| Objective: To investigate(1)the effect of different genetically modified pigs on pigto-monkey renal xenotransplantation.(2)Effects of hCD55 and h TM on pig-to-monkey renal xenotransplantation.Methods: 1.In vitro experiment(1)Blood was drawn from healthy rhesus monkey and serum Ig M/Ig G binding to,and CDC of,PBMC from wild GTKO/hCD55 、GTKO/β4Gal NT2 KO 、 GTKO/β4Gal NT2KO/hCD55 and TKO/hCD55 pigs were measured by flow cytometry.(2)Human blood was incubated with HMEC-1,GTKO/hCD55/h TM/h EPO and GTKO/hCD55 porcine vascular endothelial cells,respectively,and human thrombin and protein C were added to detect turbidity and human activated protein C.2.In vivo experiment(1)The following genetically modified pigs were used as donors: GTKO/hCD55,GTKO/hCD55/h TM/h EPO,GTKO/β4Gal NT2 KO,GTKO/β4Gal NT2KO/hCD55/h TM and TKO/hCD55,rhesus monkeys with low preformed anti-pig antibodies were screened as recipients by flow cytometry.Pig to rhesus monkey renal xenotransplantation was performed,and both primary kidneys of the recipients were resected.(2)According to the type of xenoantigen knockout of donor and whether the recipients received costimulatory pathway blocker,the recipients were divided into 4 groups: Group A(n=3)received GTKObased donor pigs,did not receive costimulatory pathway blocker;Group B(n=3),Group C(n=5)and Group D(n=1)received GTKO-based,GTKO/β4Gal NT2KO-based and TKO-based donors,respectively,and received costimulatory pathway blockers.(3)Blood of recipients was sent to the laboratory for blood routine examination,blood biochemistry,renal function and microalbuminuria.T and B lymphocyte typing and serum anti-pig antibody were detected by flow cytometry.The blood flow and resistance index of transplanted kidneys were monitored by color ultrasound.(4)The recipient was sacrificed when renal was failure,the transplanted kidney and ureter were removed,and HE staining was performed.The deposition of Ig M,Ig G,C3 c,C4c and C5b-9 in the grafts was detected by immunofluorescence,and the deposition of C4 d,CD3,CD68 and CD42 b in the grafts was detected by immunohistochemistry.Results: 1.In vitro experiment(1)Monkey serum antibody showed the lowest CDC and Ig M binding to GTKO/β4Gal NT2KO/hCD55 pig PBMC in vitro.The CDC of GTKO/β4Gal NT2 KO pig PBMC was higher than that of GTKO/hCD55 and GTKO/β4Gal NT2KO/hCD55.(2)h TM can prolong the human coagulation time and promote the production of human activated protein C.2.In vivo experiment(1)The survival time of the 4 groups was more than 2 weeks,the longest was 32 days,and all recipients did not happen hyperacute rejection.(2)Within 12 days after operation,serum electrolyte,creatinine and urea nitrogen were within normal range.Hemoglobin and urinary microalbumin decreased and increased in the early postoperative period,respectively,while platelet decreased earlier than creatinine increased.The lymphocyte counts of most recipients rebounded after surgery.(3)Among the 4 groups,9 recipients had increased anti-pig antibody,6 recipients had rebound of CD3 lymphocyte and 3 recipients had rebound of CD19 lymphocyte.the grafts resistance index increased and blood flow decreased.Before death,the grafts resistance index increased and blood flow decreased.(4)Immunohistochemistry showed that CD68,C4 d and CD42 b were deposited in all grafts,and some of the grafts were infiltrated by CD3.Immunofluorescence revealed antibody deposition(Ig M,Ig G alone or Ig M and Ig G together)in all recipient grafts,and most of the recipient grafts had C3 c,C4c and C5b-9 deposition.HE staining indicated microcirculation thrombosis and interstitial hemorrhage in the all grafts.Conclusions:(1)GTKO knockout can overcome hyperacute rejection in pig-to-rhesus monkey renal transplantation.(2)The modified porcine kidney has a good match with the recipient rhesus monkey,and can effectively maintain the electrolyte balance of the recipient rhesus monkey,and remove metabolic products in the body.(3)When acute rejection occurred,the transfer of hCD55 and h TM from donor pigs could not effectively inhibit complement activation and graft microthrombus formation,respectively.(4)GTKO/β4Gal NT2 KO and TKO as donor pigs,combined with costimulatory pathway blockers,which could not effectively prevent acute rejection,suggesting that in addition to Gal,Neu5 Gc and Sda antigens,there are other xenoantigens.Objective: To investigate whether there were antibodies bound to SLA+ class Ⅲ and class Ⅳ antigens in the post-operation sera of recipient rhesus macaques,healthy rhesus macaques,and human sera in vitro experiments.Methods:(1)Different numbers of WT pig RBC’s were incubated with normal serum for different times,after that,serum Ig M/Ig G binding to,and CDC of RBC from WT,were measured by flow cytometry.(2)The serum of 9 rhesus monkeys with elevated anti-pig antibodies after renal xenotransplantation was collected,and the serum was adsorbed with pig red blood cell(RBC)of the same genotype as that of the donor pig,and antibodies binding and CDC to PBMC were measured by flow cytometry.(3)Flow cytometry was used to detect SLA-I and SLA-DR expression on PBMC and RBC of GTKO/β4Gal NT2KO/hCD55/h TM and TKO/hCD55 pigs.(4)Serum of healthy rhesus monkeys,healthy humans,end stage renal disease and renal allotransplant recipients who were currently experiencing T cell-mediated rejection were collected.The serum was adsorbed by pig RBC,the un-adsorbed serum and the adsorbed serum antibodies binding or CDC to GTKO/β4Gal NT2KO/hCD55/h TM or TKO/hCD55 pig PBMC and RBC were measured by flow cytometry.Results:(1)Serum Ig M/Ig G binding to WT pig RBC was negative after serum antibodies being adsorbed which using more than 0.5×108 WT pig RBCs for 0.5h.(2)The serum binding to PBMC antibody and CDC of 9 recipients adsorbed by porcine red blood cells were lower than that of unadsorbed serum,and the serum binding to PBMC antibody and CDC were higher after adsorbed than before operation.(3)Different expressions of SLA-I and SLA-DR on pig PBMC may affect the binding and killing of monkey serum and human serum antibodies after adsorption,and the binding amount and killing amount of antibody were positively correlated with SLA-I and SLADR expression levels.(4)Healthy rhesus monkey and healthy serum Ig M binding to pig PBMC was positive after serum antibodies being adsorbed.The antibody binding and CDC of the adsorbed serum were significantly lower than those of the unadsorbed serum.At the same time,the unadsorbed serum was positive with porcine RBC antibody.Conclusion:(1)SLA+class Ⅲ and class Ⅳ antigens can bind to the new anti-pig antibodies in the serum of the recipient rhesus monkey.(2)SLA+class Ⅲ and class Ⅳ antigens can bind to the preformed antibodies in the serum of healthy macaque monkey and human. |
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