硫酸肝素调控组织工程肝脏血管化及其机制研究

Vascularization is the bottleneck of complex organ tissue engineering construction in vitro. Hepatic sinusoids not only provide nutrients and oxygen to the hepatocytes along the liver cords, which closely neighbored to the liver sinusoidal endothelia cells (LSECs), also formed a special vascular microenvironment by secreting a variety of cytokines, which regulate liver tissue development, homeostasis maintenance and liver regeneration after injury. There are two major key issues need to be addressed to realize vascularization of tissue engineered liver: how the LSECs and hepatocytes mutually regulate each other to reconstruct the tissue homeostasis? How the neogenerated sinusoids control the structure reconstruction and functional regeneration of the engineered liver tissue? The applicant has successfully established the strategies of preparing acellular liver biomatrix scaffold and the technology system of recellularization of the scaffold for liver tissue engineering. Moreover, our studies demonstrated that specific enzymatic modified heparan sulfate proteoglycan (HSPG) showed high affinity to LSECs and could promote angiogenesis and anti-coagulation function. In this project we propose and will validate the hypothesis that “HSPG as a spatiotemporal rheostat that guides functional vascularization and promotes liver tissue remodeling by regulation of LSECs mediated by Ang2-VEGFR2 signaling pathway”. Finally, that principle will guide us to carry out multidisciplinary research involved of stem cell biology, glycobiology, biomaterial science and bionic architecture, to understand and construct of recellularized, revascularized biomimetic liver tissue with highly anticoagulant activity.

血管化是所有复杂器官组织工程体外构筑成败的瓶颈。肝窦内皮细胞(LSEC)构成的肝血窦不仅向肝细胞索提供营养物质与氧气，还分泌多种细胞因子形成特殊血管微环境，控制肝组织的发育、稳态维持和修复再生。实现组织工程肝脏血管化需要解决2个关键问题：LSEC和肝系细胞如何相互调控达到重建组织稳态？新生血管如何调控肝组织结构重建和功能再生？申请者前期已成功建立脱细胞肝脏生物支架策略和再细胞化构建组织工程肝脏的技术体系，并证明特定酶修饰的硫酸乙酰肝素蛋白聚糖(HSPG)对肝窦内皮细胞具有高度亲和力和促进血管化及抗凝血功能。本项目中我们提出并验证假说“HSPG作为时空变阻器引导功能性血管化，通过Ang2-VEGFR2通路调控LSEC和肝系细胞反馈环路促进肝组织重塑”，并应用该原理指导我们将干细胞生物学、糖生物学、材料学与肝脏仿生构筑相结合，开展再细胞化再血管化和具有高度抗凝活性的仿生肝组织构建及其机制研究。

本项目聚焦工程化构建人工肝脏所面临的血管化重大难题，应用定量蛋白质组学、干细胞筛选培养及诱导分化等先进的细胞/分子生物学技术，阐明肝窦内皮细胞与肝脏祖细胞相互作用的关键分子和信号通路网络图谱；并利用该原理指导工程化构建脱细胞-再细胞-再血管化的人工肝组织。该项目涉及复杂脏器血管化及其机制的从基础创新研究到转化研究的路径，丰富和发展了糖生物学和组织工程再生医学知识体系，阐明复杂脏器组织工程构建中血管化的基本原理，提出了组织工程肝脏构建新策略，推动人体组织器官的修复替代的临床转化进程。. 本项目阐明了组织微环境中硫酸肝素蛋白聚糖糖链及其结合的细胞因子网络支持工程化肝脏组织血管化和结构功能重建的分子机制，并基于此构建再细胞化-再血管化组织工程肝脏。发现了特定酶修饰的硫酸肝素糖胺聚糖的促血管化作用，并利用基因工程化的内皮细胞营造肝脏微环境，实现了肝功能的大幅度提升，同时为组织工程肝脏提供了可靠稳定的种子细胞来源。在天然脱细胞直接的支持下，实现了再血管化和再细胞化的组织工程肝脏的构建，并以此为模型开展了系列疾病模型构建与机制探索研究，为药物的筛选提供了良好的体外模型。. 总之，本项目提出了一个新的机制，特定酶修饰的硫酸肝素氨基聚糖介导肝窦内皮细胞和肝上皮细胞相互作用；建立了一个新的技术体系，基于脱细胞肝脏生物支架建立再血管化再细胞化构建组织工程肝脏的新策略；开拓了一个新的视角，利用糖氨聚糖将组织工程三要素种子细胞、支架材料和三维构建整合起来，阐明血管化在复杂脏器组织工程化构建中的机制、意义和策略。