Experimental Study On Prolonging The Surivival Of Renal Allografts With Adenovirus-mediated CD154 Extracellular Domain Gene Transfection In Rats

Induction of antigen specific hyporesponsiveness even tolerance in organ transplant recipients is the foundamental way for resolving the problem of toxicity and infection in lifelong antirejection therapy with non-specific immunosuppression. In recent year, a novel strategy that modifies the grafts or cultured antigen presenting cells(APC), such as dendritic cells(DC) with some immunomodulatory genes has been developed, which renders the organ grafts hyporeponsed or tolerated by the recipients with either little side effects and little risk of systemic immunosuppression via ex vivo genetic manipulation. Promising results have been obtained in some animal transplantation model . Thus , the novel strategy offers exciting perspectives for the development of safe and effective immunotherapy in clinical organ transplantation.In this research, the recombinant hCD154 ED(Extracellular Domain)cDNA, which is believed the effective blockade of CD40-CD154 costimulatory pathway in T cell acivation, was chosen as the immunomodulator. The hCD154ED cDNA was inserted into the replication-deficient adenovirus vector to construct a hCD154 ED gene-recombinant adenovirus- Adv-hCD154ED. The effective way of transferring hCD154 ED gene into the renal allografts with Adv-hCD154ED was explored and furthermore the effect of modification of renal allografts with hCD154ED gene on the survival of renal allograft was studied in rats. The main results and consulsions were as follows:1. Construction of hCD154ED gene recombinant replication-deficient adenovirusThe hCD154ED and EGFP cDNA was inserted into the expressing vector AdEasy XL Adenoviral Vector System to construct the recombinant plasmid pShuttle - CMV -hCD154ED. The hCD154ED - IRES2-EGFP gene recombinant replication-deficient adenovirus (AdEasy-hCD154ED-IRES2-EGFP) was constructed by recombination of the plasmid pShuttle-CMV -hCD154ED and the plasmid pADEasy-1 vector in bacteria BJ5183. The recombinant adenovirus that could mediate the expression of hCD154ED in293 cell line was identified by westernblot.2. Cytotoxicity of AdvhCD154ED-IRES2-EGFP and its capacity mediating the expression of hCD154ED in cultured cellsThe cell lines PK-15 transfected by AdvhCD154ED-IRES2-EGFP. Significant difference of the survival rates of the cells were not observed between the transfected cells and the controls 2 and 4 days after transfection . The results suggest that AdvhCD154ED-IRES2 -EGFP has little cytotoxic effect on non-packaging cells. The expression of hCD154ED and EGFP in the AdvhCD154ED-IRES2-EGFP -transfected cell lines was determined by Laser Scan Confocal Microscope. The results showed that the expression rates of hCD154ED in both cell lines transfected at 37 癈 were over 95% while the expression rates and tensities in both cell lines transfected at 4"C were decreaded significantly, which suggest that the transfection temperature be the important fector influencing the tranfection efficiency and the expression of target gene. The products in supernatant of cell lines were identified by Western blot. The results showed that there were hCD154ED in the supernatant of cells, suggest that AdvhCD154ED-IRES2-EGFP has the ability mediating non-packing cells to express soluble hCD154ED, thus sets the functional foundation of further gene manipulation in organ grafts.3. AdvhCD154ED-IRES2-EGFP mediated hCD154ED gene transfection in kidneys of ratsThe transfection efficiency of AdvhCD154ED-IRES2-EGFP in kidney of BN rats was determined by immunofluorescence of EGFP. The results showed that specific fluorescence were not observed in section of kidney after perfusion of AdvhCD154ED-IRES2 - EGFP into cold preserved kidney at 4 癈 for 3h or systemic injection of AdvhCD154ED-IRES2-EGFP in vivo via renal artery for 3h. However, evident specific fluorescence was observed in sections of kidneys when the vessel of liver and the aorta and vena cava below the renal vessel were ligated and AdvhCD154ED-IRES2-EGFP was then injected systemically in vivo via the renal artery for 3h and the specific fluor...