| Liver transplantation is the most effective therapeutic approach to end stage liver diseases, but meanwhile there are many disadvantages such as the limited supply of donor livers, surgical trauma and graft rejective reaction. In resent years, researchers have studied a lot about hepatic stem cells, including discovery of the markers of hepatic stem cells and trace labeling skills to research the cells'differentiation. The gene-modifying technique of the green fluorescent protein gene transferred into stem cells is a good method of trace labeling. The interest in hepatic stem cells therapy has been increasing continuously. Compared with liver transplantation, hepatic stem cells transplantation has a lot of advantages including easy to operate, not having serious injury and being repeatable. The rat embryonic hepatic stem cells have been isolated from the embryonic day 14 fetal rat liver and transferred the green fluorescent protein gene pAcGFP1-N1 into rat embryonic hepatic stem cells successfully. In our experiment, the morphology and function of rat embryonic hepatic stem cells were researched after transfection of pAcGFP1-N1 when cultured in vitro. And the therapeutic effect of rat embryonic hepatic stem cells on acute liver injury was initially researched.There are two parts in these studies. The first part is characterization of rat embryonic hepatic stem cells after transfection of green fluorescent protein gene. After the plasmid DNA of pAcGFP1-N1 was transferred into the rat hepatic stem cells, the stem cells were selected according to different time after culture and dissociation respectively. The supernatant liquid was collected when the cells fully spread the bottom of culture flask after serial subcultivation. The content of ICAM-1 of each supernatant liquid sample was detected by ELISA. The sample was divided into two parts. In group one, suspension cells and adherent cells were separated and cultured again respectively after cultured 60 minutes, 120minutes and 180 minutes. In group two, suspension cells and adherent cells were separated and cultured again respectively after dissociated 5 minutes, 10minutes and 15 minutes. Cell markers c-Met was detected by fluorescence activated cells sorting (FACS). We found that after transfection of green fluorescent protein gene, the cells morphous was as same as before transfection, and we observed green fluorescence in a great quantity of stem cells which has been transferred by pAcGFP1-N1. In group one, the contents of ICAM-1 of the adherent cells were (3052.4?63.7)pg/ml, (1991.5?102.6) pg/ml and (1738.2?58.4 )pg/ml and of the suspension cells were (1862.7?93.1)pg/ml, (1540.0?111.4)pg/ml and (1064.8?136.2)pg/ml after cultured 60 minutes, 120minutes and 180 minutes. In group two, the contents of ICAM-1 of the adherent cells were (1864.0?91.9)pg/ml, (2003.4?116.3) pg/ml and (2964.1?139.8 )pg/ml and of the suspension cells were (1065.2?111.6)pg/ml, (1500.5?107.2)pg/ml and (1999.6?134.0)pg/ml after dissociated 5 minutes, 10minutes and 15 minutes. In group one, the proportion c-Met positive cells was 64.6% and 31.4%, adherent cells after cultured 60 minutes and suspension cells after cultured 180 minutes respectively. In group two, the proportion c-Met positive cells was 62.3% and 21.6%, adherent cells after dissociated 15 minutes and suspension cells after dissociated 5 minutes respectively. We conclude that the rat embryonic hepatic stem cells morphous have not changed after transfection of green fluorescent protein gene, the cultured cells adherent earlier and the dissociated cells suspense later express more cell adhesion molecule ICAM-1 and hepatic stem cells surface marker c-Met. This separation method can collect high purity hepatic stem cells.The second part is to investigate the therapeutic effect of the rat embryonic hepatic stem cells on acute liver injury. The model of rat acute liver injury was induced by carbon tetrachloride (CCL4) - salad oil solution. The rats of the transplantation group were injected with pAcGFP1-N1 transferred rat embryonic hepatic stem cells through femoral vein, while the control group and normal group rats were injected by normal sodium. Serous ALT, AST and TBIL were detected in the first ,fourteenth, eighteenth and thirtieth day after modeling. Hepatic tissue has been observed after stem cells transplantation through HE stain and fluorescence microscope. Compared to control group after stem cells transplantation, the liver function of the transplantation group is much better, the liver structure recovered better. Expressed green fluorescent protein has been observed in the liver tissue of the transplantation group. Conclusions are the rat embryonic hepatic stem cells have a therapeutic effect on acute liver injury and meanwhile the homing of hepatic stem cells is confirmed.
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