肝脏移植是临床上延长和挽救晚期肝病患者的唯一方法。由于供体的严重不足和移植后的免疫排斥反应，该方法的应用受到了极大限制。间充质干细胞诱导分化的类肝细胞已被证明具备一定的肝细胞功能，使用该细胞修复严重受损的肝脏，已成为临床上极具希望的技术。但是，现阶段体外诱导分化类肝细胞的方法存在肝细胞功能不完全、具有移植后免疫原性和移植后成熟分化机制不明确的问题。据此，本项目拟首先构建含白喉毒素A表达的腺相关病毒载体，感染免疫缺陷NOD/SCID小鼠后经多西环素诱导，于小鼠体内形成稳定的肝损伤环境。同时收集人类脐带间充质干细胞，在经过不同程度的体外诱导分化后注射入NOD/SCID小鼠体内，经过一段时间后比较不同体外诱导阶段的类肝细胞在体内分化和成熟进程的机制差异，找出体外诱导和体内成熟的最优化组合。同时我们希望在细胞水平提出一套类肝细胞的肝功能评价体系，为临床上晚期肝脏疾病患者的再生医学治疗提供理论依据。

Liver transplantation is the only way to prolong or save the life of patients with late-stage liver diseases clinically. However, due to the significant shortage of donor and immunological rejection after transplantation, the application of liver transplantation is constrained. Hepatocyte-like cells (i-Hep) differentiated from mesenchymal stem cells (MSC) are proven to possess a certain number of hepatocyte functions. It is a promising way to save the life of patients with liver diseases. However, at current stage, i-Hep induced from MSC may not have full spectrum of hepatic functions, immunogenicity after transplantation and uncleared maturation mechanisms after in vivo transplantation. Therefore, the current proposal will firstly construct an adeno-associated virus vector with the expression box of diphtheria toxin A, which will be used to infect the liver of immuno-deficient NOD/SCID mice to induce constant liver injury under the induction of doxycycline. In the mean time, MSC will be collected from human umbilical cord to differentiate into different forms of i-Hep in vitro. Then i-Hep with different differentiated stages will be injected into NOD/SCID mice with constant liver injury. After a certain period, the mechanistic differences of in vivo maturation among i-Heps will be compared to find out an optimistic combination of in vitro differentiation and in vivo maturation. Moreover, we want to set up a new evaluation system for the hepatic functions of i-Hep, in order to shed some light for the regenerative medical therapy of patients with late-stage liver diseases.