仿肝板结构肝组织三维培养恢复并维持肝细胞极性的机制研究

Nowadays, bioartificial liver is faced with the main problem that how hepatocytes can maintain hepatocyte phenotype and differentiated functions when cultured in vitro for long time by taking advantage of bioreactors. Hepatocyte polarity is the foundation of hepatic functions. And hepatocytes are arranged into the polarized hepatic plate structure and attain cell-cell and cell-matrix interactions within a liver. Based on the bionics, we propose the project of three-dimensional culture of liver tissue mimicking the hepatic plate structure. We conclude that liver tissue mimicking the hepatic plate structure contributes to the recovery and maintenance of hepatocyte polarity so that hepatocyte phenotype and differentiated functions in vitro will be much more resemble to the state in vivo. We are planning to construct human liver tissue mimicking the hepatic plate structure by means of microfluidic chip. Compared with the other culture patterns, we measure their hepatic functions to find a novel three-dimensional culture pattern in vitro and bioreactor that is proper to the application of bioartificial liver. Meanwhile we study the hepatocyte polarity changes to investigate the mechanism that how three-dimensional culture of liver tissue mimicking the hepatic plate structure reconstructs hepatocyte polarity and relationship between function and reconstruction of polarity. Based on the above research, we can provide guidelines for the reconstruction of hepatocyte polarity in vitro and lay a solid foundation for bioartificial liver, treatment by transplantation of hepatocyte, and study of liver pathology and physiology.

如何利用生物反应器在体外培养中长时间维持肝细胞的表型和分化功能是生物人工肝目前面临的关键问题。极性是肝细胞功能的基础，我们通过借鉴肝细胞在肝脏的极性肝板排列，以及其与非实质细胞、细胞外基质相互作用的仿生学原理，提出仿肝板结构肝组织三维培养技术研究课题，认为仿肝板结构三维培养有助于重建和维持肝细胞极性从而使肝细胞在体外培养中具备更接近于体内细胞的表型和分化功能。本项目拟利用微流控芯片技术在体外构建仿肝板结构肝组织，通过对这种培养模式下肝细胞功能进行测定并与其他体外培养模式相比较，以期得到新的适于生物人工肝应用的体外肝细胞培养模式以及新型生物反应器。同时对肝细胞极性变化进行测定，探究体外培养肝细胞极性重建的机制以及细胞功能与极性重建之间的关系，一方面为体外肝细胞极性重建研究提供指导依据，另一方面也为生物人工肝、细胞移植治疗、肝脏病理生理等进一步研究奠定基础。

体外肝细胞三维培养是肝组织工程的核心环节，肝细胞极性是肝脏生理功能的基础，只有完全仿生的三维培养，才能最大程度维持肝细胞极性和功能表型。现有三维培养研究存在不足：1.不能完全重现肝脏精细的肝板三维拓扑结构及微环境，使其活力功能无法与在体肝细胞相比；2.对肝细胞极性相关研究缺乏及相关结论没有代表性。本项目拟通过仿肝板肝组织三维培养，增设完善、标准的实验对照，对仿生状态下肝细胞活力、功能、极性系统比较，并转录组测序分析肝细胞极性相关通路改变并验证，实验结果如下：. 1.仿肝板三维培养组肝细胞在组织形态维持以及活力优于其他各组；. 2.肝细胞功能蛋白水平：仿肝板三维培养组白蛋白、α1-抗胰蛋白酶、转铁蛋白、CYP1A2、CYP2D6、谷胱甘肽-S-转移酶、安定代谢、氨代谢、睾酮代谢优于其他各组；. 3.肝功能基因表达：仿肝板三维培养组CYP2D6、CYP1A2、氨甲酰磷酸合成酶、凝血因子Ⅱ、凝血因子Ⅴ、凝血因子Ⅶ表达优于其他各组；. 4.肝细胞极性蛋白基因表达：仿肝板三维培养组紧密连接Occludin、二肽基肽酶Ⅳ、钠离子-牛磺胆酸共转运多肽、白细胞分化抗原147优于其他各组；肝细胞极性蛋白荧光染色：F组Occludin蛋白、ZO-1蛋白、MRP2 蛋白、CD147蛋白的表达优于其他各组；超微结构：仿肝板组形成更多成熟胆小管结构，优于其他各组；. 5.转录组测序分析及qPCR验证：仿肝板三维培养促进胆汁分泌通路相关改变，作用机制可能为通过核受体（法尼酯X受体、小异源二聚体伴侣受体、类视黄醇X受体）调控，调节相应胆汁摄取、外排转运体（多药耐药相关蛋白2、多药耐药蛋白MDR1和MDR3、钠离子-牛磺胆酸共转运多肽、可溶性载体OST-α）及水通道蛋白8，实现对胆汁的摄取、分泌。. 仿肝板三维培养可促进肝细胞活力、重要肝功能及肝细胞极性的改善和维持，促进胆汁分泌相关通路改变。本项目建立的相关技术体系及培养肝组织可作为一种更优的体外三维培养平台及基本单位，应用于药物研发、肝细胞极性研究、生物人工肝生物反应器设计、肝组织工程组织器官构建等研究。