Cloning And Expression Of The CXCL9 Gene And Anti-CXCL9 Antibody Attenuating Acute Rejection Of Renal Allograft

PART ONE ESTABLISHMENT OF MODE FOR ORTHOTOPIC ALLOGRAFT RENAL TRANSPLANTATION IN THE RATS WITHOUT SURGERY-MICROSCOPEObjective:To establish a feasible and stable rat model of kidney transplantation without surgery-microscopeMethods:SD and Wistar rats were used as the donors and the recipients.left kidneys were harvested as grafts after flushed with 4℃lactated Ringer solution in situ.with the hellp of extradural catheter,the donor inferior vena was anastomosed end-to-end with the recipient left renal vein.an end-to-side anastomosis of abdominal aorta was operated. connecting the donor ureter with ureterocystic flap to the recipient's bladder was conducted.Resultes:One hundred five kidney transplantation operations were performed,in which the operation success rate in the last 65 cases reached about 90.77%.The average total time operation was(130±20)min.the average time of warm ischemia and cold ischemia was less 20s and 50min respectively.a modified rat model of renal transplantation was successfully established.Conclusion:This feasible model of renal transplantation is that can be used to study the transplantation mechanism. PART TWO THE EXPRESSION ANDS SIGNIFICANCE OF CXCL9 AND CXCR3 IN PERIPHERAL BLOOD IN ACUTE RAT RENAL ALLOGRAFTS REJECTIONObjective:To evaluate the diagnostic role of chemokine CXCL9 and chemokine receptor CXCR3 mRNA in predicting the early stage of acute rejecton afer renal transplantation in rats.Methods:Renal allogeneil graft was performed using Brown Norway or Lewis rats as donors and Lewis rats as recipients.CXCL9 in serum was measured by ELISA and CXCR3 mRNA in peripheral blood lymphocytes by RT-PCR at the time points of 1day before operation and 1day,3days,5days,7days after operation.Serum creatinine(Cr) was tested simultaneously.Resultes:The level of CXCL9,CXCR3 mRNA and Cr in allograft group were significantly higher than that in isograft group(P＜0.01).There was no significant difference of CXCL9 and CXCR3 mRNA level in both allograft and isograft groups before operation,and was significantly higher on 1day,3days,5days and 7days after operation in allograft group than that in isograft group(P＜0.05).There was no difference of Cr level in allograft and isograft groups before and 1day after operation,which was significantly higher on 3days,5days and 7days in allograft group than that in isograft group(P＜0.05)Conclusion:The expression level of CXCL9 in serum and CXCR3 mRNA in peripheral blood lymphocytes are sensitive predictors for the early diagnosis of acute rejection.PART THREE PROKARYOTIC EXPRESSION AND PURIFICATION OF CXCL9 RECOMBINANT AND DEVELOPMENT OF ITS POLYCLONAL ANTIBODYObjective:To construct prokaryotic expression plasmid of CXCL9,express target protein in E.coli BL21(DE3).The target protein was used to immunize the rabbits after it was separated and purified by SDS -PAGE.then the polyconalantibody was gained.Methods:Synthesized CXCL9 gene was orientally inserted into prokaryotic expression vector pET32a(+),E.coli BL21(DE3) were transformed with pET32a(+)/CXCL9,IPTG was added to induce the fusion protein expression,the CXCL9 protein was expressed as inclusion bodies proved by solubility analysis of fusion protein,CXCL9 fusion protein was purified through Ni²⁺ affinity chromatography.The rabbits were immunized with mixtue of CXCL9 fusion protein and complete freund's adjuvant.The titer and specificity of CXCL9 Polyclonalantibody was determined by indirect elisa and western blotting.Results:CXCL9 fusion protein was obtained by the method of genetic recombination technigue,and mainly presented in clusion body of E.coli,which could be purified through Ni²⁺ affinity chromatography.Antiserum was prepared after rabbits were immunized with the purified protein.Conclusion:To construct and induce expression of pET32a (+)/CXCL9 recombinant.The high quanlity of polyclonal antibody of rat CXCL9 was made.This will be a basis of further studying the biological fuction of CXCL9.PART FOUR ANTI-CXCL9 ANTIBODY ATTENUATES ACUTE REJECTION OF RENAL ALLOGRAFTObjective:To investigated effects of anti-CXCL9 antibody on acute rejection in rat renal allografts.Methods:Acute rejection animal models after renal transplantation were established in rats,among which BN rats served as donors and LEW rats as recipients.After renal transplantation, recipients were randomly divided into groupâ… :acute rejection treated with normal rabbit serum;groupâ…¡:acute rejection treated with rabbit anti-CXCL9 serum;groupâ…¢:acut rejection treated with cyclosporine;groupâ…£:acute rejection treated with cyclosporine and rabbit anti-CXCL9 serum.Postoperative survival time were observed in four groups.Changes in transplanted kidney 5 days after operation by histopathological methods,expression of CD4⁺ and CD8⁺ T cells by immunohistochemistry,CXCR3 mRNA expression by RT-PCR,expression of IL-2 by ELISA,lymphocytes proliferation activity by 3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyl tetrazolium bromide(MTT) assay were all detected. Results:Survival of recipients in groupâ…¡(11.75±1.26)d and groupâ…£(18.25±1.70)d were significantly prolonged vs groupâ… (8.00±0.82)d, pathological scores[groupâ…¡(3.25±0.96) and groupâ…£(1.25±0.50)vs groupâ… (5.50±0.58)]were statistically different.The serum creatinine was lower levels in groupâ…¡(131.87±7.48μmol/L) and groupâ…£(59.83±7.70μmol/L) than in groupâ… (332.16±45.14μmol/L) at posttransplantation 5d.The expression of IL-2 in peripheral blood and CXCR3 mRNA in renal allografts of groupâ… were much higher than those of groupâ…¡and groupâ…£.Infiltration of CD4⁺ and CD8⁺ T cells and lymphocytes proliferation activity in groupâ…¡andâ…£were lower than those in groupâ… .Conclusion:Anti-CXCL9 antibody prohibits immune response in rat renal allografts and prolongs survival time in recipients.The underlying mechanism may be its neutralization of chemokine CXCL9 in peripheral blood,prevention of infiltration for CD4⁺ and CD8⁺ T cells and inhibition of lymphocytes proliferation activity.