胚胎肝干细胞胆向分化的分子标志特征及其调控机制

发布时间：2018-03-23 12:57

本文选题：肝脏干细胞　切入点：EMT　出处：《上海交通大学》2015年博士论文

【摘要】：肝脏干细胞是具有肝向和胆向分化潜能的细胞。作为“干细胞治疗”队伍中的重要成员,肝脏干细胞引起了人们极大的关注和需求,但肝脏干细胞的分化特性及其调控依然是制约肝脏干细胞临床治疗的应用瓶颈。为解析肝脏干细胞的分化决定和分化定向及其调控机制,在本研究中,我们利用悬浮培养法富集了胚胎妊娠期12.5天小鼠胎肝中的成球细胞,发现这群肝细胞球来源的细胞同时兼具肝脏干细胞和间质细胞的特征和分化潜能,提示其为早期胎肝中处于上皮-间质转化(epithelial-mesenchymal transition,EMT)状态的胚胎肝脏干细胞群体。该群体可分为Dlk1+和Dlk1-两个细胞亚群,鉴于膜表面型Dlk1(membrane-bound Dlk1,Dlk1M)和分泌型Dlk1(secretory Dlk1,Dlk1S)两种异构体的存在,我们随后研究发现,Dlk1+细胞同时表达Dlk1M和Dlk1S,Dlk1-细胞仅表达Dlk1S。进一步研究表明Dlk1+细胞可丢失Dlk1M转变为Dlk1-细胞,Dlk1-细胞仍保持细胞增殖能力,但其EMT程度增加,具有了胆管前体细胞样特性。深入研究发现,通过构建过表达质粒使Dlk1-细胞重新表达Dlk1M后,其分化特征得到明显的逆转,表明Dlk1M具有功能性的调控作用,并且b FGF在诱导Dlk1+细胞丢失Dlk1M的转变过程中起重要作用。此外,对Dlk1+细胞和Dlk1-细胞自噬水平的分析显示,Dlk1+细胞在转化为Dlk1-后,自噬水平降低,提示自噬可能在这一胚胎肝脏干细胞的分化调控中起作用。总之,我们的研究明晰了Dlk1随着肝脏干细胞发育的动态变化特征,Dlk1M的缺失是肝脏干细胞向胆管前体细胞分化的标志性事件,同时也揭示了在这其中多重因素对肝脏干细胞分化的调控作用,不仅为肝脏干细胞的应用提供了理论指导,也为肝脏相关疾病如胆管细胞癌等得发病机理研究提供了启示。
[Abstract]:Liver stem cells are cells with the potential to differentiate into the liver and gallbladder. As an important member of the "stem cell therapy" team, liver stem cells have attracted great attention and demand. However, the differentiation characteristics and regulation of hepatic stem cells are still the bottleneck of clinical treatment of liver stem cells. In order to analyze the differentiation decision, differentiation orientation and regulation mechanism of liver stem cells, The suspension culture method was used to enrich the globular cells in the fetal liver of 12. 5 day gestational mice. It was found that the cells derived from the hepatocytes had the characteristics and differentiation potential of both the hepatic stem cells and the interstitial cells. The results suggest that it is a group of embryonic liver stem cells in the epithelial-mesenchymal transition state of early fetal liver, which can be divided into two subgroups of Dlk1 and Dlk1-, given the existence of membrane surface type Dlk1(membrane-bound Dlk1 Dlk1M and secretory Dlk1(secretory Dlk1S. We then found that both Dlk1M and Dlk1- were expressed in Dlk1 cells. Further studies showed that Dlk1 cells could lose Dlk1M to Dlk1- cells and maintain the proliferation ability of Dlk1- cells, but the degree of EMT was increased, and the expression of Dlk1S- cells in Dlk1- cells was higher than that in Dlk1- cells, but the expression of Dlk1S- cells was higher than that of Dlk1- cells. It was found that the differentiation of Dlk1- cells was reversed after the expression of Dlk1- cells was reexpressed by constructing overexpression plasmid, which indicated that Dlk1M had a functional regulatory role. In addition, the level of autophagy of Dlk1 cells and Dlk1- cells showed that the autophagy level of Dlk1- cells decreased after transformation to Dlk1-, and b FGF played an important role in inducing the loss of Dlk1M in Dlk1 cells. This suggests that autophagy may play a role in the regulation of differentiation of embryonic liver stem cells. Our study shows that the dynamic change of Dlk1 along with the development of hepatic stem cells and the absence of Dlk1M are the iconic events for the differentiation of hepatic stem cells into bile duct precursor cells. At the same time, it also reveals the regulation of liver stem cell differentiation by multiple factors, which not only provides theoretical guidance for the application of liver stem cells, but also provides inspiration for the study of pathogenesis of liver related diseases such as cholangiocarcinoma.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R575

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