【作者】 周庆军；

【导师】 邵健忠；

【作者基本信息】 浙江大学 ， 细胞生物学， 2006， 博士

【摘要】 建立胚胎干细胞体外分化肝细胞的实验体系是开展肝脏发育的细胞与分子调控机制、胚胎发育过程中中胚层和内胚层间的相互作用规律以及肝细胞分化发育调控和相关疾病防治药物筛选等研究的重要基础，同时也可以为开展肝细胞移植、肝组织工程、生物人工肝等研究提供有效的细胞来源，因此目前该方面的研究备受关注。本文首先建立了一种拟胚体形成的新方法，在此基础上，利用不同细胞因子组合，通过拟胚体途径成功诱导了胚胎干细胞向肝细胞分化，同时建立了组蛋白去乙酰化酶抑制剂体外直接诱导胚胎干细胞分化肝细胞的体系，并发现分化过程中伴随细胞周期阻滞和细胞凋亡，在此诱导分化过程中，首次分离鉴定到一类新型的前体细胞，具有肝干／祖细胞的典型标志和双分化潜能，从而为肝脏早期发育调控的细胞与分子机制等研究提供了良好的体外模型。 当前，有关胚胎干细胞分化各种不同谱系细胞的研究多通过拟胚体途径模拟早期胚胎发育过程来进行，因此拟胚体常作为胚胎发育尤其是早期谱系决定、胚层相互诱导等研究的体外模型。我们以小鼠ES-D3细胞系为材料，利用SIM小鼠来源的STO成纤维细胞系，建立了一种饲养层细胞介导的拟胚体形成新方法。与传统拟胚体制作方法相比，具有操作简便，实验体系和结果稳定，可以大量获取拟胚体等优点，得到的拟胚体70％以上都可以发育形成成熟的囊状拟胚体，分化潜能研究显示其具有分化为神经细胞、心肌细胞和肝细胞等三胚层来源细胞的能力，在此基础上，利用aFGF、bFGF和BMP4等细胞因子组合，成功诱导了胚胎干细胞向肝细胞分化，并发现由横膈间充质分泌的BMP4可以显著增强肝细胞的分化。在早期诱导信号中，aFGF和bFGF的作用类似。 与细胞因子诱导剂相比，化学合成的诱导剂具有半衰期长、作用稳定、效应明确等特点。更适合于干细胞的长期分化诱导过程。我们采用丁酸钠和丙戊酸钠两种HDAC抑制剂作为肝细胞分化诱导剂，成功建立了胚胎干细胞体外直接分化肝细胞的实验体系，并发现HDAC抑制剂在诱导肝细胞分化过程中伴随着细胞周期阻滞和凋亡，其中CDK抑制剂p21（?）可能参与了HDAC抑制剂诱导的细胞周期阻滞。鉴于胚胎干细胞具有正常细胞和癌细胞两方面的特性，HDAC抑制剂诱导胚胎干细胞向肝细胞的分化可以作为研究组蛋白（去）乙酰化修饰调控基因表达的模型。在HDAC抑制剂诱导肝细胞分化过程中，我们还首次分离鉴定到一类新型的前体细胞，这类细胞表达肝干／祖细胞的典型标志，具有向肝细胞和胆管上皮细胞的双分化潜能，且分化得到的成熟肝细胞具有白蛋白、糖原合成，细胞色素P450活性，对CCl₄引起的小鼠肝损伤具有一定的修复作用，因此可以为肝组织工程等细胞替代治疗提供种子细胞。除与已报道的更多还原

【Abstract】 Hepatic differentiation from embryonic stem cells provides an useful in vitro model for studying hepatogenesis and liver metabolism, which is also important for the investigations of the mechanisms of liver development, the pharmokinetic and cytotoxicity screening test for drug discovery applications, and the elucidation of the molecular mechanisms and signaling pathways that regulate mutual induction between the endoderm and the mesoderm during in vivo hepatogenesis. Furthermore, hepatic differentiation from ES cells provides an efficient cell source for cell replacement therapy, such as hepatocyte transplantation, liver tissue engineering and bioartificial liver. Firstly we developed a novel method for generating embryoid bodies from mouse ES cells cultured on the STO feeder layer, and induced the hepatocyte differentiation from ES cells based on the diverse combination of growth factors required for the early liver development. Afterwards, we established a hepatic differentiation system from mouse ES cells with the induction of histone deacetylase (HDAC) inhibitors and isolated a novel hepatic stem/progenitor cell, which provides an appropriate model for the study of the cellular and molecular regulation of liver development.Presently, the most widely used method for generating various lineage cells from ES cells has been using the formation of embryoid bodies, which recapitulate many aspects of the lineage-specific differentiation programs, and the temporal and spatial gene expression patterns of early embryogenesis, therefore, the EB system provides an in vitro model for the study of embryo development and stem cells differentiation. In the present study, We described a novel method for the generation of embryoid bodies from murine ES-D3 cells based on the STO feeder layer. Compared with those traditional methods, the protocol we described is easier to perform and obtain large amounts of EBs spontaneously, of which above 70% could develop into typical cystic embryoid bodies. The results of in vitro differentiation ability evaluation revealed that the EBs could differentiate into neural-, cardiac- and hepatic-lineage cells. Based on the previous reports on embryonic liver development, we applied acidic- and basic-FGF, HGF, and BMP4 to induce hepatic differentiation of EBs in vitro, and found BMP4, secreted from septum transversum mesenchyme, acted cooperatively with FGFs from precardiac mesoderm to significantly enhance the hepatocyte differentiation, while aFGF and bFGF effected equally in the early hepatic更多还原