组蛋白去乙酰化酶抑制剂曲古菌素A联合细胞因子体外诱导胚胎干细胞分化为肝细胞的研究.docVIP

  组蛋白去乙酰化酶抑制剂曲古菌素 A 联合 细胞因子体外诱导胚胎干细胞分化为肝细 胞的研究#  5 10 15 20 25 30 35 40 朱晓波1,2，张冬梅2，张谦3，张洁2，周鸣鸣3** （1. 如皋人民医院 儿科，江苏 如皋 226000； 2. 江苏省南通大学医学院，江苏 南通 226019； 3. 江苏省南通大学生命科学学院，江苏 南通 226007） 摘要：建立细胞因子和曲古菌素 A 联合诱导小鼠 ES 细胞向肝细胞分化的 3 步法。ES 细胞 在含激活素 A 的不含白血病抑制因子的培液中培养 3 天分化为定形内胚层细胞；然后加入 酸性成纤维细胞生长因子和 TSA 联合诱导 5 天，再加入 HGF 等诱导因子培养 5 天，向肝细 胞方向分化，最后以致瘤素 M、地塞米松继续培养 5 天形成成熟肝细胞。自发分化组做阴 性对照组不加上述因子，不含 LIF 的培养液进行自发分化培养。诱导分化 18 天后，用光镜， 免疫荧光、RT-PCR 检测。结果显示诱导 18 天后，出现了大量类肝脏细胞表型的上皮细胞， RT-PCR 检测显示这些细胞表达成体肝脏细胞特异标记如酪氨酸转氨酶、白蛋白、天冬氨酸 转氨酶、甲状腺激素结合蛋白等。细胞免疫荧光也显示为甲胎蛋白、白蛋白、细胞角蛋白 18 阳性，分化细胞具有成熟肝脏细胞所具有的糖原储存、吲哚菁绿摄取和释放功能。其分 化效率用白蛋白阳性率表示，诱导分化率可达 57.38％,为肝细胞来源提供了新的方法。 关键词：胚胎干细胞; 肝样细胞; 诱导分化 中图分类号：Q952.1 Differentiation of embryonic stem cells into hepatocytes induced by a combination of cytokines and Trichostatin A Zhu Xiaobo1,2, ZHANG Dongmei2, ZHANG Qian3, ZHANG Jie2, ZHOU Mingming3 (1. Rugao Renmin Hospital,department of paediatrics , Nantong 226000; 2. Medicine school, Nantong University, Nantong 226019; 3. Life Science shool, Nantong University, Nantong 226007) Abstract: In this paper ， we presented a novel 3-step procedure to efficiently direct the differentiation of mouse ESCs into hepatocytes induced by combination of cytokines and sodium butyrate. Mouse ESCs were first differentiated into definitive endoderm cells by 3 days of Activin A treatment; next, presence of acid fibroblast growth factor(aFGF)and Trichostatin A (TSA)in the culture medium for 5 days, definitive endoderm cells efficiently differentiated to hepatocytes. After 10 days of further in vitro maturation, the morphological and phenotypic markers of hepatocytes were characterized using light microscopy, immunofluorescence and RT-PCR, respectively. Furthermore, these cells were tested about the functions associated with mature hepatocytes including glycogen storage, indocyanine green uptake and release,and the ratio of hepatic differentiation was determined by counting the albumin-positive cells，which showed that the ratio of hepatic