DNMBP对人骨髓间充质干细胞转分化肝细胞成熟的影响

Stem cell differentiated hepatocyte is a promising alternative of primary human hepatocyte, however the differentiation mechanism is still unclear, which limits the further clinical translation of the differentiated hepatocyte. During our previous research, we have established a liver humanized mouse model via transplantation of human bone marrow mesenchymal stem cell (hBMSC), and found that the transplanted hBMSC differentiated into hepatocyte like cells within three days, then these cells went through proliferation and maturation. Genes that were significantly differentially upregulated within two days were related with the regulation of liver function, among them DNMBP was found to be involved in the regulation of hepatocyte maturation. In this study, we will overexpress and knock down the gene expression of DNMBP in hBMSC, then perform the hepatogenic induction in vitro and in vivo, and evaluate the effect of DNMBP on the maturation of hBMSC-differentiated hepatocytes through the detections of human hepatocyte specific gene expressions, human albumin secretion, urea synthesis and metabolism of diazepam. mRNA sequencing and our in-house multi-omics cross-validation and network analysis will be used to analyze the transcriptome profiling at different differentiation stage after management of DNMBP, and to discover the signaling pathways and key genes that were regulated by DNMBP, finally clarify the mechanism of DNMBP on the maturation of hBMSC-derived hepatocytes. This research will provide theoretical foundation for the translation of stem cell-derived hepatocytes in regeneration medicine and clinical use.

干细胞转分化肝细胞被认为是最具前景的原代肝细胞替代细胞源，但转分化机制不清，严重阻碍临床转化应用。课题组前期已创建基于人骨髓间充质干细胞（hBMSC）移植的人肝嵌合小鼠，发现植入干细胞3天内完成转分化，经增殖后进入功能成熟期，移植后2天内特异性高表达基因与肝细胞功能密切相关，其中DNMBP参与调控转分化肝细胞的成熟。本研究拟在上述基础上，构建DNMBP过表达和敲除的hBMSC，分别进行体外肝向诱导分化和体内肝衰竭微环境下转分化研究，通过人肝细胞特异性基因表达、人白蛋白分泌、尿素合成、安定代谢能力等观察DNMBP表达变化对转分化细胞功能成熟的影响；利用转录组测序和课题组自主研发的多组学功能关联分析技术，揭示转分化不同阶段分子特征，寻找DNMBP调控肝细胞功能的信号通路及关键分子，阐明DNMBP对转分化肝细胞成熟的调控机制。为最终使用转分化肝细胞进行再生医学研究和临床应用提供理论基础。

干细胞体外诱导分化是获取肝细胞的主要途径，但转分化肝细胞功能无法达到成熟肝细胞水平，其主要问题在于转分化机制不清。本项目拟利用DNMBP调控人骨髓间充质干细胞（hBMSC）诱导分化肝细胞，但效果不佳。为深入解析分化机制，项目组利用体外诱导分化细胞模型和体内转分化动物模型，通过转录组分析，揭示干细胞转分化不同阶段分子特征，发现并验证了一批调控hBMSC转分化肝细胞的关键分子：1) 通过对hBMSC体外诱导分化肝细胞不同时间点的mRNA和miRNA测序分析发现，体外诱导20天后转录谱发生显著改变，参与固醇类代谢、脂类运输、免疫调节等肝细胞功能相关基因的表达显著上升，与多分化潜能、细胞周期等干性相关基因表达显著下调，进一步验证显示77个差异表达的miRNA中miR-26b-5p、miR-148a-3p和miR-423-3p可促进hBMSC转分化肝细胞；2) 通过对hBMSC移植肝衰竭小鼠后不同时间点小鼠肝脏内人源性肝细胞的转录组分析发现，植入后1天干性相关基因表达显著下调，植入后2天肝细胞功能相关基因表达开始上升，植入后14天39%人源性肝细胞的功能已接近原代肝细胞，进一步分析转录因子及其靶基因功能发现，TOP2A、CDT1、PCNA等转录因子参与启动肝向分化，NR1H4、FOXA2、HNF4A等转录因子参与肝细胞功能成熟，验证实验初步证实NR1H4可提升肝细胞功能。上述研究不仅丰富了干细胞相关理论知识，为干细胞临床转化提供理论基础；同时也为优化体外诱导分化、获取功能成熟肝细胞提供了新思路。. 相关研究成果已发表SCI论文5篇，作全国学术会议报告2次，获浙江省科技进步一等奖1项，受邀担任学术期刊《Expert Rev Gastroent》（IF 4.095）的审稿人。协助培养博士生1名、硕士生2名，项目负责人作为骨干获批“科技部创新人才推进计划重点领域创新团队”。