Research Of Liver Stem Cells Proliferation After Partial Hepatictomy

At present,liver transplantation has become a important way for the end-stage liver disease, however, some factors such as donor shortage, immune rejection and high treatment cost hinder its clinical application. Therefore,HTx transplantation (Hepatocyte transplantation, HTx) that has the advantage of mini invasive and fewer complications has important clinical significance. The transplantation of liver cell is including the liver cell transplantation and liver stem cell transplantation. Although the mature liver cells have some regenerative capacity and it can be reconstructed, liver cell transplantation can not produce sufficient long-term biological effects because of the cell deficiency or limited regenerative capacity. Liver stem cells have the ability to long-time amplify, and can amplify to the number of cells required to achieve clinical in vitro and can be hyperplasia in vivo after transplantation . Therefore, the liver stem cell transplantation may be the treatment of liver regeneration (Therapeutic liver repopulation).Objectives:Make a construction of SD rat liver stem cell proliferation model. Detect the stem cells proliferation in remaining hepatectomy.Methods:Establish partial hepatectomy model of liver stem cell group according to the proportion of excision. Isolate the liver cells in the liver after partial hepatectomy by type IV collagenase perfusion and culture in vitro. Joined the hepatocyte growth factor (hepatocyte growth factor,HGF) and the fibroblast growth factor (fibroblast growth factor-4,FGF-4) in RPMI1640 to induce the adherent cells. Detect the CD34 and Thy with flow cytometry. Observe the liver stem cells of each group by the content and growth. The expression of AFP and CK-18 was detected with immunofluorescence of the differentiated cells. The expression of the protein was detected with Western Blot in the differentiated cells and take a statistical analysis Results:1.Establish a rat model of hepatectomy after removal ratio of SD rat hepatic tissue.2.Obtain the cells by collagenase perfusion. The cells is round and the round adherent cells grow by colony. This shows that there exist the proliferation of the liver stem cells.3.Detected the AFP and Thy expression of liver stem cells by flow cytometry after the first 7 days.4.Observe the AFP and Thy expression in 83.4% liver resection of the rat by fluorescence microscope has the strong expression, the AFP and Thy expression in 73.1% liver resection of the rat has the followed expression, and the AFP and Thy expression in unresectable liver of the rat has the negative expression. The AFP and Thy expression after 7 days resection in rat liver has the strong expression, the AFP and Thy expression in the resection day of the rat has the followed expression, and the AFP and Thy expression after 14 days resection in rat liver has the least expression. The AFP and Thy expression of rat liver perfusion with collagenase IV for 15 minutes in the residual liver has the strong expression, the AFP and Thy expression of rat liver perfusion with collagenase IV for 10 minutes has the expression, and the AFP and Thy expression of rat liver perfusion with collagenase IV for 20 minutes has the negative expression.5.Detect the positive expression of AFP of the liver stem cells in each group by Western Blot. The AFP expression between the 83.4% liver resection and the 73.1% liver resection has no significant difference, P≥0.05. The AFP expression between the rat liver perfusion with collagenase IV for 15 minutes in the residual liver and the of rat liver perfusion with collagenase IV for 10 minutes in the residual liver has significant difference. And the former was significantly higher, P<0.05. The AFP expression after 7 days surgery in the rat liver is higher than the AFP expression in the surgery day in the rat liver, P<0.05.Concusion:1.The liver stem cells in 83.4% liver resection of the SD rat is the most, the liver stem cells in 73.1% liver resection of the rat is the second-most, and the liver stem cells in unresectable liver of the rat is the least.2.The liver stem cells after 7 days cut in rat liver is the most, the liver stem cells in the surgery day of the rat is the followed, and the liver stem cells after 14 days resection in rat liver is the least.3.The stem cells of rat liver perfusion with collagenase IV for 15 minutes in the residual liver is the most, the second is for 10 minutes, the least is for 20 minutes.