, adequate animal models that recapitulate HBV-related liver diseases are urgently required. It is difficult to establish animal models of HBV infection because HBV has a very narrow host range and exclusively infects humans. Except of chimpanzees, tupaia, and the Asian tree shrew, no other immunocompetent animals have been used to recapitulate natural HBV infection in humans. Establish a chimeric rat model of HBV infection with stem cells transplantation has been recognized a promising alternative to overcome the limits of chimeric mouse models (need human hepatocytes as transplanted cell resources, mice do not recapitulate natural HBV infection). Our previous study has demonstrated that intraportal transplantation of human bone marrow mesenchymal stem cells (hBMSCs), which were immunological tolerant and no rejection, could rescue fulminant hepatic failure pigs and rats. The implanted hBMSCs could efficiently transdifferentiate into functional hepatocytes expressing HBV infection-specific receptor sodium-taurocholate cotransporting polypeptide (NTCP). These results imply that hBMSC-derived hepatocytes in chimeric rats may susceptible to HBV infection. Based on above results, we want to develop a novel immunocompetent hBMSC/SD rat model for HBV infection using hBMSCs transplantation in a FHF SD rat induced by D-galactosamine. The liver injury, HBV susceptibility, and re-infection process of HBV infected hBMSC/SD rat model will be evaluated by immunohistochemistry, serology, genome, proteome and metabolomics analyses. Electronic microscopy, qPCR, ELISA, and immunohistochemistry will be performed to characterize HBV DNA, cccDNA, and NTCP expression, as well as HBV Dane’s particles. We also want to discover some specific molecular and to clarify the precise mechanism of HBV infection in hBMSC-derived hepatocytes through above analysis. The final results will be helpful to elucidate the complex molecular mechanisms of HBV infection, and understand HBV biological characteristics, and discover new treatment strategies of HBV- associated liver diseases.

利用干细胞移植建立人鼠嵌合肝被认为是模拟人乙肝病毒（HBV）自然感染极具前景的动物模型。课题组前期利用具有免疫耐受和无排异反应的人骨髓间充质干细胞（hBMSC）移植成功救治暴发性肝衰竭（FHF）猪和大鼠，且动物肝内大量人源肝细胞均表达HBV感染特异性受体NTCP，显示含hBMSC来源肝细胞的人鼠嵌合肝具有被HBV感染的潜能。本研究拟在前期研究基础上经尾静脉注射HBV，建立新型人肝嵌合的免疫健全SD大鼠HBV感染模型；利用该模型新分泌HBV颗粒进行再感染评价；经组织学、血清学、基因组和蛋白组学等观察模型大鼠对HBV的易感性及肝损伤过程，鉴定模型动物肝细胞中Dane颗粒和NTCP、乙肝三系、HBV DNA和cccDNA等表达，发现易感HBV人源肝细胞的特异性分子，阐明HBV自然感染hBMSC/SD大鼠的确切机制。为最终阐明HBV生物学特性、乙肝发病机制、研发和筛选抗HBV新药等提供保障。

利用干细胞移植建立人鼠嵌合肝被认为是模拟人乙肝病毒（HBV）自然感染极具前景的动物模型。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，构建了肝脏和免疫系统双人源化小鼠模型，并成功感染HBV病毒，首次模拟了人类HBV自然感染过程，克服了抗乙肝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hBMSC移植FHF FRGS小鼠构建人源化肝脏和免疫系统双嵌合小鼠hBMSC-FRGS。hBMSC移植后1-7周，小鼠血清中人ALB浓度上升至600μg/mL，移植后12周，人肝嵌合率达40-60%，小鼠肝组织中表达人ALB、AAT等肝细胞特异性蛋白及HBV感染特异性受体NTCP。植入后3周，小鼠血液、脾和肝内即可持续分离到CD45+人源性免疫细胞。. 2. 利用人肝嵌合率约40-60%的hBMSC-FRGS小鼠，成功感染4种基因型（A-D）HBV病毒。感染后8周起HBV DNA、HBsAg、HBeAg持续升高，16周后达高峰并持续至56周，且小鼠体内可检测到cccDNA，其含量与HBV DNA含量呈正相关。感染后2周起，小鼠体内IL-6、IL-4、IFN-γ等人源性炎症和细胞因子表达显著上升。感染后24周模型小鼠出现慢性乙型肝炎典型组织学特征。. 3. 利用hBMSC-FRGS小鼠模型，阐明肝细胞体内发育机制，为肝细胞感染HBV机制研究及抗病毒药物研发提供科学依据。通过对植入后不同时间点小鼠肝脏中人源性肝细胞hBMSC-Heps进行转录组测序分析，发现hBMSC从移植后1天已启动肝向分化，3天后基本完成分化，14天后功能已接近人原代肝细胞，并鉴定到24条信号通路和6个特异性表达基因可能参与调控干细胞体内分化肝细胞和功能成熟。. 上述研究成果已发表Gut(IF 17.9)等SCI论文7篇，在2017年国际干细胞年会（ISSCR）、2018和2019年美国肝病年会（AASLD）上分别做壁报交流。获美国发明专利授权1项，申请国际国内发明专利4项。