The Study On Inducement Of Rat Enal Transplantation Tolerance By Adenovirus Mediated FasL Gene

Induction of antigen specific tolerance in organ transplant recipients is the fundamental way for reaching long term survival of grafts. In recent studies, with the deeper study on cell apoptosis , people pay more and more attention on the important effect of Fas ligand(FasL) on forming immune absolve and inducing apoptosis of T lymphocyte, and pay attention on the effect of Fas/FasL mediated programmed cell death on graft immune tolerance. In this study, we use eukaryotic expression vector, the FasL gene was locally transfected , in order to explore the suppression effect to rejection of rat homotransplantation and make the foundation for further gene therapy. PART ONE ESTABLISHMENT OF RENAL TRANSPLANTATION MODEL OF RAT Objective: To establish renal transplantation model of rat. Methods: Apply modified Fisher,s renal transplantation model of rat. I orthotopic transplantation and use another suture silk to ligate the right kidney pedicle in vitro after several days. Result: Succeeded to establish renal transplantation model of rat. The achievement ratio was 84.2%. Conclusion: The rat transplantation model was used in empirical study of organ transplantation.The microsurgery skilll was adopted in our investigation by oneself. Operation achievement ratio was higher.PART TWO AMPLIFICATION OF FASL GENE Objective: To amplify FasL Gene and to sequence destination gene. Methods: After fasL gene was amplified and the DNA product was sequenced, the gene product was cut by endonuclease---Xho I, EcoR I and then was connected to pShuttleCMV, then the recombinant vector was transfected to Ecoli JM109 for further amplification, in the end , the recombinant plasmid was extracted and the cloned gene was sequenced. Result: The amplification which was followed board on FasL cDNA. It is thus evident that PCR agarose gel in 940bp. the gene product which was cut by endonuclease---Xho I, EcoR I and then was masc clone. Conclusion: FasL gene was cloned on pShuttleCMV vehicle. The product of PCR and clone gene were same with array of FasL of literature report by sequencing. PART THREE CONSTRUCTION AND CHARACTERIZATION OF FasL ADENOVIRUS RECOMBINANT Objective: To construct FasL recombinant and to amplify Ad-FasL.Methods: The cloned fasl gene was cut from pACCMVpLpA-FasL and the terminus was blunted, the FasL gene was connected to cosmid ShuttleCMV to construct shuttle plasmid, after enclose, amplification, purification, the shuttle plasmid was contransfected to 293 cell with Ad-FasL , then FasL adenovirus recombinant was successfully constructed by homogenous recombination. 293 cell was heavy amplified and was undergone clone screening and titration. Result: The Ad-FasL recombinant was successfully constructed by homogenous recombination. There was heavy amplified with Ad-FasL recombinant transfect 293 cell. Conclusion: Replication defective adenovirus Ad-FasL recombinant was successfully constructed, the virus can only be replicated by 293 cell containing E1 gene. The virus tilter was 9.33X109(pfu/ml).Ad-FasL was successfully transfected to kidney tissue and it was identified by immunofluorescence detection. PART FOUR ADENOVIRUS-MEDIATED FasL GENE TRANSFER INHIBITS RENAL-GRAFT REJECTION IN RATS Objective: To study Ad-FasL recombinant inhibits renal-graft rejection in rats. Methods: Allogeneic kidney transplants were performed between Lewis donors and Wistar recipients. The experimental rat was divided into three groups: 1. the donor kidney of Lewis rat was locally transfected by Ad-FasL and then transplanted to recipient Wistar rat. 2. the donor kidney of Lewis rat was directly transplanted to recipient Wistar rat, but after theoperation, the recipient was treated by CSA. 3. the donor kidney of Lewis rat was directly transplanted to recipient Wistar rat without any therapy .Donor kidneys were perfused in situ with Ad-5 (control), or 9×109 plaque forming units of Ad-FasL. Kidney allografts were harvested at days7 were evaluated by hematoxylin and eosin and immunohistochemical staining. Transduced allogra...