大鼠成体肝卵圆细胞分离培养及诱导分化的初步研究

Isolation,culture and differentiation of oval cells from adult rat liver

目的 通过成体大鼠肝卵圆细胞分离培养获取纯净的干细胞并观察诱导分化前后从肝卵圆细胞到肝细胞的形态变化。方法 采用AAF／PH肝干细胞刺激模型，胶原酶Ⅳ消化分离和Percoll梯度离心纯化肝卵圆细胞的方法，用地塞米松、DMSO和EGF、HGF、SCF、LIF等生长因子联合应用诱导肝卵圆细胞增殖和分化为肝实质细胞。观察肝卵圆细胞到肝细胞形态变化过程，用免疫荧光技术，Western blotting分析检测了干细胞标志c-kit和RT-PCR分析法检测肝干细胞白蛋白和CK19 mRNA表达鉴定卵圆细胞。结果 分离得到的肝卵圆细胞活度达到90％，细胞肜态包括大小和颜色呈不均质，卵圆细胞直径是肝细胞直径的1／4～1／6。诱导后出现大而圆的肝细胞，可见卵圆细胞到肝细胞的中间变化过程，新分离的肝卵圆细胞存生长因子诱导下有向肝细胞分化的特性。经c-kit免疫荧比染色照黄绿色荧光，Western blotting检测肝卵圆细胞有c-kit蛋白条带，而大鼠肝细胞及胆管细胞则未出现条带。PCR分析显示卵圆细胞有CK19和白蛋白mRNA表达。结论 新分离的肝卵圆细胞同时表达c-kit，CK19和白蛋白3种抗原，诱导分化出现一系列从卵圆细胞到肝细胞的形态学变化，这种现象证明分离的肝卵圆细胞就是肝干细胞，经诱导分化可以产生成熟的肝细胞，成为进一步研究肝干细胞生物学特性的基础。

Objective To observe the morphologic changes of hepatic stem cells differentiating into hepatic parenchymal cells by isolating and culturing oval cells from adult rat liver. Methods The oval cells that were activated by AAF/PH in Wistar rats were isolated and purified from heterogeneous liver cell population by selective enzymatic, digestion and density gradient centrifugation. The hepatic oval cells were induced to proliferate and differentiate into hepatic parenchymal cells by dexamethasone, DMSO and several growth factors. The morphologic changes from hepatic oval cells to hepatic cells were observed, c-kit, a marker of stern cells, was examined by immunofluorescence and Western blotting. The expression of albumin and CK19 mRNA of hepatic stem cells was identified by RT-PCR. Results The activity of the freshly isolated oval cells was more than 90% , the morphologic characterization including color and size was not the same, and the diameter of oval cells was 1/4 to 1/6 of the diameter of hepatocytes. The morphologic changes fi＇om hepatic oval cells to large and round hepatocytes were observed. The freshly isolated oval cells was positive for c-kil by immunofluoreseence, positive for albumin and CK19 mRNA by RT-PCR. Conclusion The morphologic changes of freshly isolated hepatic oval cells during differentiation to hepatocytes provides data of biological properties of hepatic stem cells.