| Dendritic cells (DC) are the most efficient professional antigen presenting cells (APC), and the unique APC that have the ability to activate naive T cell to a new antigen and prime primary immune response (which can be either immunologic rejection or immunologic tolerance), which monocyte and non-professional APC, such as B cell, have not. Recently, it has been described that DC can be divided into two distinct subsets, DCI and DC2, by the direction of immune response they switch on, which can be Thl or Th2 profiles. Because of the correlations of Thl and Th2 with the transplantation immune response, studying of DCI and DC2, the more initial determination procedure of immunologic response before Thl and Th2, has outstanding importance in transplantation immunology. Many cytokines can adjust strength comparison of* 9 35the two subsets of DC by their own way, as well as, alter the immunologic response manner. Granulocyte colony-stimulating factor (G-CSF) andgranulocyte-macrophage colony-stimulating factor (GM-CSF) are two of them.Aims To establish immunologic rejection model of mouse heart transplantation.To establish effective methods to separate DC from murine peripheral blood (PB) and detect DCI and DC2 subsets proportion. On the basis of it, to find the distinct biological function of G-CSF and GM-CSF in proliferating DCI and DC2 in vivo. Then to find out the different polarization of the transplantation immune response results from the distinct DC subsets regulation manner by these two cytokines. To study the modification of Thl and Th2 profiles and the alteration of transplantation immune response in vitro and in vivo. Finally, to search newsolutions for transplantation immune tolerance.Methods Two styles of mouse celiac or cervical heterotopic heart transplantation wereestablished and compared. DC were separated from PBMC by means of density gradient centrifugation and complement-dependent killing of non-DC cells by cytotoxic monoclonal antibodys, including granulocyte, monocyte, macrophage, T cells, B cells and NK cells. After purification, DC were stained with FITC-conjugated CDllc and PE-conjugated CD8a. Two coloured flow cytometry were used to detect DCI, DC2 and the odds of DC1/DC2. On the basis of these two methods, we explored the deviation of DC1/DC2 by optional dosageof recombinant human G-CSF (rhG-CSF), and observed the alteration of absolute number of peripheral DC1 and DC2 and the odds of DC1/DC2, after subcutaneously injection of 10 n g rhGM-CSF or rhG-CSF everyday for 6 days. Then experimental group of BALB/c mice were injected with C57BL/6 mice PBMC to make pre-sensitization. 6 days later, IFN- Y and IL-4 excreted by peripheral CD4+ T cells were detected by means of ELISA. And in vitro one-way mixed lymphocyte reaction (MLR) were performed to find out the alteration of transplantation immune response. Finally, we observed the alteration of transplantation immune response in mice heart transplantation model betweenBALB/c and C57BL/6 mice.Results Animal model for transplantation immunity Two styles of mice celiacor cervical heterotopic heart transplantation were established successfully and compared. Under available conditions, the former success rate was higher, but the latter cost less time.Separation of DC Using our DC separating method, We can get the best CDllc+ cells purity as 83.1% ?.1%. Because CDllc is regarded as the classification marker of mouse DC, it can represent the purity of DC, which reaches the request of general immunologic experiment.Alteration of absolute number of DC1 and DC2 and DC1/DC2 Both rhG-CSF and rhGM-CSF could alter balance of peripheral DC subsets significantly, but the results were opposite. RhGM-CSF significantly increasedSB i i 1Rthe absolute number of peripheral DC1 from (38.3 ?9.3) ?06/L to (116.8+25.6) X 106/L (P <0.01), but did not affect that of DC2 [(10.3+2.9) X 106/L vs. (7.8 ?4.4)X 106/L, P >0.05], DC1/DC2 rised significantly from 17.9 + 7.6 to 3.9+1.2, (.P<0.01). On...
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