| Objective In this experiment, we mainly use PLNCX2-FLAG-CTLA4 Ig gene packaged into retroviral transfection of guinea pig kidney locally, throughing the expression of CTLA4 Ig to block CD28-B7 costimulatory pathways,creating immunosuppressive microenvironment, and combining with Sinomenine to inhibit mouse peripheral blood CD4+T cell proliferation, both by studying its heterogeneous explore synergies hamster kidney transplant rejection factor-related apoptosis-Fas/Fas L expression in response, so that the clinical treatment of renal transplant rejection in kidney transplantation and the clinical application of Xeno-renal transplantation will be provided new methods and ideas in future.Methods Male SD rats(12-14 weeks old), male guinea pigs(12-14 weeks) each 40, were randomly divided into four groups(Each group of SD rats, guinea pigs were 10). Experiment were divided into four groups, Group A: empty retroviral vector gene transfer+saline Blank control group; Group B: empty retroviral vector gene transfer+Sinomenine group; Group C: CTLA4 Ig retroviral gene transfer +saline group; Group D : CTLA4 Ig retroviral gene transfer+sinomenine group. Surgical methods forfirst partial CTLA4 Ig retroviral gene transfected into guinea pig kidneys, then the whole guinea pig kidneys were transplanted into SD rat heterotopic neck, where the left kidney as kidneys were transplanted kidney, right kidney will be cut under used to detect the expression of CTLA4 Ig. SD rats in each group were observed after graft survival time by mouse survival time; using HE staining to detect the Pathological grade of SD rats in each group after renal transplantation; monitoring creatinine values after 48 hours check by tail vein blood, at the same time monitoring 48 hours urine; CTLA4 Ig expression of the cut right kidney were detected by Western blotting; Western blotting and immunohistochemistry were to detect the expression of murine renal Fas/Fas L protein.Results 1. Creatinine and urine output monitoring Postoperative 48 h tail vein pumping serum creatinine values: Group B, C creatinine values were(9.20±0.83) mg/L and(9.00±0.71) mg/L, significantly were lower than in group A(15.00±1.00) mg/L(P<0.05), while group D mice transplanted creatinine was lowest(4.20±0.84) mg/L, there was a significant(P<0.05) difference compared with the other three groups. Postoperative 48 h urine values: Group B, C mice urine values were(11.80±1.09) ml and(11.60±1.14) ml, significantly higher than in group A(6.40±0.55) ml(P<0.05), while group D transplanted rat urine maximum amount of(19.20±0.81) ml, there was a significant(P<0.05) difference compared with the other three groups. 2.The survival time of Kidney transplant and recipient miceGroup A rats graft survival time was(5.60±0.89) d, the survival time of recipient mice was(6.50±0.87) d; Group B, C graft survival time was(10.45±0.85) d,(10.85±0.80) d, by the survival time of mice(12.40±0.86) d,(12.80±0.81) d, were longer than in group A(P<0.05), where D group graft and recipient mice survived the longest, respectively(14.80±0.85) d and(17.75±0.87) d. compared with the other three groups, the difference was statistically significant(P<0.05). 3. HE staining Each group renal pathological HE staining test results show: A group of postoperative renal tissue HE staining showed a large number of lymphocytes, Banff09 allograft nephropathy grading rationale for IIB-III level, prompted a strong reaction graft rejection. Group B, C graft also has Lymphocyte infiltration, but the expression intensity is lower than in group A, Banff09 kidney transplant rationale for IIA-III grade level. and group D only has a small graft lymphocytes, Banff09 transplanted kidney disease management for IA-IIB grade level, significantly lower than the other three groups. 4. Western blotting Kidney was transplanted into recipient mice after neck, CTLA4 Ig expression of the cut right kidney were detected by Western blotting in different experimental groups, which only group C, D were detected the expression of CTLA4 Ig, group A, B without CTLA4 Ig expression. Each group after renal transplantation rejection, Western blotting detecting the expression of Fas/Fas L in various renal tissues were visible. Group A in which the expression is the strongest, Group B, C were comparedwith A, the expression of Fas/Fas L were significantly decreased, However, little difference between the expression of group B, C, while group D was the weakest, there was a significant(P<0.05) difference compared with the other three groups. 5.Immunohistochemistry Immunohistochemical detection of renal transplantation in each group Fas/Fas L protein expression show: Group A renal tissue Fas/Fas L expression were tested brown, strong expression of strong positive rate of 100%. Group B, C Fas expression in renal tissue strong positive rate was 60% and 40%, respectively, Fas L expression strongly positive rates were 40% and 60%. Group D graft Fas/Fas L was no strong positive expression, Fas expression was weakly positive in four cases, one case of positive; Fas L expression was weakly positive in three cases, one case was positive. Significantly lower than the control group(P<0.05).Conclusion 1,CTLA4 Ig has the Function of anti-rejection in Xeno-renal transplantion of mouse, so that Fas/Fas L expressions are decreased, prolonging graft survival time. 2,Sinomenine has strong immunosuppressive effects, it can reduce xenograft mouse kidney transplant rejection, so that Fas/Fas L expressions are decreased, prolonging graft survival time. 3,CTLA4 Ig and Sinomenine have Synergistic effects of anti-rejection, Meanwhile applying CTLA4 Ig and Sinomenine can significantly reduce Xeno-renal transplantion of mouse rejection,Reduceing the expression of Fas/Fas L, significantly prolonging graft survival time. |
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