Hepatic Oval Cells Exhibit Endothelial/Mesenchymal Plasticity Though EMT In Local Microenvironment

Part One:Isolation, identification and cultivation of hepatic oval cells in the rat 2-acethylaminofluorene/partial hepatectomy modelObjectives:To establish the proliferation of hepatic oval cells model of 2-AAF/PH in rat, to investigate the methods of isolation, identification and cultivation of hepatic oval cells.Methods:Rats were fed 2-acethylaminofluorene daily with a dose of 15mg/kg, the fifth day 2/3 partial hepatectomy was performed under the anesthesia of 10% chloral hydrate withdrawal 2-AAF, the sixth day 2-AAF was administered daily again for one week.2-AAF was replaced by normal saline in the control group. Rats were sacrificed by cervical dislocation after anesthesia at the day point of 2,6,10,12,16 and 21 after the surgery, each time point with 3 rats to draw the proliferation curve of hepatic oval cells. The rat with a peak of hepatic oval cells proliferation was chose, using the modified two step collagenase perfusion and gradient centrifugation to isolate hepatic oval cells, and identifying by flow cytometry, immunofluorescence and transmission electron microscopy. Results:Hepatic oval cells proliferation was successfully induced in 2-AAF/PH model, and the proliferation peak presented in the tenth day after surgery, no hepatic oval cell in the control group. Freshly isolated cells showed a cobblestone growth, flow cytometry showed OV6 positive cells accounted for 85.36%±7%, immunofluresence staining showed cells expressed hepatic oval cell markers of OV6, AFP, CK19, ALB, C-kit, transmission electron microscopy showed characteristics of immature cells of large nuclear cytoplasm ratio and a little organelles in cytoplasm.Conclusions:Hepatic oval cells proliferation was successfully induced in 2-AAF/PH model, and the proliferation peak presented in the tenth day after surgery, highpurity hepatic oval cells were collected by the modified two step collagenase perfusion and gradient centrifugation, which has laid the foundation for the future study of hepatic oval cells.Part Two:The epithelial-mesenchymal transition promotes transdifferentiation of subcutaneously implanted hepatic oval cells into mesenchymal tumor tissueObjectives:To establish the model of transplanting hepatic oval cells by subcutaneous injection into nude mice, and to study the plasticity of hepatic oval cells.Methods:Hepatic oval cell line of LE/6 was transplanted subcutaneously into 50 female nude mice and possible neoplastic changes were observed, HepG2 cells were used for positive control with inoculating subcutaneously in 5 nude mice. The characteristic of neoplastic was identified by HE, immunohistochemistry, transmission electron microscopy, fluorescence in situ hybridization, confocal and Western blot.Results:22 of 50 nude mice generated subcutaneous tumors (44% positive rate), all the mice in control group had subcutaneous tumor formation (100% positive rate). HE staining showed the neoplastic structure was more like a mesenchymal tumor, and which was completely different with HepG2 tumor. Immunohistochemistry showed the tumor expression of mesenchymal tumor markers, such as vimentin, SMA, desmin and S-100, TEM found high light dense body in the cytoplasm which was a specific marker of mesenchymal tissue. FISH confirmed the tumor from inoculation of LE/6 cells. Confocal showed Snail and Vimentin, Snail and N-cadherin co-expression. Western blot showed Snail, Vimentin and N-cadherin high expression, and low expression of E-cadherin.Conclusions:Hepatic oval cells subcutaneously implanted into nude mice can transdifferentiate into mesenchymal tumor tissue. EMT involved in this process of transformation. Hepatic oval cells have a strong plasticity in the local environment.Part Three:The transdifferentiation of hepatic oval cells subcutaneously transplanted of mesenchymal tumor tissue into the liverObjectives:Mesenchymal tumor tissue derived from subcutaneously implanted hepatic oval cells implanted into liver, to observe if the mesenchymal tumors can transdifferentiate into liver tumor tissue in long-term.Methods:Hepatic oval cells subcutaneously implanted into nude mice formed mesenchymal tumor tissue, then transplanted into the liver parenchyma of 20 female nude mice, and observed the changes in the abdomen of nude mice. The control group directly used hepatic oval cells injection into nude mice liver. Using HE, immunohistochemistry and TEM to identify the properties of the formation tumor in liver.Results:3 nude mice formed tumor in liver in 20 inoculated mesenchymal tumor tissue (15% tumor formation rate), no tumor formation in control group. HE staining showed the liver tumor containing the majority of mesenchymal cells and small amout of abnormal epithelial-like cells. Immunohistochemistry showed that mesenchymal cells expressed Vimentin and SMA, while the abnormal epithelial-like cells expressed hepatocyte markers HepParl. TEM showed abnormal epithelial-like cells were more like hepatoma cells.Conclusions:Hepatic oval cells subcutaneously implanted into nude mice formed mesenchymal tumor tissue, then transplanted into the liver parenchyma can partially convert to liver cancer cells, and form a liver sarcoma. The transformation mechanism is closely related with EMT and MET. The local microenvironment played an important role on the transdifferentiation of hepatic oval cells. Hepatic oval cells transdifferentiation through repeated EMT and MET could become liver cancer stem cells.