Abstract:

Objective To investigate the in vitro culture and characteristics of stem cells of hepatic oval cells in rats. Methods Rats were treated with 2-acetylaminofluorene and carbon tetrachloride to establish the models of hepatic oval cells, two-step collagenase perfusion technique and flow cytometry sorting were used to separate and purify oval cells, and cell culture and amplification were performed. The ultrastructure of hepatic oval cells was observed by transmission electron microscopy, and the expression of stem cell markers OV-6, c-kit and Thy-1.1 were detected by immunocytochemistry. The expression of c-kit, hepatic cell marker (HNF-4α and AFP) and bile duct cell marker (CK-7 and CK-19) gene in oval cells was detected by RT-PCR. The expression of OV-6 and AFP protein was determined by Western blotting. Results Obvious oval cell proliferation was observed in rat liver tissues, distributing as cluster and gradually extending to liver parenchyma. These cells could be cultured in vitro up to 40 d, with small size and ovoid nuclei. It was observed under transmission electron microscopy that the cell diameters ranged from 5 μm to 10 μm, and there were high nucleus/cytoplasm ratio and few organelles, including mitochondria and endoplasmic reticulum. It was revealed by immunocytochemistry that there was positive staining of OV-6, c-kit and Thy-1.1 on cell surface. RT-PCR analysis indicated that there was expression of c-kit, HNF-4α, AFP, CK-7 and CK-19 gene in oval cells. Western blotting demonstrated that there was continuous expression of OV-6 protein, and the expression of AFP protein gradually increased. Conclusion Hepatic oval cells derived from rat livers possess a high potential to proliferate in vitro, while maintaining the bipotential nature of stem cells.

Key words: oval cells, proliferation, in vitro, stem cells