HNF4α基因修饰脐带MSCs/永生化肝细胞共微囊移植在急性肝衰竭治疗中的作用机制研究

Immortalized hepatocytes are one of the ideal materials in the treatment of hepatocyte transplantation for acute liver failure, but the environment of inflammation in acute hepatic failure seriously affect the viability, function and differentiation of immortalized hepatocytes. Through previous research we discovered and confirmed the expression of HNF4α induces efficient differentiation of immortalized hepatocytes, HNF4α genetically modified human umbilical cord MSCs (hMSCs-HNF4α) can enhance the immunomodulatory and anti-inflammatory effects of hMSCs. In this project, we will explore the regularity of co-culture between hMSCs-HNF4α and immortalized hepatocytes, establish the most suitable co-encapsulation system for hMSCs-HNF4α and immortalized hepatocytes. The therapeutic effect on liver failure was studied by co-encapsulation intraperitoneal transplantation.The co-encapsulation transplantation of hMSCs-HNF4α and immortalized hepatocytes is expected to increase intercellular contact, improve the environment of inflammation, improve the viability, function and differentiation of transplanted immortalized hepatocytes, promote the proliferation and differentiation of hMSCs-HNF4α in microcapsules, promote liver regeneration and improve liver function.The objective of this research is to clear the regularity and interaction between hMSCs-HNF4α and immortalized hepatocytes during the co-encapsulation transplantation, solve the cell source and security problems of hepatocytes transplantation and lay a solid foundation of theory and practice for clinical application.

永生化肝细胞是急性肝衰竭肝细胞移植理想材料之一，但肝衰竭炎症内环境严重影响永生化肝细胞的活力、功能及分化状态。通过前期研究我们发现并证实了肝细胞核因子4α（HNF4α）表达可诱导永生化肝细胞高效分化，HNF4α基因修饰人脐带MSCs（hMSCs-HNF4α）可增强hMSCs免疫调节和抗炎作用；本课题探索hMSCs-HNF4α与永生化肝细胞共培养的规律，建立微囊化最适共培养体系并行共微囊腹腔移植，研究其对急性肝衰竭的治疗作用，以期通过hMSCs-HNF4α与永生化肝细胞共微囊移植增加细胞间的接触，改善肝衰竭炎症内环境，提高移植永生化肝细胞的活性、功能及分化状态，促进hMSCs-HNF4α在微囊内的增殖与分化，促进肝再生，改善肝功能。实验目的是明确hMSCs-HNF4α与永生化肝细胞共微囊移植过程中的相互作用和规律，解决肝细胞移植存在的细胞来源及安全性问题，为临床应用奠定坚实理论与实践基础。

肝细胞移植是治疗肝功能衰竭的有效方法，但肝衰竭炎症内环境严重影响肝细胞的活力、功能及分化状态；干细胞已被证实具有强大的分化潜能、自我更新能力、免疫调节作用以及靶向治疗功能，有望在急性肝衰竭炎症反应调节治疗中发挥重要作用，本课题探索肝细胞/干细胞共微囊移植在急性肝衰竭治疗中的作用及内在机制。课题组前期研究证明转染HNF4α能够增加永生化肝细胞中肝特异性蛋白的水平，本课题首次研究HNF4α基因修饰的人源性脐带间充质干细胞对成人原代肝细胞的作用及两者共微囊微联合移植在急性肝衰竭中的治疗作用。通过慢病毒转染构建HNF4α过表达的干细胞，与人肝细胞共微囊并移植到小鼠体内。移植后构建急性肝衰竭模型，检测小鼠肝脏损伤程度及炎症水平。建立巨噬细胞系极化模型和D-gal急性肝细胞损伤模型，观察其对巨噬细胞的极化以及肝细胞损伤的影响；采用蛋白芯片分析过表达HNF4α-MSC与MSC细胞因子的含量差异，通过GO富集分析推测HNF4α-MSC可能的保护机制。研究结果发现HNF4α-MSC能够明显增加人原代肝细胞的合成和分泌功能，HNF4α-MSC和肝细胞共移植可通过促进巨噬细胞从M1型向M2型极化来缓解肝脏的损伤程度及炎症水平。蛋白芯片分析结果表明HNF4α 转染能够明显增加保护作用的细胞因子的水平，GO富集分析结果表明这些因子可能通过激活ERK1/2来发挥作用。研究表明HNF4α-MSC和肝细胞共移植可以通过促进巨噬细胞从M1向M2极化来缓解急性肝衰竭，HNF4α过表达促进MSC分泌的细胞因子在其治疗作用中发挥重要作用。本项研究从肝细胞/干细胞的获取、分离培养，两者之间的比例、相互作用，两者微囊化培养及共微囊移植等方面展开了系统深入的研究，并进一步阐明了肝细胞/干细胞共微囊移植缓解急性肝衰竭免疫炎症内环境的内在机制，为今后临床应用奠定了坚实的理论与实践基础。