脱细胞肝组织支架诱导人尿源性干细胞肝定向分化及肝组织结构构建的研究

Human hepatocytes proliferation in vitro and liver tissue reconstruction are important parts of liver cell-based therapy and tissue engineering, however human primary hepatocytes are scarce source. Most of current researches try to acquire the non-liver derived stem cells invasively and promote their directional differentiation into hepatocytes. Urine-derived stem cells (USCs) that can be obtained noninvasively possess a high capability for proliferation, multi-potent differentiation and paracrine properties. Our research group has isolated USCs and made it mass amplification successfully as well as revealed its myogenic and potency of epithelial induction differentiation. We also have attempted to manufacture porcine decellularized liver tissue scaffolds which can be used to culture human hepatocytes with three-dimensional dynamic cultures. This project is aimed at developing new type seeded human urine-derived cell that can be obtained noninvasively and used in treatment of liver regeneration therapy. We will further optimize both normal and injured porcine decellularized liver tissue scaffold, combining with the hepatocyte directional induced factors and three-dimensional dynamic culture system to optimize the extracellular microenvironment of the seeded USCs, induce the directional differentiation of human USCs into hepatocytes in vitro and promote the reconstruction of structure and function of liver-like tissues, and further reveal the effect and regulatory mechanism of component, structure and physic-chemical property of the decellularized liver tissue scaffolds in the directional differentiation of stem cells to hepatocyte-like cell. Preliminary exploring the new theory and technology of new type liver cell-based therapy and liver tissue engineering.

人肝细胞体外增殖与组织重建是肝脏细胞治疗和组织工程重要环节，然而人原代肝细胞来源困难。目前研究较多的是通过有创操作获得非肝源性干细胞并促进其向肝细胞定向分化。人尿源性干细胞（USC）可无创获取，拥有高度增殖能力、多谱系分化潜能和旁分泌功能。本课题组已成功分离并批量扩增了USC，并揭示了其肌性和上皮性诱导分化潜能；同时已尝试用于人肝细胞培养的猪肝组织脱细胞支架的制备及其三维动态培养。本项目旨在开发新型可无创获取的人尿源肝再生治疗用种子细胞，将在前期研究基础上，拟进一步优化正常及肝损伤猪脱细胞肝组织支架，联合肝细胞定向诱导分化因子、三维动态培养，优化USC种子细胞的细胞外微环境，在体内外诱导USC肝定向分化并促进肝脏样组织结构和功能重建；揭示脱细胞肝组织支架的组成、结构及理化性质对干细胞向肝样细胞定向诱导分化的影响及其调控机制；初步探索新型肝脏细胞治疗和肝组织工程的新理论与技术。

终末期肝脏疾病患者众多，肝脏移植由于受到器官来源和费用昂贵的限制， 难以广泛开展。肝脏组织工程是构建可移植肝脏的一条创新型途径。其核心内容是通过肝组织工程支架材料、肝脏种子细胞和相应的细胞生长调节因子，仿生构建肝再生微环境促进肝组织结构及功能重建，开发可供移植的组织工程肝脏。.本研究利用人尿源性干细胞作为种子细胞，在体外通过仿生构建具有三维超微结构的肝组织特异性细胞外基质和肝细胞诱导生长因子，促进尿源性干细胞向肝细胞样细胞诱导分化，维持肝细胞表型和功能特征，并在体内长期存活、增殖重构组织结构并形成类肝样组织。在国家自然科学基金资助期间，课题组取得成果主要包括:.1.研究脱细胞肝组织直接取材及制备技术对脱细胞肝组织支架理化性能的影响，优化了脱细胞肝组织支架制备技术。研究发现，水洗法及水洗涤+PAA法制备的细胞外基质有利用细胞的生长，能够为体外肝组织再生提供良好的生物支架作用。.2.从健康成人尿液中成果分离出尿源性干细胞（USC），并在体外实现了USC向肝样细胞定向分化的诱导。课题组目前已熟练掌握从成人新鲜尿液样本中分离USC的实验方法，并制定了实验的标准流程，大大提高了USC细胞分离的成功率，分离出的USC在体外具备较高的增殖活性。.3.在脱细胞肝组织支架的三维培养条件下，适时添HGF/FGF/OSM/Dex等诱导因子，可观察到USC向肝细胞的定向分化，充分证明了USC向肝样细胞分化的可能性，也证明了本研究制备的脱细胞肝组织支架能够在体外构建适于USC生长分化的三维微环境，为进一步研究肝组织再生、生物人工肝以及肝脏疾病的细胞治疗提供了实验基础和理论依据。.本项目融合生物医学、材料学和工程学的理论与技术，开发人USC 源肝组织细胞模型，并从组织、细胞、分子水平揭示并完善尿源性干细胞向肝样细胞定向诱导分化相关的材料设计、微环境构建的基本理论与技术。该研究还将推进药物代谢、药物毒性等评价用肝细胞模型的创建以及肝再生疗法等研究开发领域的发展，具有重大的社会和经济效益。