胚胎干细胞向胆管上皮细胞定向分化的体外实验研究

目的探讨胚胎干细胞（ES细胞）体外定向分化为胆管上皮细胞（BE细胞）的诱导条件和分化规律。方法选用小鼠ES细胞,首先进行拟胚体分化,然后在培养系统中按不同时间段分别添加转化生长因子（TGF）、酸性成纤维细胞生长因子（aFGF）、肝细胞生长因子（HGF）和上皮生长因子（EGF）等,使ES细胞向BE细胞方向分化。用免疫细胞化学等方法检测BE细胞标记物细胞角蛋白7（CK7）、细胞角蛋白19（CK19）,γ-谷胺酰转肽酶（GGT）的表达,观察其表达时间和空间分布规律。结果ES细胞分化第10天,在拟胚体细胞分化群落中出现一种环状结构并有CK7和GGT表达,分化第13天时有CK19表达,三者的表达都随培养时间延长而增强;光镜下阳性细胞结构符合立方上皮细胞形态学特点。结论ES细胞在特定培养条件和细胞生长因子的作用下,可以定向分化为BE细胞并可以形成类胆管样结构。

Directional differentiation of embryonic stem cells into biliary epithelium cells in vitro: an experiment with mice

OBJECTIVE: To investigate the mechanism and regulation of differentiation from embryonic stem (ES) cells into biliary epithelium (BE) cells and to find a new source f of BE cells for liver engineering. METHODS: ES cells of BALB/c mice (BALB/c-ES) and ES cells of mice of the line 129 (D3-ES) were cultured in the medium without LIF for 5 days with the result that embryoid bodies (EBs) were developed from the ES cells. For directional differentiation, the EBs were plated onto a 24-well gelatin-coated tissue culture dish and some growth factors, such as transforming growth factor (TGF), acid fibroblast growth factor (aFGF), hepatocyte growth factor (HGF) and epidermal growth factor (EGF) etc were added into the medium successively in the experiment group but not in the control group. The differentiation status was observed by inversion microscope dynamically. The BE cell markers, such as cytokeratin 7 (CK7), cytokeratin 19 (CK19) and gamma-glutamyltransferase (GGT) were detected by immunocytochemistry (ICC) and histochemistry. RESULTS: After the culture for 5 days the ES cells developed into many EBs in the medium without LIF. In the EBs cells clusters, many circular structures appeared on the differentiation day 10. On the days 10 and 13 ICC, CK7 and CK19 began to be expressed respectively. GGT began to be expressed at day 10. CK7, CK19 and GGT were also expressed in the ES cells of the control group, however, appeared remarkably later. CONCLUSION: ES cells can differentiate into BE cells under specific culture condition and the action of growth factors. Such ES cell differentiating system can provide BE cells and may serve as a good new source of differentiated cell types for liver engineering.