细胞表面糖蛋白在骨髓间充质干细胞体内分化为肝细胞中的作用机制研究

The effects of stem cell transplantation and stem cell based liver tissue regeneration have been widely recognized on the treatment of hepatic failure. Cell surface glycoproteins have an important role in stem cell differentiation as potential key regulatory proteins. The similar regulatory mechanism has not yet been examined in stem cell differentiation into hepatocytes. Our previous study had rescued fulminant hepatic failure (FHF) pigs and mice by human bone marrow mesenchymal stem cell (hBMSC), with several liver specific glycoproteins identified in hBMSC-derived hepatocytes (hBMSC-Hep). Cell surface glycoproteins are key molecules as induction signal receptor in stem cell. And the apparence of specific glycan expressions is closely associated with the multipotential differentiation of stem cell. However, the mechanisms of glycoproteins in regulating hepatogenic differentiation remain to be investigated. Therefore, the aim of this study is to characterize the mechanisms of cell surface glycoprotein during stem cells differentiation into hepatocytes. Fluorescence Activating Cell Sorter (FACS) will be used to select the hBMSC-Hep differentiated in FHF mice. The qualitative and quantitative proteomics analysis of glycoproteins will be performed by a combination of hydrophilic interaction liquid chromatography and multiple fragmentation tandem mass spectrometries after the enrichment of cell surface glycoproteins in hBMSC-Hep. The regulatory mechanisms of glycoproteins will be observed in the specific pathways during hBMSC differentiation and verified by relevant expression of glycosyltransferases and glycosidases. The final results will be helpful to develop novel cell surface markers to evaluate the properties of hBMSC-Hep and clarify the precise molecular mechanisms of glycoproteins during hBMSC differentiation in vivo.

干细胞体内分化机制研究是干细胞临床转化的关键问题。细胞表面糖蛋白作为表面抗原、受体、酶或运载蛋白在干细胞分化调控中起关键作用，但其在分化为肝细胞中的作用未见报道。课题组通过人骨髓间充质干细胞（hBMSC）移植成功治疗FHF猪与小鼠，发现大量hBMSC分化的人源性肝细胞参与宿主肝脏再生与修复，并在干细胞分化过程中鉴定到多种肝脏特异的功能糖蛋白（如肝细胞特异性受体NTCP）。但糖蛋白结构与含量的动态变化信息缺乏，糖蛋白调控机制尚不明确。本研究拟在前期研究基础上，深入研究干细胞体内分化为肝细胞的动态过程。利用流式细胞术分选hBMSC移植后体内分化多个时间点的人源性肝细胞，采用梯度溶解结合亲水富集法获取细胞表面糖蛋白，应用质谱技术系统表征并筛选人源性肝细胞标志物。采用糖蛋白-凝集素受体配体识别系统、糖基转移酶调控等阐明糖蛋白在干细胞分化中的作用，最终为干细胞在宿主体内的分化机制研究提供理论基础。

暴发性肝衰竭（fulminant hepatic failure, FHF）病死率高达80%，除肝移植外无有效治疗方法。以干细胞为基础的再生医学为重症肝病的治疗开辟了新途径。然而，干细胞向肝细胞分化的机制尚不清楚，其在临床一线治疗的应用仍存在困难。本研究基于人骨髓间充质干细胞(hBMSC)移植救治FHF小鼠构建人源化肝脏嵌合小鼠模型，获取干细胞移植后1天、2天、3天、7天、14天的小鼠的肝脏组织。肝组织样本进行免疫组化分析显示肝组织中CD29、CD90等干细胞的标志物阳性率逐步降低，而白蛋白，牛磺胆酸转运蛋白NTCP等阳性率逐步升高，结果显示干细胞在肝衰竭小鼠中快速分化为类肝细胞。通过肝细胞灌流技术与流式细胞术分选获得小鼠肝脏中的人骨髓间充质干细胞来源的类肝细胞（hBMSC-Hep)，并进行组学鉴定。采用生物信息学技术对干细胞连续多个分化阶段的hBMSC-Hep分子特征进行差异分析，建立hBMSC-Hep细胞的特异性标志物。进一步采用功能关联网络分析GSLA技术，对分化各个阶段之间的差异基因进行功能分析。D1与D2差异基因分析显示，干细胞启动分化过程表现为改变细胞极性，大量与细胞分化相关的基因与通路在D2高表达。D2与D3的差异基因主要集中在细胞周期调控方面。其中主要有RNA转录相关的启动因子，RNA生物合成功能的正调控等方面。D3与D7的差异基因分析显示，83个关键通路被富集，细胞开始进入大量细胞增殖阶段,同时开始出现酒精代谢功能，毒素代谢功能等肝脏生化功能途径。D14高表达的基因预测出大量肝脏功能性途径，表明D14干细胞已经分化成较为成熟的类肝细胞。此外，本研究还对hBMSC分化为肝细胞过程中的miRNA的调控机制进行研究，并采用miRNA模拟物mimics或抑制剂inhibitions研究miRNA调控分化的机制，发现miR-148-a-3p、miR-26p-5p、miR-423-3p等有促进干细胞分化为肝细胞的能力。