Construction Of Recombinant Adenovirus Vector Expressing CD40Ig And The Study Of Its Effects On The Induction Of Rat Heart Allograft Tolerance

At present, organ transplantation is one of the most reliable methods that can cure the exhausted organs resulting from irreversible functional damages. Under the development of the medical technology, the advancement of basal medical science and the application of new immunosuppressive agents, research of organ transplantation developed quickly, and survival time of transplanted organ is prolonged obviously. But there are still a great number questions unsolved, such as the dysfunction of transplanted organs resulting from chronic immune rejection, and neoplasm, infection coming from the non-specific immunosuppression of immunosuppressive agents. Therefore, how to induce immunologic tolerance, avoid immune rejection, reduce or unuse immunosuppressive agents and has become the goal that every medical worker wants to fulfill.According the theory of Immunologic tolerance, this project constructed a recombinant adenovirus vector that can express human CD40Ig and observed the effects of Adenovirus mediated CD40Ig gene transfer combined with donor specific cell transfusion on induction of heart allograft tolerance using mouse strains with fully MHC barrier. The conclusions are following:1. Adenovirus shuttle plasmid containing human CD40Ig recombinantfusion gene was constructed and linearized by Pme I, then cotransfected with Ad backbone plasmid named pAdeasy-1 into E coli. strain BJ5183 for preparing recombinant adenovirus plasmid pAd/CD40Ig. After that it was transfected into 293 cells, in which intact Adenovirus Ad/CD40Ig was packaged.2. SDS - PAGE electrophoresis and Western-Blot analyses proved that these vectors are the right recombinant Ad/CD40Ig that can express human CD40Ig. 48 hours after injection of these vectors into rats through caudal vein, The expression of CD40Ig could be detected in vivo by ELISA analysis. About two weeks later, the expression level reached culmination. In vivo, the expression of CD40Ig could last more than 30 days.3 Heart allograft was transplant into left neck of the recipient rats as following pattern: the carotis communis artery of recipient-the aorta of allograft. the left jugularis external vein of recipient-the left pulmonalis sinistra artery.4. Ad/CD40Ig could prolong the survival time of allograft, but could not suppress the reaction of MLR and DTH to donor's spleen cells.5. Combining with donor specific bone marrow cells transfusion or low dose MMF application, Ad/Cd40Ig could prolong the survival time of allografts obviously. Recipients of this group show a low response to donor specific spleen cells when DTH and MLR are observed.