大鼠肝干细胞体外诱导分化的实验研究

目的 分离成体大鼠肝干细胞,观察体外培养条件下肝干细胞分化为肝细胞和胆管细胞的过程以及形态学变化.方法 采用AAF/PH肝干细胞刺激模型,通过胶原酶Ⅳ分步消化分离和Percoll梯度离心纯化的方法得到肝干细胞,联合应用地塞米松、DMSO和EGF、HGF、SCF等生长因子诱导肝干细胞增殖并分化为肝实质细胞.结果 分离得到的肝干细胞呈卵圆型,直径是肝细胞的1/4～1/6,免疫荧光检测显示其表达c-kit和OV6两种干细胞抗原,PCR分析显示其表达CK19和白蛋白mRNA.体外诱导培养条件下细胞首先分化成大而圆的成熟肝细胞,继而出现分支样的胆管细胞,而且可见从卵圆形细胞到成熟肝细胞的中间变化过程.结论 新分离的肝干细胞同时表达c-kit、OV6、CK19和白蛋白多种抗原,诱导分化条件下可见从体积较小的卵圆形肝干细胞到体积大许多倍的肝细胞的形态学变化过程.

Studies on the induction and differentiation of rat's hepatic stem cells in vitro

Objective To isolate hepatic stem cells from adult rats, and to examine the process and morphologic changes of isolated hepatic stem cells differentiated into hepatic cells and bile duct epithelial cells under in vitro culturing conditions. Methods AAF/PH was used to stimulate the oval cells. Selective enzymatic digestion and density gradient centrifngation were employed to isolate and purify these cells from heterogeneous liver cell population. The hepatic stem cells were induced to proliferate and differentiate into hepatic parcnchymal cells by the addition of dexamethasone, DMSO and several growth factors. The morphologic changes of hepatic oval cells to that of hepatic cells and bile duct epithelial cells were examined. Immunofluorescence method was used to detect the hepatic stem cells c-kit and the ex pression of OV6 antigen. The albumin of hepatic stem cells and the CK19 expression were identified by RT PCR analysis. Results The activity of freshly isolated oval cells were over 90%, with different colors and size The diameter of oval cells was about 1/4-1/6 that of hepatocytes. Large and round hepatocytes appeared first during differentiation, and the cell morphology changed when the oval cells differentiated to hepatocytes. Freshly isolated oval cells was positive for c-kit and OV6 with irnmunofluorescence staining, and RT-PCR analysis indicated that the albumin and CK19 mRNA were expressed in the new isolated cells. Collusion The oval cells could be induced to differentiate into a variety of cell lineages including hepatocytes and bile duct epithelial cells in the presence of DMSO and several growth factors. Several antigens such as c-kit, OV6, CK19 and albumin may be expressed in the freshly isolated hepatic stem cells, and the course of morphologic changes may be observed under in vitro conditions of induction and differentiation.