Influence Of CTLA4Ig And CD40Ig Retroviral Genes Transfection On The Rejection Of Xenogenic Rat Renal Allografts

【Objective】Through PLNCX2-FLAG-CTLA4Ig and PLNCX2-HA-CD40Ig genespackaged by retroviral transfected into the kidney of guinea pig to blocking thecostimulatory pathway of CD28-B7and CD40-CD40L,observe the induction ofxenogenic renal allografts immune tolerance by CTLA4Ig and CD40Ig localtransfection between guinea pigs to SD rats, and finding a new way to prevention andcure the rejection of xenogenic renal allografts.【Methods】Forty male SD rats(12-14weeks old) and forty male guinea pigs(12-14weeks old)were randomized into four groups, Surgical methods is stransfer the retrovirals ofCTLA4Ig and CD40Ig genes into the renal of guinea pigs,and then transplanted theleft renal into the neck of SD rats, the right renal preservated under the ice.The firstgroup is the control group with retrovirals of empty carrier (A Group); The secondgroup is thre transfection group with retrovirals of CD40Ig genes(B Group);Thethird group is thre transfection group with retrovirals of CTLA4Ig genes(C Group);The fourth group is the transfection group with retrovirals of CTLA4Ig and CD40Igenes(D Group). The Westen bloting detected retrovirus transfection efficiency of theguinea pig ‘s right kidney. Observe the survival time of transplated renal and SD rats.To detect the level of rejection reaction with HE staining,to detect the expression ofBax protein by immunohistochemical, to detect the expression of bcl-2/Bax mRNA byRT-PCR, and detect the Apoptosis rate of the transplated renal by flow cytometry.【Results】1.Western blottingAfter transfected the CD40Ig or CTLA4Ig on the transplated renal, CD40Ig and(or)CTLA4Ig expressed in its organization. No CD40Ig and CTLA4Ig expressed in groupA, CD40Ig expressed in group B, CTLA4Ig expressed in group C, CD40Ig andCTLA4Ig were expressed in group D.2.The survival time of the transplated renal and SD ratsThe survival time of transplanted renals of group A were（8.1±2.1）d，the survivaltime of SD rats were(9.5±2.3)d. The survival time of transplanted renals of groupB、 C、D were(13.1±2.2)d、（12.4±2.6)d and（16.6±3.0)d，the survival time of SD rats ofgroup B、C、D were(14.4±3.1)d、（14.5±3.3)d and（18.8±2.7)d, obviously longer thangroupA(P<0.05)，The survival time of transplanted renals and SD rats of group Dwere the longest than other three groups，the difference was statistically significant（P<0.05).There was no significant statistical different between B and Cgroups(P>0.05).3. HE stainingThe HE staining of A group showed a large number of lymphocytes, it’spathological grading of Banff07were IIB-III, prompted a strong reaction aboutkidney transplant rejection. The B、C group’s expression of lymphocytes were lowerthan A group，and the pathological grading of Banff07were IIA-III, but the D group’sexpression of lymphocytes were the least，the pathological grading of Banff07wereIA-IIB.4. ImmunohistochemicalA group of transplanted kidney’s expression of Bax were tested tan,the strongpositive rate of100%, the strong positive rate of group C and group B,were60%and80%, D group had one case of a strongly positive reaction, a weak positive reaction in4cases, strong positive rate was20%(P <0.05).5. RT-PCRThe Bax mRNA expression of B、C、D group’s transplanted kidney tissue weresignificantly lower than A group, the lowest expression of transplanted kidney tissueabout Bax mRNA was D group.The expression of bcl-2mRNA were contrary, Dgroup was the highest expression, A group was the lowest (P <0.05). There was nosignificant statistical different between B and C groups(P>0.05).6.The apoptosis rate of transplated renalsThe apoptosis rate of B、C groups were (24.34±2.78)%and (25.21±2.69)%,significantly lower than group A (54.69±6.25)%(P <0.05), the lowest rate ofapoptosis was group D (15.03±1.21)%, there was a significant difference comparedto theA、 B、C groups (P <0.05). There was no significant statistical different betweenB and C groups(P>0.05).【Conclusion】1.CTLA4Ig and CD40Ig retroviral gene transfection kidneys were localized extend graft’s survival time.2.Meanwhile transfected by CTLA4Ig and CD40Ig genes can significantlyreduce the renal xenograft rejection, prolong the survival time of grafts.