中文摘要：

肝干/祖细胞是肝细胞和胆管上皮细胞（biliary epithelial cells,BECs）共同的前体细胞,为了对这一前体细胞的分化情况进行研究,利用报告基因来监测肝干/祖细胞的分化走向.首先,通过PCR方法从肝癌细胞系HepG2的全基因组中克隆了细胞角蛋白19（CK19）启动子片段,构建了CK19启动子调控的海肾荧光素酶和红色荧光蛋白（red fluorescent protein,RFP）双报告载体（pSicoR-CK19-hrl-mrfp）.其次,将上述慢病毒载体转染肝干/祖细胞后,通过流式细胞分选获得稳定转染的细胞株.再次,将上述细胞株与表达＂上皮形态发生素＂（Epimorphin,EPM）的PT67细胞共培养后,整合有pSicoR-CK19-hrl-mrfp表达载体的肝干/祖细胞不仅形态发生了变化,而且排列为二维环状结构,另外还检测到由CK19启动子启动表达的海肾荧光素酶和RFP,细胞形态和基因表型都证明肝干/祖细胞经诱导已经分化成为BECs.与此形成对照的是肝干/祖细胞与不表达EPM的PT67细胞共培养后,没有观测到上述的变化.所以,CK19启动子调控的双报告载体不仅可以实时地显示肝原始细胞在不同的诱导环境下的分化走向,而且还可以定量地检测CK19启动子活性的变化情况.总之,这一载体的成功构建将为研究肝干/祖细胞的分化提供了便捷的工具,同时也有助于筛选可诱导肝干/祖细胞定向分化的分子.

英文摘要：

Liver progenitor cells are co-precursor cells of hepatic cells and bile duct epithelial cells, a reporter gene was used to research the differentiation of liver progenitor cells. First of all, the cytokeratin 19 promoter segment was cloned from hepatocellular carcinoma cell line HepG2 and then following the promoter a renilla luciferase and a red fluorescence protein＇s fusion gene were inserted to finish the double report lentiviral vector. Second, the liver progenitor cells were transducted with lentivirus, and then GFP positive cells were enriched by flow cytometry sorting. Third, the GFP positive liver progenitor cells were co-cultured with PT67 cells which could express the molecule-epimorphin for 5 days. Then, it was found that the stable transducted liver progenitor cells＇ shape were not only transformed and arranged as cord like structure, but also renilla luciferase and red fluorescence protein which enhanced by CK19 promoter were detected. So, these results proved that the liver progenitor cells had been induced to bile duct epithelial cells. The vector enhanced by CK19 promoter can monitor the differentiation of liver progenitor cells in different environment. In a word, this lentivirus vector can help us study the differentiation mechanism of liver progenitor cells, and scan the molecules which can do a help in differentiation.