Experimental Study On The Hyporesponsiveness Of Foxp3-transferred CD25～+CD4～- T Lymphocytes

Organ transplantation is one of important managements for the treatment of endstage organ failure. On the other hand, allograft rejection is still the key factor influencing the survival of allafts. Although allograft rejection is partly relieved by immunosuppressants, but recipients'life quality are severely affected and at the cost of immunecompetance by it。It has become the focus in the transplantation field that how to induce specific immune tolerance in the recipients to the donor organ.T lymphocytes play an important role in the allograft rejection. The aim of immune therapy is to establish the allograft immune tolerance as natural self-tolerance, but not the systematic non-specific immune suppression. Antigenic specific Foxp3 transduced T cell produced by allotypic antigen-reactive T lymphocytes, or adopted Treg cells can be used in the therapy to re-establish self-tolerance.ObjectiveTo transferred the Foxp3 gene into CD25~+CD4~- T lymphocytes and induce it expression, then infuse it to autoimmune disease model to re-establish self-tolerance. It will be the basis of controling antigen specific immunity reaction, direction location guiding T regulatory lymphocyte in vitro, derivning transplantation tolerance and treating rejection.Methods1. To construct eukaryotic coexpression plasmid pmFoxp3-IRES2-EGFPFoxp3 cDNA was extracted from Balb/c mouse monocyte suspension by RT-PCR. After purified, Foxp3 PCR products were inserted into pMD18-T plasmid. The positive clones were identified by blue/white colour screening. After amplified, recombinant plasmids were extracted. Recombinant plasmids pMD18-T-Foxp3 and pIRES2-EGFP wer cut by restriction enzyme EcoRⅠand Bgl II. Purpose fragments were collected and connected. After transducing the competence cell DH5αwith connected products, recombinant plasmids pmFoxp3-IRES2-EGFP were amplified and extracted.