本文選題：肝臟干細(xì)胞　切入點(diǎn)：EMT　出處：《上海交通大學(xué)》2015年博士論文

【摘要】：肝臟干細(xì)胞是具有肝向和膽向分化潛能的細(xì)胞。作為“干細(xì)胞治療”隊(duì)伍中的重要成員,肝臟干細(xì)胞引起了人們極大的關(guān)注和需求,但肝臟干細(xì)胞的分化特性及其調(diào)控依然是制約肝臟干細(xì)胞臨床治療的應(yīng)用瓶頸。為解析肝臟干細(xì)胞的分化決定和分化定向及其調(diào)控機(jī)制,在本研究中,我們利用懸浮培養(yǎng)法富集了胚胎妊娠期12.5天小鼠胎肝中的成球細(xì)胞,發(fā)現(xiàn)這群肝細(xì)胞球來(lái)源的細(xì)胞同時(shí)兼具肝臟干細(xì)胞和間質(zhì)細(xì)胞的特征和分化潛能,提示其為早期胎肝中處于上皮-間質(zhì)轉(zhuǎn)化(epithelial-mesenchymal transition,EMT)狀態(tài)的胚胎肝臟干細(xì)胞群體。該群體可分為Dlk1+和Dlk1-兩個(gè)細(xì)胞亞群,鑒于膜表面型Dlk1(membrane-bound Dlk1,Dlk1M)和分泌型Dlk1(secretory Dlk1,Dlk1S)兩種異構(gòu)體的存在,我們隨后研究發(fā)現(xiàn),Dlk1+細(xì)胞同時(shí)表達(dá)Dlk1M和Dlk1S,Dlk1-細(xì)胞僅表達(dá)Dlk1S。進(jìn)一步研究表明Dlk1+細(xì)胞可丟失Dlk1M轉(zhuǎn)變?yōu)镈lk1-細(xì)胞,Dlk1-細(xì)胞仍保持細(xì)胞增殖能力,但其EMT程度增加,具有了膽管前體細(xì)胞樣特性。深入研究發(fā)現(xiàn),通過(guò)構(gòu)建過(guò)表達(dá)質(zhì)粒使Dlk1-細(xì)胞重新表達(dá)Dlk1M后,其分化特征得到明顯的逆轉(zhuǎn),表明Dlk1M具有功能性的調(diào)控作用,并且b FGF在誘導(dǎo)Dlk1+細(xì)胞丟失Dlk1M的轉(zhuǎn)變過(guò)程中起重要作用。此外,對(duì)Dlk1+細(xì)胞和Dlk1-細(xì)胞自噬水平的分析顯示,Dlk1+細(xì)胞在轉(zhuǎn)化為Dlk1-后,自噬水平降低,提示自噬可能在這一胚胎肝臟干細(xì)胞的分化調(diào)控中起作用�？傊�,我們的研究明晰了Dlk1隨著肝臟干細(xì)胞發(fā)育的動(dòng)態(tài)變化特征,Dlk1M的缺失是肝臟干細(xì)胞向膽管前體細(xì)胞分化的標(biāo)志性事件,同時(shí)也揭示了在這其中多重因素對(duì)肝臟干細(xì)胞分化的調(diào)控作用,不僅為肝臟干細(xì)胞的應(yīng)用提供了理論指導(dǎo),也為肝臟相關(guān)疾病如膽管細(xì)胞癌等得發(fā)病機(jī)理研究提供了啟示。
[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.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R575

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