Mechanical Research On Induction Of Oval Cell Proliferation By Curcumstantial Factors

PartⅠIsolation, culture and characterization of hepatic oval cells from adult mice treated with N-2-acerylaminofluorene diet and partialhepatectomyObjectives: To modify a reliable method for induction, isolation and culture of murineadult hepatic oval cells and characterize the cultured cells.Methods: Oval cell response was stimulated by treatment with N-2-acetylaminofluorene(2-AAF) diet and partial hepatectomy. A modification of a two-step perfusion protocol andisopycnic centrifugation was used to isolate oval cells from livers. Immunofluorescenceand staining of glycogen were used for characterization of oval cells.Results: In this model, there was obviously proliferation of oval cells in livers ofmice. Using this method, isolated oval cells were heterogeneous and represented all subsetsof hepatic oval cell population. Cells in culture had been passaged upwards of 20 times andhad been cultured continuously for 3 months. Isolated cells were characterized as oval cellsby the methods of immunofluorescence, RT-PCR and staining of glycogen.Conclusions: The method using 2-AAF diet and partial hepatectomy was also suited forinducing proliferation of murine hepatic oval cells. Murine cultured oval cells, togetherwith this reliable method for isolation and primary culture of oval cells would provide auseful tool for investigating hepatocarcinogenesis of oval cell. PartⅡRole of Toll-like receptor 2 signaling pathway in inducing theproliferation of oval cells by AFB1Objective To investigate the proliferation of oval cells induced by AFB1, the expressionand role of Toll-like receptor 2 signaling pathway in inducing oval cell proliferation by theAFB1.Methods Peritoneal injection of AFB1 was used to made the model of AFB1 affecting onmice. ALT and AST were measured to represent the degree of the liver injury. Theproliferation of oval cells in the liver was detected by immunohistochemistry. The mRNAexpressions of TLR2, MyD88, HGF and AFP in the liver were detected by real time- PCR.The protein expressions of TLR2, MyD88 and NF-κBp65 were detected by the method ofWestern Blot. The expressions of cytokines, TNF-α, IL-1, IL-6 and HGF were detected bythe method of enzyme linked immunosorbent assay(ELISA).Results Compared with the controls, serum levels of ALT and AST were elevated at 3hours, which indicated there was liver injury in the mice treated with AFB1. Serum levelsof ALT and AST were peaked in 30 days and lasted at high levels to 60 days(P<0.05).There were no oval cell proliferation in the livers of the control. Following treatment withAFB1, the mouse liver showed that increased number of small, ovoid, basophilic cells in 7days and which was lasted to 60 days. These small basophilic cells stained positively forthe co-expressed marker of oval cells and the biliary cells, CK19. Morphologically, thesecells were consistent with oval cells. In this course, the expressions of TLR2 pathway andsubsequent cytokines were elevated in the livers(P<0.05).Conclusion AFB1 could induce severe liver injury and induce the oval cell proliferationin the livers of mice. Activation of TLR2 pathway and followed release of cytokines mightplay an important role in inducing the oval cell proliferation by AFB1. TLR2 signalingpathway might play an important role in the proliferation of oval cell and oval cell'shepatocarcinogenesis. PartⅢChanges of gene expression in oval cells and hepatocytes induced by circumstantial factorsObjective To investigate proliferation of oval cells induced by AFB1 and HBV, and investigate the changes of gene expression in oval cells and hepatocytes induced by AFB1 and HBV, using the method of microarray analysis.Methods. Forty male Balb/c mice and forty HBV transgenic mice were randomly divided into four groups: the control group, AFB1-treated group, HBV transgenic group and AFB1+HBV-treated group, there were twenty mice in each group. Quantitative analysis of hepatitis B surface antigen, semiquantitative analysis of hepatitis B e antigen, qualitative analysis of hepatitis B surface antibody, hepatitis B e antibody and hepatitis B core antibody were used to characterize the HBV transgenic mice. ALT and AST were measured to represent the degree of the liver injury. The proliferation of oval cells in the liver was detected by immunohistochemistry. Six months after the models were made, the mice were sacrificed to isolate oval cells and hepatocytes from the livers. The changes of gene expression were measured with the method of microarray analysis.Results (1) Serum HBsAg concentration in HBV transgenic mice was 250.00 IU/mL (normal<0.05 IU/mL), HBeAg was 1.66 S/CO(normal <1.00 S/CO), HbsAb, HbeAb and HBcAb was negative. (2) There was no death in the control group (0/20), and death rates were elevated in the other three groups( 6/20 and 30% in AFB1-treated group, 2/20 and 10% in HBV transgenic group, 12/20 and 60% in AFB1+HBV-treated group), the differences were statistically significant(P<0.05). The differences between AFB1+HBV-treated group and AFB1-treated group as well as HBV transgenic group were also statistically significant(P<0.05). (3) comapared with serum ALT and AST levels in the control mice, serum ALT and AST levels were elevated in AFB1-treated and HBV transgenic group(P<0.05), and peaked in AFB1+HBV group(P<0.05), which indicated there were severe liver injury in the mice of these three group. (4) There were no oval cell proliferation in the livers of the control. Following treatment with AFB1,HBV and both , the mouse liver showed that increased number of oval cells in the livers(P<0.05). (5) compared with the control, there were 1577 genes having changing expression in oval cells isolated from AFB1 group, 805 genes in hepatocytes. There were 3467 genes having changing expression in oval cells isolated from HBV group, 1196 genes in hepatocytes. There were 4846 genes having changing expression in oval cells isolated from AFB1+HBV group, 2542 genes in hepatocytes. These genes were involved in multiple signaling pathways, such as Notch, Wnt, PI3K-AKT. In addition, there were much more oncogenes whose expression were elevated in oval cells than in hepatocytes, much fewer anti-oncogenes whose expression were elevated in oval cells than in hepatocytes.Conclusions Treatment with AFB1,HBV and both could all induce the proliferation of oval cells in the livers, and could induce severe liver injury and elevated deaths. HBV could induce inflammatory reaction in the mice, but could induce liver injury and induce the proliferation of oval cells by other ways. Treatment with AFB1,HBV and both could all induce could all induce abnormal gene expression in oval cells. The abnormal expression of oncogenes and anti-oncogenes might play important roles in transformation of oval cells to tumor cells. HCC might be originated from the transformed oval cells.