人脐带干细胞诱导的类肝细胞在小鼠肝内的成熟分化机制研究

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.

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。

依照申请书与计划书，项目实施过程中首先在已有文献报道的基础上，对主流的类肝细胞分化方法进行了改良，采用两步法得到了分化自人类脐带间充质干细胞的类肝细胞。通过一系列检测方式的组合对其肝功能进行了验证。接着构建了pAAV-rtTA-DTA质粒，通过尾静脉高压注射的方法将其注射至NOD/SCID免疫缺陷小鼠体内。发现口服多西环素能够显著诱导小鼠肝细胞大量死亡，和其他药物配合能够构建稳定的急性肝衰竭模型。然后将分化好的类肝细胞和没有分化的原始干细胞分别通过尾静脉注射至急性肝衰竭小鼠体内，发现两种细胞增殖和分化过程及对受体老鼠肝细胞功能的恢复差异不显著，说明至少对于急性肝衰竭而言，在体外不经过分化，直接输入原始干细胞可能更为安全。最后详细研究了细胞在体外培养情况下抗逆境（如氧化应激损伤和炎症损伤）的能力和体内移植后修复肝脏组织的分子机制，发现抗氧化剂小分子（如依达拉奉和Zeaxanthin dipalmitate）够通过MAPK-PKC- Nrf2通路或miR-210有关通路调节其内源抗氧化水平，增加其体外抗逆境能力和移植治疗NOD/SCID急性肝衰竭的效率。.本项目执行期间共以第一作者或通讯作者发表SCI论文4篇，中文核心论文1篇，申请专利1项。依托本项目的研究成果，已经成功申请主持了2项后续研究课题，包括广东省重大科技专项和深圳市知识创新计划基础研究项目。培养1名硕士研究生。获得广东省高等学校“千百十工程”校级培养对象、广州市珠江科技新星和广东省优秀青年教师等人材项目奖项。.