| The liver is the largest gland in animals and one of the most important organ. Liver function is extremely complex, Known as in vivo "chemical factory". Therefore, the liver disease is serious threat to human health, and is one of human beings leading cause of death due to illness. There is still no effective treatment for a variety of end-stage liver disease caused by various reasons. Orthotopic liver transplantation (OLT) has become the final treatment, but the shortage of the liver donor and immune rejection limit the application of liver transplantation. Hepatoeyte transplantation (HCT) is a second treatment for a variety of end-stage liver disease and have more advantages with respect to OLT, but is also facing the problem of the same cell source. At present, as one of in vitro support treatments, bioartificial liver (BAL) also have acheived little Progress because of the problem of liver cells source. How to further address the issue of source of liver cells, the number and vitality has become a key problem for HCT and BAL treatment. Embryonic stem cells (ES cells) with unlimited proliferation and differentiation of three germ layers is an important seed cells for regenerative medicine, and brings new hope for various injuries and degenerative diseases. Establishing an effective technology of differentiation is an important direction of ES cells research, which provide the desired cell source for the repair of tissue injury and disease. Studies have shown that ES cells can be successfully differentiate into liver cells, which offers the potential unlimited cell source for a variety of end-stage liver disease. However, ES cells differentiation into hepatocyte is in infancy. At present, by simulating the mechanism of liver development, employing the related cytokines or compounds, genetic modification and co-culture to differentiate ES cell into hepatocyte, which is complex technically, inefficient and costly. In our study, we used mouse embryonic stem cell line ES-D3 cells as research object, we induced ES cells to differentiate into hepatocyte-like cells using an adherent culture system with a two-step induction. we tested the morphology, gene expression, protein molecular markers and cell function and other aspects of ES-D3 cells derived hepatocyte-like cells; By the model of acute liver injury with carbon tetrachloride in mice we established, we transplanted the hepatocyte-like cells into spleen of injury liver model, it can intergrate into the liver after homing into the injury liver tissue. Finally, we tested the potential of human fetal liver cell HL-7702 cells in vivo assay method as a source of BAL using the established model of acute liver injury in mice with carbon tetrachloride.The study is divided into six parts. In the first, second and third parts, we cultured the mouse embryonic stem cell line ES-D3 cells and induced to differentiate into hepatocyte-like cells, at the same time, we tested the morphology, gene expression, protein molecular markers and cell function and other aspects of ES-D3 cells derived hepatocyte-like cells, in addition to it, study also showed sodium butyrate induced apoptosis and EGF can inhibit apoptosis; In the four parts, we established a mouse model of acute liver injury with carbon tetrachloride by gavage method; In the fifth parts, we transplanted the DAPI labelling ES-D3-Hep cells into injury liver tissue through the spleen injection method, showed that ES-D3-Hep cells can intergrate into the liver after homing into the injury liver tissue. In the sixth parts, we tested the potential value of human fetal liver cells HL-7702 as a cell source of BAL by peritoneal transplantation using the established model of acute liver injury with carbon tetrachloride. The main results are as follows:In the first part we cultured mouse embryonic stem cell line ES-D3 cells in serum-free and feeder-free culture systerm by based on the combination of gelatin coating and KNOCKOUT-DMEM and KNOCKOUT-SERUM REPLACEMENT. By comparing the combination of differential adhesion and simple passage, simple passage on the the effect of MEF feeder cells removal, the results showed that the effect of combination of differential adhesion and simple passage to remove feeder cells is better than the simple passage, which laid the foundation for the follow-up differentiation. We differentiated the mESC-D3 cell into hepatocyte-like cells using sodium butyrate, hepatocyte growth factor and dexamethasone under the established chemically defined conditions in 19 days by using an adherent culture system with a two-step induction. The mESC-D3 cell gradually differentiated into hepatocyte-like cells, morphology of light microscopy and transmission electron microscopy observed that the differentiated cells possess the morphological structure of hepatocyte characteristics.In the second part, we detected the expression of some marker genes of ES-D3 cells derived hepatocyte-like cells by RT-PCR. Meanwhile, we tested the apoptosis and cell cycle distribution in the first phase of the process of induction of differentiation using sodium butyrate. The results showed that the ES-D3 cells derived hepatocyte-like cells expressed marker genes of AFP, ALB, TTR and AAT, the cells of spontaneous differentiation slightly expressed marker genes of AFP and ALB, not express late markers TTR and AAT. The results indicated that the ES-D3 cells derived hepatocyte-like cells possess the gene expression characteristics of hepatocyte and comprised of hepatocyte-like cells with different stages of maturity. After 7 days of Induced differentiation, NaB induce apoptosis and a large number of the cell cycle arrest in S phase. the proportion of S phase cell cycle can significantly reduce and the proportion of G0/G1 phase cells increases by the addition of EGF, indicated that EGF can inhibit cell apoptosis Induced by NaB.In the third part, we detected the expression of some marker protein of ES-D3 cells derived hepatocyte-like cells by immunofluorescence analysis, while hepatocyte function were identified.The results showed that the ES-D3-Hep cells express hepatocyte-specific marker proteins ALB, AFP, CK8 and CK18, while some cells of spontaneous differentiation group also expressed ALB and AFP, but did not express CK8 and CK18. Differentiation rate of hepatocyte of differentiation groups is 37.27% and 41.67% respectively, no significant difference between the two groups, but compared with spontaneous differentiation group, the difference was significant. ES-D3-Hep cells specific function tests showed that the ES-D3-Hep cells of differentiation groups possess the metabolic function of ICG, while only a small number of cells in spontaneously differentiated group possess the metabolic function of ICG; PAS experiments showed that ES-D3-Hep cells synthesized glycogen in differentiation groups, and less or slight staining in spontaneous differentiation group; These results suggest that the ES-D3-Hep cells express hepatocyte-specific marker proteins, but also have hepatocyt specific cell function.In the fourth part, we established a stable model of acute liver injury in mice with carbon tetrachloride using gavage method, by observing the behavior of the state in mice, mortality, liver pathological changes, histological pathological changes and in ALT and AST activity changes.5μl/g dose of carbon tetrachloride is optimal.In the fifth part, we transplanted the in vitro DAPI labeled ES-D3-Hep cells into mice with acute injury liver by spleen injection method to observe whether ES-D3-Hep cells can possess the real support for the function of hepatic metabolism. The results show that, DAPI labeling rate is up to 100%, trypan blue staining viability after labeling cells have no significant difference compared with unlabeled cells, suggesting that DAPI is a good marker of in vitro Transplantation tracer; In 10 days after cell transplantation, the survival of mice were observed, the survival rate of cell transplantation group is higher than the solvent group, but the difference was not significant; Cell transplantation showed some scattered blue fluorescent spots by observing liver frozen sections, indicating that the transplanted The ES-D3-Hep cells can intergrate into the injury liver tissue.In the sixth part we transplanted intraperitoneally established immortal human fetal hepatic cell line (HL-7702) to CCl4-treated acute liver injury mice to determine whether the cells can provide life-saving metabolic support during acute liver injury induced by CCl4-treatment. The results showed that the levels of blood ammonia were lower and liver albumin were higher respectively (P<0.05) after HL-7702 cell transplantation than those non-transplanted controls at day 3 and 7 respectively. Histological examinations showed the transplantation group were also better than the control at day 7 after transplantation, but there were no difference at day 14 after transplantation. In conclusion, the established immortal human fetal hepatic cell line is a promising cell source for providing life-saving metabolic support and a bioartificial liver (BAL) device in the treatment of acute liver injury. |
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