经门静脉移植人骨髓间充质干细胞治疗暴发性肝衰竭的分子机制研究

Fulminant hepatic failure (FHF) is a potentially life-threatening condition for which liver transplantation is the only effective treatment. However, a shortage of donor organs for transplantation results in the death of many patients waiting for liver transplantation. Hepatocyte transplantation provides a promising alternative. However, it has rarely produced therapeutic effects because mature hepatocytes cannot be effectively expanded in vitro, and the availability of hepatocytes is often limited by shortages of donor organs. Thus, recent studies have focused human bone marrow mesenchymal stem cells (hBMSCs) that could be easily isolated using non-invasive procedures to yield hepatocytes in vitro and in vivo. The hepatocyte differentiation capacity of BMSCs has been characterized in vitro and in vivo. Some groups have already started transplanting autologous bone marrow cells into patients with chronic liver fibrosis or cirrhosis. However, little is known about the use of hBMSCs to treat FHF in animal models or in patients with FHF, even though such studies would be clinically important. Furthermore, because of difficulties in tracking transplanted hBMSC-derived hepatocytes in patients, the roles of BMSCs in liver regeneration have not been fully elucidated. Our previous studies showed that FHF-derived BMSCs demonstrate a hepatic transcriptional profile and express many of the same genes expressed by hepatic progenitor cells, which suggests that extrahepatic stem cells, especially BMSCs, may be a resource for hepatocyte repopulation and can play an important role in liver regeneration. Thus, we want to investigate whether the intraportal transplantation of hBMSCs is a safe and effective method to prevent FHF in a large animal (pig) model. Based on above results, the D-galactosamine induced FHF pig will be used as an animal model for observation of the effectiveness of hBMSCs transplantation. Fluorescence in situ hybridization will be used to detect human Y-Chromosome for observing the role of cell fusion. Immunohistochemistry will be used to analyze human hepatic specific markers (like albumin, hepatocyte specific antigen) positive cells in FHF pigs for determining the role hBMSCs of transdifferentiation. Microarray and the latest high-throughput microRNA sequencing technology will be performed to characterize the specific expression and transcriptional profiling during the period of hBMSC transplantation. The specific antibody chips contains more than 200 kinds of cytokines, Inflammatory chemokines and apoptosis factors will also be used to observe the effects of hBMSCs on anit-inflammatory, apoptosis, immune regulation and paracrine role of specific pathways. The final results will be helpful to clarify the precise molecular mechanisms on hBMSCs participate the liver regeneration in FHF, and further provide a scientific basis for hBMSC treatment of FHF early clinical application.

原位肝移植是目前公认治疗暴发性肝衰竭(FHF)的唯一有效方法，人骨髓间充质干细胞(hBMSC)移植为FHF治疗提供了诱人前景。课题组前期研究发现，BMSC极有可能是FHF肝细胞短时间内再生与修复的重要来源，最新研究证实，经门静脉移植hBMSC可有效阻止FHF猪的死亡。为此本研究拟在前期工作基础上，进一步观察经门静脉移植hBMSC至D-半乳糖胺所致的FHF猪的确切疗效；利用免疫组化与细胞原位示踪术观察hBMSC在宿主肝内转分化和细胞融合作用；利用全基因组芯片扫描和最新的高通量测序技术对植入细胞来源的人肝细胞和猪肝组织进行表达谱和microRNA转录谱分析；结合最新的包含200余种细胞因子的抗体芯片和代谢组学技术，全面分析hBMSC参与宿主抗炎症(凋亡)、免疫调节和旁分泌等作用与特异性调控通路，阐明hBMSC参与FHF肝脏再生与修复的精确机制。为hBMSC治疗FHF早日应用于临床提供科学依据。

暴发性肝衰竭（FHF）病死率高达80%，除肝移植外无其他有效治疗方法，基于干细胞移植的肝脏再生医学为FHF治疗开辟了新方向，然而干细胞作用机制不明等问题严重阻碍了临床转化。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FHF猪的研究基础上，继续对移植治疗作用机制、hBMSC转分化肝细胞分子特征及调控规律、体外构建类肝器官等进行了深入研究，并取得系列创新成果：. 1) 首次利用转录组、蛋白组、代谢组等多组学功能关联分析技术系统阐明了hBMSC移植治疗FHF的作用机制。发现植入干细胞可明显抑制FHF 所致的致命性细胞因子风暴，并在 7 天内恢复 FHF 宿主内环境，精确定量证实此时猪肝脏中的人源性肝细胞约占 4.5%，表明早期干细胞增殖转分化的作用有限。移植干细胞早期主要通过旁分泌作用抑制炎症介质分泌、调节免疫反应等来改变宿主对 FHF 损伤的响应，最终促进宿主自身肝脏再生修复。并发现Delta-like ligand 4 (DLL4)在干细胞移植治疗 FHF 过程中对肝组织修复发挥了关键作用，其疗效在FHF模型猪和模型鼠中均得到验证。该研究不仅重新认识了干细胞移植作用机制，更为基于干细胞作用的单分子或分子鸡尾酒疗法的临床转化提供了科学依据。. 2) 利用高通量细胞因子芯片分析发现hBMSC体外分化肝细胞高表达TIMP-4和FST，且体内移植实验证实hBMSC来源肝细胞（hBMSC-Hep）呈TIMP-4和FST阳性表达。显示TIMP-4和FST可作为干细胞转分化至成熟肝细胞的新分子标记物。. 3) 利用高通量二代测序技术建立不同分化状态下hBMSC-Hep特异的mRNA谱和miRNA谱，发现630个持续上调基因、1082个持续下调的基因和77个显著变化miRNA的靶基因多与细胞周期和分化发育有关。进一步深入研究上述差异表达分子及相关信号通路可为优化干细胞肝向分化提供新的思路和方法。. 4) 利用全肝脱细胞支架和自主研发的三维循环灌流生物反应器建立了体外类肝器官构建技术平台，已初步实现了全肝脱细胞支架的再细胞化和血管化，为重要生命器官体外工程化构建提供了理论依据和技术支撑。. 上述研究成果已发表 Gut（IF14.92）等 SCI 论文 7 篇，申请国际国内发明专利 4 项，获国家科技进步一等奖 2 项。