Abstract:

BACKGROUND: Fetus liver-derived liver stem cells, as cell transplantation donor, have advantages in source. However, difficult culture, easy to differentiate following passage and immature tracing localization technique restrict liver stem cell transplantation.

OBJECTIVE: To observe whether primary cultured liver stem cells after tranfection by electroporation with pEGFP-C1 plasmid can stably express green fluorescent protein (GFP).

METHODS: Mechanical separation and enzymatic digestion method were used to isolate liver stem cells from fetal Sprague Dawley rat liver tissues following pregnancy for 13.5 days. Half-amount medium replacement was used for purify isolated fetal liver cells following cultured in specific medium. Immunofluorescence technique was used to identify adherent cells. The plasmid pEGFP-C1 was identified correctly by eletrophoresis and then transfected into fetal liver stem cells．

RESULTS AND CONCLUSION: The isolated fetal liver stem cells adhered to the culture plastic and presented round or oval cells after 24-hour cultivation in vitro. Isolated cells were almost identical circular after 2-day cultivation. At 3 days, cells grew into a colony which was constructed by 3 or 5 cells, with cell diameter of 6-10 μm; at 5 days, cell colony became enlarged and was composed of 10 dense, round cells of various sizes with clear boundary. At 8 days, they grew like epithelium cell. At 12 days, cells became big, extended like fried egg, with irregular forms, particles in cytoplasm, grew slowly. Following passage, there were no significant changes in cell amplification speed. Cells still presented epithelium-like shape at the passage 3. The adhered cells at day 5 following primary incubation were positively for human stem cell factor receptor CD34 and cytokeratin 19 using immunofluorescence technique. Green fluorescence was observed in many stem cells which has been transfected by pEGFP-Cl．This study successfully established liver stem cells with GFP.