| Backgrounds and aimsThe researches of bone marrow stem cells(BMSCs) were only focused on the treatment of hematological diseases (Leukemia, thalassemia etc) several decades ago. But with more and deeper studies the mulitilinear potential of BMSC was observed, which brought about the up-surge of the research on BMSC. Researchers found that BMSC could not only differentiate into the cells of the same germinal layer, but also other germinal layers. In 1999 a study showed that bone marrow is a potential source of hepatic oval cell which gave people a new thought on the treatment of liver disease -treating them with bone marrow cells. But there were still many problems unsolved. For example, although many researches showed respectively that BMSCs with different phenotypes could differentiate into the cells with hepatocyte characters in some conditions, did they have the equal potential ? No one knew. In addition, the mechanism of orienting differentiation of BMSCs was still a riddle which limited the clinical applications.hi this subject we were to define whether or not there were some difference in the differentiating potential of the three representative kinds of BMSCs into hepatocyte under some conditions so that we could screen cells which easily differentiate into hepatocyte, if existed. In addition, we aim to probe into the effect and mechanism of the two growth factors on the differentiation by detecting some signals of hepatic linear cells and the mRNA of the corresponding receptors. Materials and MethodsThe bone marrow cells got from sternum or ilium of healthy volunteers were devided into three groups: group A, bone marrow mononuclear cells; group B, CD117+cells; and group C, CD117"CD45" cells. The cells in group A were separated by gradient centrifugation, B and C by magnetic cell sorting method 0 and the sperating effects were detected by hematoxylin and DAPI respectively. Cells in each group were put into the DMEM medium with HGF, FGF4, HGF + FGF4 or no growth factor respectively. The markers of hepatic linear cells were examined on day 0, 14, 21 and 28 by immunocyte chemistry and PAS (periodic acid-schiff) in every group. C-met and FGFR2 mRNA was examined on day 0, 7 and 14 induced by HGF+FGF4 or no growth factor by RT-PCR in group C and the newly isolated cells in group A and B. Results1 The bone marrow mononuclear cells seperated by gradient centrifugation had homogenous size under inverted phase contrast microscope and no bi-nuclear or multi-nuclear cells were found by hematoxylin.2 The activity of cells separated by magnetic cell sorting was higher than 95% by trypan blue staining. The purity of CD 117+ cells was 99%.3 The cells began to be adherent from the 24th hour and the number of adherent cells increased with time. The cell grew in clonal manner.4 The effect of only HGF or only FGF4 had no significant difference, but cells grew a little faster when HGF and FGF4 were added together.5 The markers of hepatocyte linear appeared on the cells in all the three groups cultured with growth factors, but did not appear without growth fator.6 The positive percent of every hepatocytic marker at the same time point had no statistically difference among three groups, and as well as among HGF, FGF4, HGF+ FGF4 (P>0.05).7 By RT-PCR, low level mRNA of c-met and FGFR2 was detected in all the three kinds of newly isolated bone marrow stem cells and cells cultured without growth factor for 7 days and 14 days. But higher in cells cultured with medium containing HGF +FGF4. Conclusion1 The gradient centrifugation is fit for the separation of bone marrow mononuclear cells.2 The purity of cells separated by magnetic cell sorting is very high. The activity of these cells wasn't influenced by the seperating procedure.3 Bone marrow mononuclear cells, CD117+ cells, CD117"CD45"cells have the equal potential of differentiating into hepatocyte.4 Both HGF and FGF4 can induce bone marrow stem cell to differentiate into hepatocyte singly or coordinately.5 C-met a...
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