Heterotopic Segmental Intestinal Xenotransplantation From GTKO/hCD46 Transgenic Pig To Cynomolgus Monkey

The shortage of donor organs is the main factor restricting the development of organ transplantation.Pigs were considered the most likely to become organ donors for clinical application for their similar organ size,easy feeding and rapid reproduction.Alpha-1,3 galactose(a-gal)was a natural antigen expressing on the surface of porcine endothelial cells.Alpha-gal could be recognized by pre-existing antibodies in human beings.These antibodies would activate complement and lead to rapid endothelial cell injury,hemorrhage and thrombosis within minutes to hours after transplantation?This rejection was called hyperacute xenograft rejection(HAXR),which occurs during several minutes to twenty-four hours after organ reperfusion.In 2002,scientists succeeded in cultivating alpha-1,3 galactosyltransferase knockout(GTKO)transgenic pigs.Then,genetically modified with GTKO and hCD46 KI(human CD46 knock-in)pigs were also successfully cultivated.These pigs carry no a-gal antigen but express human complement regulatory protein(CD46),which can inhibit the activation of the complement system,so as to avoid the occurrence of HAXR.At present,there were no reports on intestinal xenotransplantation from GTKO/hCD46 transgenic pigs to cynomolgus monkeys.It was hypothesized that HAXR could be lighteded or even avoided when the intestine from GTKO/hCD46 transgenic pig was transplanted into cynomolgus monkey.In this study,established a heterotopic,segmental pit-to-cynomolgus monkey intestinal xenotransplantation model.The graft donors were hybrid pig and GTKO/hCD46 transgenic pig respectively.The vital signs,blood coagulation function,intestinal mucosal barrier function,changes of immunologic function and xenograft rejection was monitored.We aimed to find out whether small bowel from GTKO/hCD46 transgenic pig could lighten or escape from HAXR,and expound some mechanism of HAXR in intestinal xenotransplantation,as well as provide a theoretical biasis for clinical organ transplantation to find a new source of organ donors.Part ?Establishment of hybrid pig-to-cynomolgus monkey heterotopic segmental intestinal xenotransplantation modelObjectives:To investigate the procedures for establishing a stable model of intestinal xenotransplantation from hybrid pigs to cynomolgus monkeys,with the purpose of providing a good experimental tool for the xenograft rejection study in intestinal xenotransplantation.Methods:Hybrid pigs were used as organ donors and cynomolgus monkeys as recipients.During perioperative period,dynamic monitoring of blood pressure,ECG monitoring of vital signs and ventilator assisting breathing were administrated on recipients.The procedure of segmental heterotopic intestinal xenotransplantation in recipients was performed as following:the donor's anterior mesenteric artery was anastomosed end-to-side to the recipient's infrarenal abdominal aorta,and the donor's tributary of anterior mesenteric venous was anastomosed end-to-side to the recipient's infrarenal inferior vena cave.Subsequently,both the proximal and distant end of graft intestine was stayed ligated.The changes of vital signs of recipient and occurrence of rejection were recorded.Results:Heterotopic segmental intestinal xenotransplantation surgery were performed in 5 cynomolgus monkeys.The perioperative vital signs of these 5 recipients were stable.The average length of transplanted intestine was 52.0±5.7 centimetres.The success rate of vascular anastomosis was one hundred percent.The modified Park score of transplanted intestine 15 minutes after reperfusion was significantly higher than donor intestine before transplantation.Several hours after reperfusion blood supply dysfunction and hemorrhage of intestion and mesentery was observed.The intestinal xenograft survival was 152±71.6 minutes.Conclusions:A stable,repeatable model of heterotopic segmental intestinal xenotransplantation from pig to cynomolgus monkey was established.The graft appeared typical hyperacute xenograft rejection during several hours after transplantation.This model would provide an ideal tool for further research on intestinal xenotransplantation from transgenic pigs to cynomolgus monkeys.Part ?Comparison of xenograft rejection after heterotopic