Construction Of Replication-defective Adenovirus Carring Human CTLA4-Ig Gene And FasL Gene And Their Tolerance Induction In Rat Renal Allografts

Objectives: (1) Construction the replication-defective adenovirus carrying human CTLA4-Ig gene and FasL gene ; (2) The plasmids pAdTrack-CMV-hCTLA4-Ig and pSuCMV-FasL were seperatedly transfected into 293 cells in vitro and observed the expression of CTLA4-Ig gene and FasL gene; (3) Construction models of orthotopic allograft renal transplantation between SD donors and Wistar recipients; (4) Perfusion in situ with Ad-CTLA4-Ig gene and Ad-FasL gene into the recipient kidneys through the renal artery during cold preservation , observed the survival time of the kidney transplantation in rats and searched the possible mechanism of immune tolerance induced by transfecting CTLA4-Ig gene and FasL gene therapy. Methods: (1) The plasmid pGEM3ZF-hCTLA4-Ig containing CTLA4-Ig gene was cut with HindⅢand XbaⅠ, the human full-length CTLA4-Ig cDNA was obtained at the same time, then subcloned it into the replication-defective adenovirus vector pAdTrack-CMV which was also cut with HindⅢand XbaⅠligated by DNA ligase, and the acquired recombinants were subsequently transfected into E coli strain DH53 for preparing recombinant, the replication-defective adenovirus plasmid named pAdTrack-CMV-hCTLA4-Ig; (2) The sequence of FasL gene was acquired by PCR from J28-FasL.It was ligated into the replication-defective adenovirus vector pSuCMV which was digested by NheⅠand SaⅡusing DNA ligase and the acquired recombinant was then transfected into E coli strain DH5 for preparing recombinant replication-defective adenovirus plasmid named pSuCMV-FaslL. The polymerase chain reaction(PCR) and RT-PCR were used to determine whether there were integration of CTLA4-Ig gene or FasL gene and the expression of mRNA in the plasmids and examined the sequence of CTLA4-Ig and FasL; (3) The human renal 293 cells were transfected by pAdTrack-CMV-hCTLA4-Ig and pSuCMV-FasL seperatedly in vitro. The expression of CTLA4-Ig and FasL in these cells were determined with RT-PCR and Westarn blot; (4) The models of orthotopic allograft renal transplantation between SD donors and Wistar recipients were established using "the cuff technique"with some modifications. The rats were randomly divided into 4 groups: The Group A, without any treatment; Group B, treated with Ad-EGFP (1.0×109pfu/ml); Group C, treated with Ad-CTLA4-Ig(1.0×109pfu/ml); Group D, treated with Ad-CTLA4-Ig(1.0×109pfu/ml) and ad-FasL (1.0×109pfu/ml). After kidney transplantations, the kidney and peripheral blood were obtained to determine the kidney function and the level of IL-2,IL-10, IFN-γusing enzyme-linked immunosorbent assay(ELISA). The expression of CTLA4-Ig and FasL in the kidney was examined by immunohistochemistry. The apoptosis in lymphocytes was evaluated by the terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end-labeling (TUNEL) method. The pathological character and ultrastructure of the grafts were observed through microscope and electron microscope. Meanwhile, the post operation survival time of each group was recorded. Results: (1) The combinants of pAdTrack-CMV-hCTLA4-Ig or pSuCMV-FasL were transfected into 293 cells through Lipofectamine 2000,the titer of virus in their supernatant reached 8.95×109 pfu/ml and 7.3×109 pfu/ml seperatedly. It was proved by PCR and RT-PCR that there were the integration of CTLA4-Ig gene or FasL gene and the expression of mRNA. The sequence of CTLA4-Ig or FasL gene was verified by GENECOME Co. (2) The human kidney 293 cells were transfected by pAdTrack-CMV-hCTLA4-Ig or pSuCMV-FaslL, after 24h, the expression of CTLA4-Ig and FasL in these cells were proved by RT-PCR and Westarn blot; (3) After some modifications in the renal transplantation with "the cuff technique", the time of operation was reduced significantly, the success rate of renal transplantation improved remarkably; (4) At 7 days after renal transplantation, the levels of creatinine in serum were significantly higher in Group A and B than in Group C and D; The detection of pathology suggested that severity of acute reject reaction after 7 days was grade ⅡB-Ⅲin Group A and Group B, grade 0-ⅠA in Group C and Group D; The expression of CTLA4-Ig and FasL in the kidney examined by immunohistochemistryshowed that there was no expression of CTLA4-Ig of FasL in Group A and Group B, but there was expression of CTLA4-Ig in Group C, expression of CTLA4-Ig and FasL in Group D; The result of TUNEL and electron microscope proved that there was apoptosis in lymphocytes of kidney tissue in Group D; The result of ELISA showed there was a higher level of IL-2 and IFN-γin Group A and Group B, the level of IL-10 was higher in group C and D; The survival time of rats were 8.17±1.17 days in Group A, 8.00±1.55 days in Group B, 31.33±6.77 days in Group C, 64.67±6.41 days in Group D, the survival time in Group A and B were shorter than in Group C and D, but the survival time in Group D was longer than in Group C. Conclusions: (1) The generated replication-defective adenovirus vector carried pAdTrack-CMV-hCTLA4-Ig or pSuCMV-FaslL could express CTLA4-Ig or FasL effectively; (2) The vector carried pAdTrack-CMV-hCTLA4-Ig or pSuCMV-FaslL could express CTLA4-Ig or FasL effectively in 293 cells; (3) The orthotopic allograft renal transplantation between SD donors and Wistar recipients, which was considered to be an ideal model in studying immune tolerance of transplantation, is a combination with high rate of acute rejection; (4) The gene therapy of transfected CTLA4-Ig or FasL gene could induce immune tolerance of renal transplantation in rats, the mechanism of CTLA4-Ig gene was the blockade of costimulatory signals and FasL gene was the inducing apoptosis of activated T cell, and the deviation of Th1/Th2 type cytokines may be the involved mechanism, the combination transfection of these two gene could produce a better result.