| The liver is the largest human visceral organs, with bile secretion, digestion and metabolism of fatty, synthesis and secretion of amino acid, the synthesis and storage of glycogen, the storage and filtering blood, detoxification and elimination of waste, the secretion of serum albumin to maintain a stable internal environment and other important functions. Liver function is one of the basic elements of security of population health. For malignant liver diseases such as liver cancer, so far the most effective treatment and prevention the exacerbation of operation is to remove the cancerous portion of the liver, but it will greatly reduce the patient’s liver function, thus to promote liver regeneration in the damaged liver is important. Compared with other organs of vertebrates, liver has greatly ability of regeneration, in normal liver cells is not split the silent, but liver cells have strong proliferation ability and can quickly enter the cell division cycle after liver injury. Liver by operation was performed in2/3rats or mice can recover100%of liver weight in7-10days. When the proliferation of hepatocyte is inhibited or liver damage, a portion of the liver precursor cells may participate in the repair of liver regeneration, but the identification and isolation of precursor cell still has difficult up to know. Therefore, to establish a greater degree of even fully liver injury animal model is of great significance. Identification of endogenous precursor cells and regulatory mechanism in liver regeneration process is very mportant. The liver has high regenerative capacity, but it is not clear whether most biliary cells (particularly larger cholangiocytes) transdifferentiate into hepatocytes in regenerating liver. We investigated how this process might contribute to liver regeneration in zebrafish.In this study, we use zebrafish as a model organism to explore the cell sources and mechanisms in liver regeneration, first we construct a transgenic zebrafish whose liver can be killed specificity induced by Mtz. We found that the liver can be induced to cell death close to100%and return to normal regeneration after withdraw the Mtz. By BOD1PY FL C5and glycogen color PAS reaction assays, we find the new regenerated liver cells have the ability to fat metabolization, bile acid secretion and glycogen storage. Therefore zebrafish liver injury model we established is a functional regeneration model.When the liver cells were injuried specificly, bile duct coarsens, contraction and a small part of the bile duct cells begain to express hepatic cells markers in regeneration, later, all of the bile duct cells lose their normal morphology and express the specific markers of hepatocyte, suggesting that the bile duct played an important role in liver regeneration. EdU labeling experiments found that a large number of proliferation cells are mainly concentrated in the bile duct cells. And at the beginning of regeneration, the proliferation of bile duct is very low, while the TUNEL experiments failed to detect the apoptosis of bile duct cells in the regeneration, so bile duct cells maintain integrity in the process of liver regeneration. In order to further determine whether the bile duct cell transdifferentiate into hepatocyte, we use Cre-Loxp systems to track the destiny of bile duct,70%new hepatocyte regeneration are derived from the bile duct cells. These phenomena indicate after liver damage during regeneration, the bile duct cells changed their morphology, transdifferentiate into liver cells and play a major contribution on liver regeneration.In order to further define the function of bile duct cells during liver regeneration, inhibiting Notch signal to specifically reduce the development of bile duct system in the early embryonic development, liver regeneration is impaired after the development of bile duct system was significantly inhibited. At the same time, we use a bile duct mutant to further prove the roles of biliary cells on liver regeneration. In addition, in zebrafish embryos with biliary toxicity by drug treatment found that even weak toxicity also inhibits bile duct cells to transdifferentiation. At the beginning of regeneration, bile duct cells began to express some early endoderm and liver specific markers, which also show that the bile duct cells will be accompanied by a process of dedifferentiation to an intermediate of precursor cells.At the same time, we used fluorescence in situ hybridization coupled antibody staining found early endodermal transcription factors are up regulated in bile duct cells, sox9b also upregulated in bile duct cells in liver regeneration. There is no proliferation in bile duct but the expression of sox9b has been raised after liver damaged. In sox9b mutants, we found that the bile duct cells to the hepatocyte transdifferentiation is restrained and liver regeneration slowed down, indicating that sox9b on bile duct cell transdifferentiation is important. Sox9b is one of the target gene of Notch signal pathway, when the Notch signaling suppressed, the transcription of sox9b is also suppressed. At the same time, we can detect the up regulation of another Notch target gene Hes5in the biliary system during the Mtz treatment, indicating that Notch mediated liver regeneration through the sox9b to participate in the transdifferentiation of bile duct cell.In summary, we have successfully constructed the liver injury model, identied the signal mechanisms associated with liver damage and firstly show that almost100%of zebrafish liver injured, it can be recovered to normal regeneration; we also revealed in the process of regeneration and repair of damaged liver, the transdifferentiation of mature biliary duct cells play a very important role; at the same time we validate that the transdifferentiation of bile duct cells is dependent on Notch signaling pathway. Study on the sources and mechanisms of liver regeneration can provide some clinical significance for liver disease. |
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