| The graft versus host disease (GVHD) and graft rejection is the major lethal complications associated with allogenic bone marrow transplantation (allo-BMT). Killer inhibitory receptor(KIR), which expressed on the surface of NK, some T cell, mononuclear phagocyte or B cell, can recognize the specific major histocompatibility complex class I (MHC I) molecular of self tissue to conduct negative signals and inhibit T cell and NK cell activation. And then, the secretion of inflaming factors and expression of TCR molecular decreased. Some reason of rejection is the donor cells don't express the proper MHC I molecule matching the receptor and the NK cell and T cell are activated to attack the donor cell. In the same way, without the KIR matched with receptor MHC I, the donor immunocyte couldn't establish the negative signal between the donor and receptor. As a result, the lymphocytes of donor are attacked and the GVHD occurs.In this research, the genes of MHC I and KIR from receptor mice (B ALB/c) were transferred into the bone marrow and spleen cells of donor mice (Cs7BL/6) and expressed on the hematopoietic and immunologic cells of donor. And then the allo-BMT was carried out on BALB/c mice. The bi-directional specific combination of KIR and MHC I molecule would establish the negative signals to killer cells, induce the specific immunologic tolerance of individual and prevent the GVHD and the graft rejection.Method1. The coding gene of Ly49A and H-2Dd, which were respectively the KIR and MHC I of BALB/c, were cloned by RT-PCR, and the retrovirus expressing vectors of pMSCV-Ly49A (puro+) and pMSCV-H-2Dd (neo+) were constructed.2. The two reconstructed retrovirus expressing vectors were respectively transfected into PT67 cell, packed to infective virus. The Cs7BL/6 mouse bone marrow cell and spleen cell were infected by the two kinds of reconstructed retrovirus, and the gene expression of extracecullar Ly49A and H-2Ddwere detected by immunofluorescence and flow cytometry.3. The hematopoietic function, immune function and tumor-killing function of gene-transferred cells were evaluated with CPU-Mix culture, lymphocyte proliferation, mixed lymphocyte culture and killing activities of CTL and NK.4. The gene-transferred cells were transfused respectively in the Cs7BL/6 (H-2b ) -BALB/c(H-2d) allograft GVHD model and graft rejection model. The mean survival time (MST) and the symptoms of GVHD were observed and the IL-2 level of serum after transplantation, the ratio of bone marrow chimera were measured in order to analyze the effect of genes transfer of KIR and MHC I molecule on GVHD and rejection.Results1. The restriction endonuclease digestion and DNA sequencing analysis showed the reconstruction of retrovirus expressing plasmids of pMSCV-H-2Dd (neo+) and pMSCV-Ly49A (puro+) were successful.2. The two reconstructed plasmids were packed to infective virus particles in incasing cells PT67 with a tite of about 5.8xl05-3.4xl06cfu/ml. These two kinds of retrovirus were co-infected in Cs7BL/6 bone marrow cell and spleen cell, and the two kinds of external molecules on cell surface all did express proved by immunofluorescence. The percentage of spleen cells of expressing both Ly49A and H-2Dd is 48.3+3.7% at 2 days after infection, and that of bone marrow cells is 13.6+2.3 %. After antibiotics screening of 4 weeks, they reached 62.4+3.9% and 18.1+2.6% respectively.3. The mixed colony-forming unit (CPU-Mix ) culture showed that the hematopoietic function of virus infected Cs7BL/6 mice bone marrow cells had no statistical difference with the non-infected C57BL/6 cells. The virus infection had no effect on the response ability of lymphocytes to the ConA stimulation. The mixed lymphocyte culture showed the reconstructed retrovirus infected Cs7BL/6 micebone marrow cells had a remarkably weaker effect to BALB/c mice spleen cells on the stimulation intensity or the response ability extensity than the non-infected and empty-vector-infected Cj7BL/6 cells. The i...
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