segmental pig-to-cynomolgus monkey intestinal xenotransplantationbetween GTKO/hCD46 transgenic pig donor and hybrid donor Objectives:To establish a heterotopic segmental intestinal xenotransplantation model from GTKO/hCD46 transgenic pig to cynomolgus monkey.Observe the differences of coagulation function,intestinal mucosal metabolism,xenograft rejection,and the survival time of intestine between transgenic donor group and hybrid donor group.Discover the advantage of GTKO/hCD46 transgenic pig over hybrid pig as intestine donor.Methods:The hybrid pig-to-cynomolgus monkey intestinal xenotransplantation model in Part ? was regarded as control group.While in the transgenic group,the donor was GTKO/hCD46 transgenic pig.The coagulation function of recipients was detected by both routine coagulation test and thromboelastography.The occurrence of xenograft rejection was evaluated by histopathological examination.The mitochondrial structure of intestinal mucosal epithelial cells was observed by transmission electron microscope.The intestinal energy metabolism was measured by high performance liquid chromatography(HPLC).Results:Heterotopic segmental intestinal xenotransplantation surgery were performed in 5 cynomolgus monkeys,with the graft intestine harvested from two GTKO/hCD46 transgenic pigs.Coagulopathy occurred after transplantation in both groups.However,conventional coagulation parameters and TEG results were much better in transgenic group than in control group.At 30 minutes and 120 minutes after reperfusion,modified Park score of intestine was significantly lower in transgenic group than that of the control group.At 30 minutes and 120 minutes after reperfusion,intestinal mucosal ATP and AMP contents were significantly higher in transgenic group than those in the control group.Intestinal xenograft survival analysis indicated that intestinal xenograft survival in the transgenic group was significantly higher than the control group.Conclusions:Coagulopathy occurred in both GTKO/hCD46 transgenic pig-to-cynomolgus monkey intestinal xenotransplantition model and hybrid pig-to-cynomolgus monkey model.The intestinal xenograft survival in the GTKO/hCD46 transgenic pig donor group was significantly higher than the hybrid pig donor group.However,HAXR was still not overcomed even if the intestine was harvested from GTKO/hCD46 transgenic pig.Part ?Study on the mechanisms of GTKO/hCD46 transgenic pig donor reducing hyperacute xenograft rejectionObjectives:The aim of this study was to observe the changes of humoral immunity on recipients in transgenic group,detect the relative content of intestinal epithelial tight junction proteins,and observe natural antibodies,complement and fibrinogen deposition in the intestinal graft.We also aimed to confirm that GTKO/hCD46 transgenic pig intestine can reduce hyperacute xenograft rejection,and tried to clarify its role.Methods:Based on the intestinal xenotransplantation models in Part I and Part II,the humoral immune and cell immune function of recipients in both groups was detected before and after transplantation.Western blot analysis was used to detect the tight junction proteins content.Intestinal permeability was determined by plasma D-lactate levels.Deposition of fibrinogen,immunoglobulin IgA and complement C3 was investigated by immunofluorescence microscopy.Results:At the time of 30 minutes after reperfusion,the percentage of neutrophils in recipients in transgenic group was significantly lower than that of control group.Serum IgM,C3,C4 concentration was significantly higher than that of control group.No obvious change trend of blood lymphocyte ratios in both groups.AT the time of 30minutes and 2 hours after reperfusion,the content of tight junction protein Occludin and ZO-1 expression were significantly higher than those in control group,and the plasma D-lactate content was significantly lower than that of the control group.IgA,C3 and Fib deposition in intestine graft in both groups.However,deposition in control group was more serious than the transgenic group.Conclusions:Although compared with the hybrid pig small intestine,GTKO/hCD46 transgenic pig small intestine had lightened HAXR,however,identification of xenogeneic antibodies was not completely inhibited,activation of complement system was not completely inhibited either.