Study On Differentiation Of Bone Marrow Cells Into Hepatic Lineages In Vivo And Vitro

Backgrounds:At present there hasn't been an appropriate therapeutic method for liver failure of various causes. Orthotopic liver transplantation (OLT), artificial liver and hepatocyte transplantation have been used to clinic for many years, but they can't be spreaded because of many limits such as absence of donor, long-term taking immuno-suppressive drugs and high cost. So it's urgent to search for more effective and economical therapeutic methods.Since 1990s the clinical application of haematopoietic stem cells (HSCs) has been increasingly popular in such fields as Mediterranean anemia, Leukemia and hematopoietic reconstitution of patients with solid tumors treated with large dose of chemotherapy and/or irradiation. On research of HSCs, people found that bone marrow stem cell (BMSC) had the ability of multipotential differentiation, under some microenvironment, it could differentiate into other types of mature cells such as nerve cell, skeletal muscle cell and so on, so it is hypothesized that BMSC might be a complex cell population which has different potential of differentiation.In 1999 it was reported that BMSC could differentiate into hepatocytes. So if autologous bone marrow cells could be induced to proliferate and differentiate into hepatocytes, we could use autologous bone marrow transplantation (BMT) to compensate for liver function, simultaneously resolve the problem of absence of donor and immunorejection. Therefore, it has wide prospect for the application of BMSC.Recent reports demonstrated that a population of p2m~/Thy-l+ bone marrow cells expressed hepatocyte-specific markers such as albumin, AFP and their mRNA, when these cells were induced in vitro , they would express hepatocyte-specific phenotype of CAMS.2, so this population of cells may represent bone marrow derived hepatic stem cells (BDHSC). Further researches are needed to elucidate the properties of these cells.In the study of the differentiation of bone marrow cells into hepatocytes, irradiation was generally used to destroy the bone marrow of recipients, which made donor-derived bone marrow easily home and proliferate in the medullary cavity of bone of recipients. In the 2-acetylaminofluorene (2-AAF)-CCl4 model, it has been confirmed that bone marrow cells could differentiate into hepatocytes and expressing-AFP progenitor cells. And additional protocols confirmed that with small dose of whole body irradiation bone marrow cells could also differentiate into hepatocytes under the condition of little or none hepatic injury. Though the differentiation of bone marrow cells into hepatocytes in vivo had been confirmed, the mechanism of this process was still uncertain even if the hypothesis that some cytokines and hepatic microenvironment may take part in the process of differentiation.In this study three kinds of animal models were used to explore the differentiation of bone marrow cells into hepatic lineages by cross-sex BMT in vivo under different conditions, and analyze the effects of 2-AAF on differentiation rate of bone marrow cells.Two dimensional polyacrylamide gel electrophoresis (2D-PAGE) combining with mass spectrum (MS) for proteome research has been widely used at present. This technology can identify the proteomic difference of different cells or tissues under different conditions and has important significance on the research of cell differentiation, tumors and aging. So we initially established 2D-PAGE in our lab. The total proteins of liver tissues in 2-AAF-partial hepatectomy (PH) model before and after hepatectomy wereseparated by 2D-PAGE and different proteins were identified by image analysis, among these different proteins it was expected some factors such as cytokines, which can cause the activation of hepatic stem cells and the differentiation of BMSC into hepatic lineages, could be found. Materials and Methods:Expression of AFP and its mRNA of bone marrow cells were detected by immunohistochemistry (IHC), reverse transcription polymerase chain reaction (RT-PCR) and western immuno...