| At present, more and more patients with late organic failure had been saved by transplantation. But the survival time of the transplantations in majority of the patients remains limited because of graft rejection. CsA and FK506 are the common immunosuppressive agents without immune specificity in clinic practice. The patients who received the antirejection treatment by using these drugs will have more chances of fatal infection and tumor. Moreover, the immunosuppressive agents are unable to solve graft rejection thoroughly. The specific immune tolerance to graft antigen is an ideal approach to the prevention of graft rejection and a research hotspot in the field of transplantation.Dendritic cells (DCs), which are potent professional antigen presenting cells (APC), play an important role in immune system because of their abilities to activate naive T cells and initiate a primary immune response. Recently, the studies have showed that the immature DCs display tolerance-inducing activities, but they exhibit their immunogenicity after maturation. Donor-derived immature DCs modified by the effective immunoregulation factors may be of great benefit to immunosuppression because of the maintenance of immature DCs in vivo and the persistent expression of immunoregulation factors in the precise microenviroment where alloantigens presentation occurs and T-cell responses are initiated.a-melanocyte stimulating hormone(MSH) is a endogenous neuropetide which restrain many acute and chronic inflammatory responses by inhibiting the production of inflammatory cytokines(IL-l, TNF, IL-6, MIF, etc ), chemokines (IL-8, MCP-1, etc), nitric-oxide synthase and NO. MSH, however, induces the production of immunosuppressive cytokine IL-10. a-MSH is able to downregulate the adhesion molecule expression of monocytes, macrophages and vascular endothelial cells, and inhibit the proliferation of T cells, the secretion of IL-2 and the expression of CD3,CD25 and CD28. The expression of melanocortin receptor-1 (MC-1R) ,which is one of the receptors for a-MSH , was recently found on human peripheral blood-derived monocytes and DCs. The studies showed that the expression of the costimulatory molecules CD86 and CD40 were downregulated on DCs in the presence of a-MSH. In addition, a-MSH is a small rapid-metabolic regulatory peptide without antigenicity and accumulated toxicity in human body, all of which indicated that a-MSH had strong capacity of immunoregulation .The vectors which are used in gene therapy include adenoviral (Ad) vectors, retroviral (RV) vectors, adeno-associated virus (AAV) vectors, herpes simplex virus (HSV) vectors and hybrid viral vector. But few vectors are suitable for transfecting DC because many vectors with the target genes induce immune response to the virus antigens and the target gene productions in vivo, and lead to attack the virus vector transduced DCs, which are transfused into the body. The studies have proved that the AAV vectors with no pathogenicity and low immunogenicity in human body are able to infect both proliferating cells and quiescent cells, and maintain the functions of the transgene products and the long-term stable expression of the target genes which integrate into host genome. Therefore AAV vectors are the ideal vectors for the gene transfer to DC.The present study was to modifie the DCs with a-MSH gene (a-MSH-DC) by using the AAV vectors, determine the characteristics, the phenotypes and the functions of a-MSH-DCs and investigate the effects and mechanisms of a-MSH-DC on the graft rejection.Part I Phenotypic characteristics and functions of bone marrow derived immature dendritic cells modified by a-MSH geneThe plasmids, pAAV-RC and pHelper plus pAAV-a-MSH or pAAV-hrGFP were transferred to HEK293 packing cell line to obtain the supernatant containing rAAV carrying a-MSH and green fluoresent protein (GFP) genes (rAAV-a-MSH), and rAAV carrying GFP gene (rAAV-GFP), respectively. Then the rAAVs were purified. DCs were generated from bone marrow cells of BALB/c mice in the presence ofrecombina...
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