利用搭载三维微肝器官的京尼平修饰去细胞化生物支架构建具有胆管网络的组织工程肝脏的机制研究

Significant advances have been made in tissue engineering liver (TEL) based on decellularization and recellularization strategy. In preliminary studies, we have proved that the heparinized decellularized liver scaffold (DLS) could improve proliferation of primary hepatocyte spheroids, and reconstruction of vascular system while long-term maintenance of specific functions, but failed to form any bile duct. In fact, reconstruction of bile duct network in vivo has been the emphasis and difficulty in TEL. Given that induced pluripotent stem cells (iPSC) show strong ability of proliferation and differentiation, and genipin demonstrates the characteristics of improving proliferation and migration of biliary epithelial cells, and promoting bilirubin metabolism, we will generate three-dimensional hepatic organoids with micro bile duct structures derived from iPSC, primary rat hepatocytes and endothelial cells using rocker culture system, and recellularize genipin crosslinking DLS with hepatic organoids to construct bioengineered hepatic tissue, then implant into omentum majus in liver-injured rats. This study aims at the feasibility of reconstruction of bile duct network in bioengineered hepatic tissue and its effectiveness of the treatment on liver injury. Based on this, the influences and molecular mechanisms of extracellular matrix microenvironment from different modifications on cell engraftment, distribution, differentiation and proliferation of hepatic organoids will be further explored, so as to get a convenient, economic and efficient strategy to construct TEL. This study is expected to provide a new thought for bile duct network reconstruction in TEL, to prolong the survival time further, and pave the way for precise treatment for patients with liver disease in the future.

在肝移植替代治疗方案中，基于去细胞-再细胞化策略的组织工程肝脏（TEL）研究进展迅速。本课题组在前期研究中发现肝细胞聚球体与肝素修饰去细胞化肝脏支架材料（DLS）在体内能够快速构建具有血管网络和特异性功能的工程肝，但未实现肝内胆管网络的重建，胆汁淤积终将引起工程肝内细胞坏死而慢性失功。体内重建胆管网络是TEL的重点和难点。鉴于诱导性多能干细胞（iPSC）具有强大的增殖和分化能力，京尼平修饰DLS具有促进胆管细胞增殖和迁移及促进胆红素代谢的特点，本项目拟用iPSC定向分化的胆管微结构、肝细胞和内皮细胞混合震荡培养形成的三维微肝器官对京尼平修饰DLS再细胞化，构建工程肝，研究其在肝损伤动物体内重建胆管网络的可行性和治疗肝损伤的有效性。在此基础上深入研究工程肝所处微环境及其对细胞定植、分布、分化、增殖的影响及分子机制。本研究有望为TEL重建胆管网络提供新的思路，进一步延长TEL移植后的存活时间。

肝脏替代治疗是目前终末期肝病最有效的治疗措施，基于去细胞-再细胞化策略的组织工程肝脏（TEL）研究进展迅速，拓宽了肝脏替代治疗的研究领域。鉴于诱导性多能干细胞（iPSC）具有强大的增殖和分化能力，京尼平修饰DLS具有促进胆管细胞增殖和迁移及促进胆红素代谢的特点，课题组在本研究中首先对iPSC定向分化为胆管样细胞，并与原代肝细胞和内皮细胞混合震荡培养形成的三维微肝器官。进而采用Triton-SDS灌注法制备去细胞化肝脏生物支架材料（DLS），并行京尼平交联修饰。在体外用三维微肝器官对京尼平DLS进行再细胞化构建组织工程肝，将其异位植入四氯化碳（CCl4）诱导肝损伤大鼠模型的大网膜并吻合空肠。结果表明经过诱导iPSC形态发生上皮-间质转化样变化，细胞增殖减慢和无法分化的细胞死亡增加。诱导后期因细胞形态变化、表型改变且没有足够的空间继续生长而卷曲成具有腔管样结构的团块。H&E染色分析显示在移植后10-14周微肝组织内部各种细胞数量明显增加，细胞排列逐渐规则，组织成致密的聚集体，有管腔结构分布列其中和移植物外周。对植入的微肝组织进行胆管细胞特异性标志物免疫荧光染色（CK7/SOX9），显示出微肝组织内细胞表达胆道上皮的特征。阳性细胞主要分布于微肝组织边缘，多为立方状体细胞，排布形成管腔结构，说明移植物在体内形成具有胆管管腔结构的功能性组织工程肝脏。鉴于微肝组织能够表现出肝脏特异性功能，这些微肝器官移植物在CCl4诱导的肝损伤中表现出治疗潜力。这些结果为含有胆管网络的组织工程肝脏从构建到移植提供了一种经济可行的方法，并进一步延长其在体内的存活时间，具有一定的临床应用潜力和价值。