作　　者： ; ; ; ; ; ; ; ; ;

机构地区： 华中科技大学同济医学院附属协和医院

出　　处： 《生物化学与生物物理进展》 2004年第6期518-522,共5页

摘　　要： 探讨了肝细胞在胚胎干细胞 (EScell)体外诱导分化系统中成熟分化的条件、机制及其鉴定方法 .利用TGF ,bFGF、HGF等细胞生长因子进行BALB/c小鼠ES细胞向肝细胞方向的定向诱导 .利用反转录聚合酶链反应 (RT PCR)、免疫细胞化学 (ICC)和放射免疫法 (RIA)动态检测肝细胞特异性基因和蛋白AFP ,ALB ,G6P ,TAT ,CK8,CK18等在培养体系中的表达 ,并测定肝细胞的尿素合成功能 ,最后测定肝细胞分化率 .结果 ,肝细胞特异基因AFP ,ALB ,G6P和TAT最早分别于第 3、 9、 11、 13天表达 ,肝细胞特异蛋白AFP ,CK8,CK18和ALB最早分别于第 7、 9、 9和 11天开始表达 .第 12天开始检测到尿素出现 ,浓度为 8 3μmol/L ,并随培养时间延长而浓度逐渐增加 .最后 ,测得生长因子诱导组肝细胞的分化率为 32 % ,对照组肝细胞分化率为 8% .说明肝细胞可以在ES细胞体外诱导分化系统中出现并成熟分化 ,bFGF、HGF、OSM等可以明显提高细胞分化率和成熟度 ,有望成为解决肝功能替代疗法中细胞来源问题的新希望 . The mature differentiation of ES cells into hepatocytes and its differentiation ratio were studied in vitro. First, the BALB/c mouse ES cells were cultured for 5 days to develop into EBs in EBs culture medium. Then, the cell growth factors such as transform growth factor (TGF), basic fibroblast growth factor ( bFGF) and hepatocyte growth factor (HGF) were added into the medium to induce the differentiation of EBs cells into hepatocytes. During the culture medium, the reverse transcriptase-polymerase chain reaction (RT-PCR), immunocytochemistry (ICC) and radioinummoassay (RIA) were used to determine the expression of hepatic genes and proteins, such as (x-fetoprotein (AFP albumin (ALB), glucose-6-phosphatase (G6P), tyrosineaminotransferase ( TAT), cytokeratin 8 (CK8) cytokeratin 18 (CK18) and urea, to analyse the hepatic differentiation. At last, the hepatic differentiation ratio was also determined by counting the ICC-positive cells. In results, the mRNA of AFP, ALB, G6P and TAT didn't respectively expressed till at day3, day9, day11 and day13. AFP, ALB and urea in culture medium were not detected till at day8, day11 and day12 with a concentration of (3.4 +/- 0.6) mug/L, (0.21 +/- 0.04) mg/L and (8.3 +/- 1.4) mumol/L. Hepatic proteins such as AFP, ALB, CK8, CK18 didn't respectively expressed in cytoplasm till at day7, day9, day9, day11 and ICC-positive cells had morphological structures consistent with mouse primary hepatocytes. Hepatic differentiation ratio was determined at day19 with ratio of 32%. In conclusions, hepatocytes could be obtained in ES cells differentiation system and get matured with differentiation ratio of 32% when growth factors such as TGF, bFGF and HGF added into culture medium. This may produce a new resource of hepatocytes in liver engineering and hepatocytes transplantation for treating hepatic failure.

关 键 词： 胚胎干细胞 肝细胞 定向分化

领　　域： [生物学]