| Objective : Analyzing the difference of natural anti-pig antibodies level in transplant-related population or rhesus monkey.And analyzing the Influence of natural anti-pig antibodies level for pig-rhesus monkey xenotransplantation.discussing the clinical significance of natural anti-pig antibodies for pig-human kidney xenotransplantation.To provide experimental basis for clinical study of xenotransplantation.Methods :(1)Detecting natural anti-pig antibodies levels in transplant-related populations by FCM: blood samples were collective from the second affiliated hospital of Hainan medical university,including healthy volunteers(n=20),ESRD patients(n=20),DBD(n=20)and transplantation recipients who is occurring TCMR(n=20).Isolating serum from these sample,testing they Ig M/Ig G binding and CDC with PBMC or RBC which both from the WT,GTKO,GTKO/β4Gal NT2 KO,GTKO/CMAHKO and TKO/h CD55 pigs by flow cytometry,which pigs were breed by the Chengdu Clonorgan Biotechnology company;(2)Detecting natural anti-pig antibody level of rhesus monkey by FCM: Collecting20 serum samples of rhesus monkey,testing their Ig M/Ig G binding and CDC with PBMC which from the WT,GTKO/h CD55,GTKO/β4Gal NT2 KO,and GTKO/β4Gal NT2KO/h CD55/h TM pigs by flow cytometry,which pigs were breed by the Chengdu Clonorgan Biotechnology company;(3)The influence of natural anti-pig antibody for the effect of pig-monkey kidney xenotransplantation in vivo study: choosing a GTKO/β4Gal NT2KO/h CD55/h TM pig as donor,and 2 rhesus monkeys as recipient.Using anti-CD38 monoclonal antibody reduced the level of natural anti-pig antibody in rhesus monkeys before transplantation.After immunological induction,completing 2 cases of pig-rhesus monkeys kidney transplantation,and resecting both of the monkey’s natural kidneys.The renal function and immune status of the recipient were evaluated by routine laboratory examination and immunological detection.Exploring the pathological injury types of graft loss by HE staining,immunofluorescence and immunohistochemistry.Comparing these results with previous research to analyze the effect of reducing the natural anti-pig antibody level for effect of the xenograft kidney transplantation.Results:(1)Levels of natural anti-pig antibodies in transplant-related populations:volunteer Serum with WT,GTKO and GTKO/β4Gal NT2 KO Ig M binding are168.95(121.12,267.68)、60.30(40.10,224.99)、93.44(40.44,204.94),respectively.And Ig G binding are 45.35(24.45,108.03),15.72(8.71,44.89),23.82(5.84,67.91),respectively.They both were the highest among the four groups,and there were significant differences with the other groups(P < 0.05).And CDC level of volunteer Serum with these pig PBMCs are 95.68%(90.56%,96.67%),48.18%(15.33%,74.14%),14.36%(9.32%,45.07%),respectively.However,the antibody binding levels and CDC of GTKO/CMAHKO and TKO/h CD55 with four populations serum were lower,and there was no significant difference among the groups(P > 0.05).The CDC levels(Calculated value,negative meaning no cell killing)of recipient sera to TKO/h CD55 pigs PBMC in the four groups were healthy volunteers 4.93%(2.42%,7.06%),ESRD patients 5.37%(0%,7.92%),DBD 0.82%(-3.07%,4.46%)and Allo-TCMR recipients 1.33%(0.53%,3.69%).All the natural anti-pig antibodies in different populations were mainly Ig M(P < 0.05).The difference of RBC antibody binding level in different groups and gene pigs was basically consistent with that of PBMC;(2)Levels of natural anti-pig antibodies in rhesus monkey: The CDC level of different rhesus monkey serum with PBMC of two GTKO/β4Gal NT2KO/h CD55/h TM pigs was the lowest,were respectively 3.01%(1.88%,5.98%)and 3.61%(2.76%,8.94%),and significantly lower than that in other gene pigs(P < 0.05).The level of Ig G binding is 102.04(31.73,195.85)and Ig M binding is 52.65(31.96,148.96)in serum of WT pigs were significantly higher than those of other genes pig(P< 0.05);(3)Effect of pig-monkey kidney xenotransplantation: The natural anti-pig antibodies Ig G with different gene pigs were all decreased before the operation,and the level of CDC was also decreased,but there was no obvious decreasing trend of Ig M.2 cases of pig-NHPs kidney transplantation were succeed that both recipients were no obvious abnormality in Renal appearance and no hyperacute rejection during the operation.And the recipients survived for 17 and 29 days,respectively.Early postoperative renal function and electrolyte metabolism were basically normal.However,platelet first increased and then decreased significantly before the increase of serum creatinine.anti-CD38 monoclonal antibody was given after the increase of circulating antibody,and no decrease in level of induced anti-pig antibody was observed.The pathological examination of the transplanted kidney indicated that the experiment was terminated caused by ABMR.There is different deposition level of C4 d,C5b-9 and Ig G in kidney and ureter.Conclusions:(1)Although healthy volunteers,transplant-related population and NHP have natural anti-pig antibodies,these antibodies do not have specificity of gene modification pig.(2)Anti-pig antibody mediated rejection is the main factor affecting the long-term survival of the graft in pig-monkey kidney xenotransplantation.(3)Anti-CD38 monoclonal antibody can reduce the natural anti-pig antibody level and CDC.However,it does not effectively reduce the injure of early rejection to pig-monkey kidney xenotransplantation.The reason remains to be further studied. |
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