Study On The Protective Effect Of HCD55 In Islet Xenotransplantation

Background and objective: Islet transplantation is a crucial treatment of type I diabetes(T1DM),but it faces the problem of donor shortage.Islet xenotransplantation with porcine islet cells as the donor is an effective way to solve this problem.Instant blood-mediated inflammatory reaction(IBMIR)is induced after porcine islet cell transplantation through the hepatic portal vein,resulting in massive loss of islet cells in the early stage after transplantation.Complement activation is one of the key factors causing IBMIR.Human decay acceleration factor(h DAF/hCD55)is a complement regulatory protein with the function of inhibiting complement activation.Taking porcine islet cells expressing hCD55 as donors may be beneficial to reduce the occurrence of IBMIR.Based on the GGTA1 gene knockout(GTKO)and GTKO/hCD55 Bama minipigs prepared by the research group in the previous stage,this study aims to explore the effect of hCD55 on inhibiting complement activation and reducing IBMIR in islet xenotransplantation.Methods: 1.GTKO and GTKO/hCD55 porcine islet cells were isolated by the modified Ricordi digestion method,Dithizone(DTZ)staining was used to assess the islet cell purity,and the viability of islet cells was detected by fluorescein diacetate(FDA)/propidium iodide(PI)staining.2.To identify the function of isolated islet cells,the insulin secretion function was measured by stimulating islet cells with(high/low)glucose in vitro,porcine islet xenotransplantation was performed in T1 DM severe combined immune deficiency(SCID)mice in vivo to test its blood glucose correction effect on T1 DM SCID mice.3.To verify the expression of hCD55 on GTKO/hCD55 porcine islet cells,the nucleic acid level was identified by polymerase chain reaction(PCR)and reverse transcription PCR(RT-PCR),the protein level was identified by flow cytometry and tissue immunofluorescence.4.The effect of hCD55 on inhibiting complement activation and reducing cytotoxicity was studied in vitro by complement dependent cytotoxicity test(CDC)and complement deposition test.5.Streptozotocin was used to model T1 DM in rhesus monkeys,GTKO and GTKO/hCD55 porcine islet cells were transplanted through the hepatic portal vein,and then the release of specific porcine C-peptide at 15 minutes,30 minutes and 4 hours after transplantation of the two genotypes of islet cells was compared,and the effect of hCD55 on reducing IBMIR was evaluated in vivo.Results: 1.DTZ staining of GTKO porcine islet cells showed that the purity was83.00±6.71%,FDA/PI staining showed that the viability was 86.25±2.50%,the content of insulin secreted by high glucose stimulation was 2.92±0.32 times than that after low glucose stimulation,and four of the five transplanted T1 DM SCID mice returned to normoglycemia.2.DTZ staining of GTKO/hCD55 porcine islet cells showed the purity was 81.67±7.64%,FDA/PI staining showed that the viability was 87.50±3.54%,and the content of insulin secreted by high glucose stimulation was 2.01±0.13 times as low glucose stimulation,and 80% of the five transplanted T1 DM SCID mice returned to normoglycemia.3.RT-PCR result showed that GTKO/hCD55 porcine islet cells expressed hCD55 m RNA,flow cytometry and tissue immunofluorescence showed that hCD55 protein was expressed on GTKO/hCD55 porcine islet cells.4.CDC results showed that compared with GTKO porcine islet cells,GTKO/hCD55 porcine islet cells significantly reduced cytotoxicity(P<0.05).5.Complement deposition test results showed that compared with GTKO porcine islet cells,the deposition of human C3 c complement and human C5b-9 complement on GTKO/hCD55 porcine islet cells were significantly decreased(P<0.01,P<0.0001,respectively).6.In the pig to T1 DM rhesus monkey islet xenotransplantation experiment,compared to the transplantation of GTKO pig islet cells,the specific pig C-peptide released by the recipients transplanted with GTKO/hCD55 pig islet cells at 15 min,30 min,and 4 h after transplantation were reduced.Conclusion: hCD55 is expressed on GTKO/hCD55 porcine islet cells.hCD55 plays an effective role in inhibiting complement activation and reducing the damage of human complement to porcine islet cells and tends to alleviate the occurrence of IBMIR,which has certain significance for the selection of donor genotypes and functional evaluation in preclinical islet xenotransplantation.