Research Of Wnt/β-Catenin Signal In The Process Of Oval Cell Proliferation Activated By Human Umbilical Cord Mesenchymal Stem Cells In Rats With Acute Liver Failure

Objective:Through intravenous injections of human umbilical cord mesenchymal stem cells and physiological saline in rats which were induced of acute liver failure respectively, we are aimed to identify the differences in liver functions, histology, proliferation of oval cells and the expression of Wnt/β-catenin signaling pathway between two groups. We are aimed to reveal that hUCMSCs may activate the liver regeneration in rats with acute liver failure, and to explore the possible mechanisms, which may provide theoretical basis for clinical application of hUCMSCs.Methods:(1)To induce acute liver failure, rats were intraperitoneally administered an injection of 0.9 g/kg of a hepatotoxin, D-galactosamine (Gal-N). Intravenous infusion of 2×106 hUCMSCs 24 hours postinduction is regarded as the hUCMSCs Group. In contrast, intravenous infusion of physiological saline 24 hours postinduction is regarded as vehicle Group. To evaluate the liver function of each group, the levels of alanine transaminase (ALT) and aspartate transaminase(AST) were measured. Liver histology and survival rates of both group were examined.(2)The expression of oval cell’s markers,including CK19 and EpCAM, were evaluated by immunohistochemistry (IHC) analysis, by which we can observe the proliferation of oval cells on day 3、5and10 after transplantation of hUCMSCs or physiological saline. Compare the numbers of oval cells in both groups.(3)The relative expression of Wnt/p-catenin signal related genes including Wnt2^ Wnt7a^ Wnt7b and EpCAM were determined by quantitative real-time polymerase chain reaction (qRT-PCR) analysis. The expression of β-catenin in both groups were evaluated by IHC and Immunofluorescence(IF) analysis.Results:(1)Liver function analysis revealed that the levels of ALT and AST in hUCMSCs group decreased significantly compared with those in control group at 24 and 48 hours after transplantation. Liver histological data showed that transplantation of hUCMSCs alleviated liver damage and improved liver repair obviously. There were statistical significance between two groups in respect of pathological grade (T=32.5, P<0.01). Survival analysis showed that death rate of hUCMSCs group was significantly lower than that of control group.(2) IHC analysis revealed that the activation and proliferation of oval cells reached the peak on the fifth day and some had expanded into the parenchyma of liver. In addition, oval cells in hUCMSCs group were significantly more abundant than those in control group on that day (t=9.1531,P<0.01).(3) qRT-PCR analysis revealed that the expression of Wnt2、Wnt7a、Wnt7b and EpCAM in hUCMSCs group were respective 2.4 folds (t=3.950, P=0.0168),3.1 folds (t=4.846,P=0.0084),3.0 folds (t=2.888, P=0.0447),1.7 folds (t=3.219, P=0.0323) higher than control group after 24 hours of transplantation. IHC and IF analysis revealed that β-catenin was mainy accumulated in the cytoplasm and nuclear in activated oval cells. β-catenin was more abundant in hUCMSCs group.Conclusion:Our results demonstrated that transplanting hUCMSCs into acute liver failure rats could improve the liver proliferation and alleviate the liver damage and death rate. The possible mechanism of this process might be involved in the activation of Wnt/β-catenin signal, which led to the activation and proliferation of oval cells.