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Background: Bone marrow stem cells (Bone Marrow Stem Cells, BMSCs) is a kind of pluripotency and self-replication in the early undifferentiated cells, its easy separation, purification and amplification, cell therapy is the ideal source of stem cells [1 -3]. Bone marrow stem cells are hematopoietic stem cells (hematopoietic stem cells, HSCs) and mesenchymal stem cells (mesenchymal stem cells, MSCs) into two categories have been confirmed. In recent years, with the increase in bone marrow stem cell research and in-depth and found that bone marrow stem cells can differentiate into only with the germ layers, but also across other cells to differentiate into mesoderm. Liver stem cells are currently considered the existence of a body of self-proliferation and differentiation potential of stem cells. Currently on the bone marrow stem cells induced to differentiate into hepatocytes more coverage, but on the bone marrow stem cells into liver stem cells induced to differentiate reported was small, so this experiment so as the starting point, in vitro bone marrow stem cells into hepatic stem cell transformation studies. Liver disease is a common clinical disease, while traditional treatment for end-stage liver disease limited efficacy, orthotopic liver transplantation (orthotopic liver transplantation, OLT) is recognized as a more effective means of treatment, but donor shortage, technical complexity and difficult universal application; bioartificial liver cell transplantation in the treatment compared with orthotopic liver transplantation in the treatment, with a simple, repeatable, a multi-purpose liver, liver cells and can be cryopreserved low immunogenicity, graft loss, or an immune Receptor after exclusion, etc, in addition, the liver cells in vitro can be genetically modified, is a good vector for gene therapy, the liver cell transplantation is considered ideal method of treatment. Restricted due to liver cell source, so stem cell research using stem cell transplantation and / or stem cell engineering production of liver cells to treat various liver diseases provide the possibility. Objective: ① experimental simulation liver development microenvironment in vitro rat bone marrow stem cells into hepatic stem cell differentiation; ② explore the in vitro rat bone marrow stem cells into the liver stem cell viability and the induction of differentiation characteristics; ③ right induced differentiation of liver stem cells morphology and function identification; ④ stem cell transplantation in the treatment of liver preparation for the experiment. Methods: Rats under sterile conditions and bilateral femur tibia, rinse marrow cavity, to obtain bone marrow by density gradient separation of bone marrow stem cells were isolated and cultured, induced and non-induced group established group, the application of hepatocyte growth factor (Hepatocyte Growth Factor, HGF) 25ng/mL, fibroblast growth factor -4 (Fibroblast Growth Factor-4, FGF-4) 10ng/mL, stem cell growth factors (Stem Cell Factor, SCF) 10ng/mL co-induction, the induction section 0 , 7,14,21,28 days, Morphological changes were observed; using radioimmunoassay supernatant AFP (alpha fetoprotein, AFP) concentration; immunocytochemical staining alpha-fetoprotein (AFP), cytokeratin protein 19 (cytokeratin19, CK19), albumin (albumin, ALB) expression; PAS staining of glycogen glycogen determination induced expression; flow cytometry-induced cell AFP, ALB percentage of positive cells. Results: ① induced culture of bone marrow stem cells are ovoid or rounded liver stem cell-like change; ② radioimmunoassay cells to induce alpha-fetoprotein concentration increased gradually and reached the peak of 14d followed a downward trend, with non- induction group, with the exception of 0,7 d no difference, the remaining differences at each time point were significantly (P lt; 0.05); ③ immunocytochemistry assay to induce the expression of cell-specific alpha-fetoprotein, cytokeratin 19 and white protein; basic non-treated group did not express three proteins; ④ P AS glycogen staining to 21d and 28d in the induction of glycogen in cells expressing positive; ⑤ flow cytometry Rule 14d induction group ratio of alpha-fetoprotein-positive cells 66.20%, induction of 28d albumin-positive cells was 80.14 percent ratio. Conclusion: ① by density gradient centrifugation and adherent culture method can successfully isolated bone marrow mononuclear cells, and the growth vigor and purity are good. ② cell seeding density (5-10) × 105 个 / cm2 most conducive to cell growth; culture medium FBS concentration of 10% -15%, the bone marrow stem cells the most favorable. ③ HGF, FGF-4, SCF build joint induction system, suitable for induction of bone marrow stem cells into hepatic stem cell differentiation, and the three mutual coordination role in the induction of differentiation of the whole process. ④ bone marrow stem cells can differentiate into hepatocytes in vitro stem-like cells, induced cells secrete alpha-fetoprotein, cytokeratin 19, albumin characteristic feature. ⑤ liver and bone marrow stem cells can be used as seed cells for tissue engineering major sources. Bone marrow stem cells readily available, easy to operate, easy to survive in vitro, making it an excellent seed cells for tissue engineering.
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