大鼠肝卵圆细胞体外分离培养和诱导分化为肝细胞的初步研究

Derivation, characterization and differentiation in vitro of hepatic oval cells of adult rats

目的探索体外诱导成年Wistar大鼠肝卵圆细胞(HOCs)分化为肝细胞的可行性。方法雄性Wistar大鼠36只,建立肝卵圆细胞增殖模型,采用两步法灌注和Percoll密度梯度离心法分离纯化HOCs,行体外培养并添加HGF、OSM和FGF4诱导其分化。结果每只模型大鼠肝脏体外分离可获得约1.34×105/mlHOCs,细胞为圆形、椭圆形或多角形,大小为正常肝细胞1/6～1/3,核质比例大,2周后可呈克隆样增殖生长。所获HOCs胞浆和胞膜表达干细胞标志Thy-1及C-kit,其原始细胞标志AFP呈阳性表达;能稳定传代,在HGF、OSM和FGF4刺激下形态逐渐发生改变,细胞伸展且体积渐增大,贴壁能力减弱,诱导14天后分化细胞Alb表达明显阳性,且随着诱导时间延长阳性率逐渐升高;诱导分化的细胞胞浆G-6-P及PAS染色均呈阳性。结论HOCs在体外一定条件刺激下可诱导分化为肝细胞。

Objective To establish a proliferating model of hepatic oval cells （HOCs） with adult Wistar rats, isolate and culture in vitro the HOCs, and to approach the possibility of inducing the HOCs differentiated into hepatocytes. Methods Rats were fed with 0. 070/00 wt/wt ethionine, On day 8, 2/3 partial hepatectomy （2/3 PH） was performed. Isolate, harvest and purify HOCs by type Ⅳ collagenase perfusion with semi in situ 2 step method and Percoll density gradient method. Epidermal growth factor （EGF） and hepatocyte growth factor （HGF） were added to complete Williams＇ medium E （WE） to culture the HOCs HOCs was induced differentiation with HGF, oncostatin m （OSM） and fibroblast growth factor4 （FGF4） into hepatocytes. Results The concentration of cell after purification was about 1. 34× 10^5/ml. Most cells were small, about 1/6-1/3 the size of normal hepatocyte. Ovoid, elliptical or polygonal in shap, the nucleus cytoplasm ratio was relatively large, and clone-like proliferation appeared 2 weeks later. LSCM revealed positive expression of Thy- 1 and （;kit in cytoplasm and membrane of HOCs. ICC showed AFP in cytoplasm of HOCs. Stimulated by inducing system, the shape of HOCs changed gradually. The volume enlarged and cells lost their adherence ability. ICC indicated apparent positive stain of cytoplasm Alb 14 days after differentiated induction, and the positive ratio increased along with the extension of induction duration. Cytochemical tests in dicated brown or black sediment with G-6-P staining and red particles with PAS staining, respectively. Conclusion The proliferation model of rats＇ HOCs was established after ethionine feeding and 2/3 PH. HOCs can be obtained with type IV collagenase perfusion and Percoll density gradient isolation and purification. Clone proliferation can be achieved through culturing HOCs in vitro. Under certain conditions, HOCs can be induced and differentiated into certain typical hepatocyte phenotype.