| BACKGROUND: Bone marrow stem cell is a kind of earlier period non-differentiated cell which possesses many kinds of differentiation potencies and the capability of self-copy, self-renew. Bone marrow stem cell is covenient to segregate, purify and amplification. Therefore, it is thought Bone marrow stem cell is a kind of ideal stem cell resource for cell therapy. It has been certificated that there are two major kinds of stem cell in the bone marrow: Hematopoietic stem cell (HSCs) and Mesenchymal stem cells (MSCs). Recent years with more and deeper studies the mulitilinear potenial of Bone marrow stem cell was observed, which brought about the up-surge of the research on Bone marrow stem cell. Researchers found that Bone marrow stem cell could not only differentiate into the cells of the same germinal layer, but also other germinal layers. Such as under proper induction conditions Bone marrow stem cell can transdifferentiate into multilineage cells, including neurocytes, cardiac muscle, lipocyte, bone, cartilage, skeletal myscle and hepatocytes, advanced research shown Bone marrow stem cell can also differentiate into hepatic stem cell. At present, it is well-known that hepatic stem cell is a kind of stem cell which has the capability of self-duplication and the multi-directional differentiation potencies in vivo. As the researching results shown, hepatic stem cells can be found in the adult liver.The oval cell, as progenitor cell, can differentiate into hepatocyte and bile duct epithelium cell, the oval cell can take part in hepatic tissue repair in injury of liver .Now the study of which Bone marrow stem cell induce to differentiate into hepatocytes has become a researching topic in the world, but reports of which Bone marrow stem cell transdifferentiate into hepatic stem cell seldom have.therefore, the experiment depend on that for cut-in point,to study rat bone marrow stem cells differentiation into hepatic stem cells in vitro.Hepatic diseases are clinical common diseases and frequently encountered diseases. For the terminal stage of all liver diseases, the therapeutic effects of tradition therapeutics are limited, while, OLT (orthotopic liver transplantation) is the most effective method to treat acute liver failure, end-stage and metabolic liver diseases at present, with widely clinical application,but there are still some difficulties, such as insufficient liver donor , Transplantational technique complicated and other reasons. Compared with OLT, bioartificial liver and hepatocyte transplantation have many advantages: Non-surgical procedures; Repetitive; A signal donor could potentially provide hepatocytes for several patients; Hepatocytes have low immunogenicity and may be cryopreserved for later use; and it has little influence on donors if failed or immunologic rejection. Hepatocytes could also be genetically modified in vitro and used for gene therapy. Therefore, hepatocyte transplantation is ideal therapeutics method.The source of hepatocytes are limited, achievements of stem cells in biomedical field provide a new way for treating liver diseases.OBJECTIVE:①This study stimulated the liver microenvironment, to induce rat bone marrow stem cells to differentiate into hepatic stem cells in vitro;②Explored the feasibility of differentiation of the rat bone marrow stem cells into hepatic stem cells in vitro and the time phase characteristics in the differentiation progress;③To identify the morphous and function of differentiated hepatic stem cells;④To prepare for hepatic stem cells transplantation therapy.METHODS: rat Bone Marrow Stem Cells were collected from the aspirates from femurs and shinbones of SD rats by density gradient centrifugation. induction group and non-induction group were divided into,adult rat BMSCs were treated with or without 25 ng/mL recombinant mouse HGF,10ng/mL recombinant human FGF-4 and 10ng/mL recombinant rat SCF,on days 0, 7, 14, 21 and 28 after induction,The morphologic characteristics of the cells were observed;The concentration of AFP in culture clear supernatant liquid was determined by radioimmunoassay (RIA);The expressions of AFP,CK19 and ALB protein were detected by immunocytochemical technique; glycogen stain test by Periodic acid Schiff(PAS) method;The ratio of AFP-positive cells and ALB-positive cells were tested flow cytometric analysis (FACS).RESULTS:①The bone marrow stem cells after being induced showed orbicular-ovate and round transformation of hepatic stem cell-like.②Radioimmunossay showed that alpha fetoprotein levels were increased in the inducton group, reached a peak at day 14,and then decreased.There were significant differences between the induction group and the non-induction group at days except 0 and 7 (P<0.05).③Immunohistochemical method showed that alpha fetoprotein, cytokeratin19 and albumin were positive in the induction group ,but negative in the non-induction group.④Postive expression of glycogen were detected on 21th and 28th day of induction;⑤Flow cytometry showed that the ratio of alpha fetoprotein-positive cells was 66.2% on the 14th day of induction in the induction group, and that of ALB-positive cells was 80.14% on the 28th day of induction in the induction group.CONCLUSIONS:①By Ficoll density gradient centrifuge and monolayer cultured bone marrow mononuclear cell can successfully be isolated from bone marrow blood in vitro.and cells obtained were of higher viability and purity.②The cells density of (5-10)×105个/cm2 is fit for bone marrow stem cells growth; DMEM low glucose medium containing 10%-15% fetal bovine serum was very suitable for culture and proliferation of rat bone marrow stem cells in vitro.③Bone marrow stem cells can differentiate into the cells with hepatic stem cells phenotype and function in the presence of HGF, FGF-4 and SCF, and three kinds of cytokines each other coordination in the whole induction process.④Bone marrow stem cells can differentiate into hepatic stem cell-like cells in vitro. Bone marrow stem cells after induction have the characteristic functions of secretion of alpha fetoprotein, cytokeratin19 and albumin.⑤It is indicated that BMSCs provide us a new type of seed cells source for liver tissue engineering.
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