| Aim: To investigate the repopulation and differentiation of recipient derived bone marrow stem cell in transplanted livers, and the influence of intraportal transfusion of recipient derived bone marrow stem cell on allograft survival of orthotopic liver transplantation.Methods: The two-cuff orthotopic liver transplantation model was performed, which female Wistar rats were used as donors, female SD rats as recipients. Then recipient derived bone marrow stem cells were transplanted from SD (male) into livers of Wistar (female) rats. Recipient rats were divided into 5 groups: blank-control group(Group A), D-hanks solution group(Group B), whole bone marrow cells group(Group C), Mesenchymal stem cells( MSCs) group(Group D), Hematopoietic stem cells(HSCs) group(Group E). Group A (n=8) performed liver transplantation only. Group B (n = 8) received intraportal D-hanks solution. Groups C(n = 8), D(n = 8) and E (n = 8) underwent intraportal transplantation of whole bone marrow cells, MSCs and HSCs respectively(SD male, 5 X 105/ml, 0.5ml). All groups received subtherapeutic immunosuppression (Cyclosporine A 0.25 mg/kg/d) for 14 days from lday before the operation. Liver repopulation and differentiation were assessed 60 days or 90 days after the transplantations using in site hybrid histochemistry (ISHH ) (for rat Y-chromosome specific Sry gene-positive bone marrow stem cells) and the double staining technique combining ISHH with alpha-fetoprotein (AFP) or Albumin Immunohistochemistry(IHC). The blood alanine transaminase(ALT), total bilirubin(TBil) were observed 1 week after the transplantation, liverpathological changes and survival time were also observed.Results: (1) Histologically, recipient derived bone marrow stem cells (SD, male) were noted throughout the transplanted livers. Quantitatively, DNA probe to the Sry region of the Y-chromosome was identified as nuclear-positive BCIP/NBT signal (blue coloring).The percentages of BCIP/NBT positive cells (The repopulating rate) 60 days after the transplantation in group C, D and E were 62.70 +1.79%, 62.50 + 1.60% and 32.38 + 1.41 % . Similarly, the percentages 90 days after the transplantation was 63.88+1.99% in group C, 63.50+2.27% in group D and 32.88 + 1.55% in group E respectively. No significant difference was found between group C and group D (P>0.05 ), but the percentages in group C and group D were larger than group E significantly (P<0.05) . And the difference between the groups 60 days after the transplantation and 90 days after the transplantation, was not statistically significant. (2) AFP or Albumin IHC was identified as cytoplasm -positive DAB signal (brown coloring) to prove recipient derived bone marrow stem cells have the capacity to differentiate into hepatocytes. Sry region of the Y-chromosome (nuclear blue coloring) and AFP or Albumin(cytoplasm brown coloring) double labeled cells were observed in group C, D and E. The percentages of Sry and AFP double staining positive cells in group C, D and E 60 days after the transplantation were 51.62+1.16%, 55.53+2.32% and 23.28+1.55%, and the percentages of Sry and Albumin double staining positive cells 60 days after the transplantation was 55.34+ 1.36%in group C, 56.83+2.32% in group D and 25.78 + 1.55% in group E. Similar results were obtained in 90 days after the transplantation. The percentages of of Sry and AFP double staining positive cells in group C, D and E were 54.13 + 1.58%, 54.60 + 1.85 % and 25.26+2.67 %, and the percentages of Sry and Albumin doublestaining positive cells in group C, D and E were 54.26+2.58%, 55.10 + 2.45% and 26.16 + 1.67%. No significant difference was found between group C and group D (P>0.05) , but the percentages of the double staining positive cells in group C and group D were larger than group E statistically (P<0.05) . And the percentages of the double staining positive cells in groups 60 days after the transplantation were the same as the groups in 90 days after the transplantation (P>0.05. (3)The group C, D and E showed long-term graft survival (MST>180 d...
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