Experimental Study On Preventing Rejection In Rats Pancreaticoduodenal Transplantation Using ImDC Treated With NF-κB Decoy Oligodeoxynucleotides

Part 1:The establishment of pancreaticoduodenal transplantation model in ratsObjective To establishment of pancreaticoduodenal transplantation model in ratsMethods SD rats were used as experimental animals.The donor pancreaticodudodenal is excised with en bloc technique while a fine needle with a infusion was introduced into the aorta distal and perfused continiously until bloodless with 7-8 ml of cold(4℃)saline,then the graft including stomach and part colon was clipped.The portal venous of the graft was anastomosed with the recipent's left renal vein by a cuff technique.End to side anastomosis was performed for artery employing 8-0 nylon sutures, a side to side anastonosis was made between the graft duodenum and the host jejunum.Results The 24h survival rate was 87.7%after allotransplantation of pancreaticoduodenal.The donor harvesting time was 40±3min,and warm ischemia time was 2±1min;The recipient's operation time was 55±4min,and the cold ischemia time was 50±2min.Conclusions This modal can be used in experimental study on physiological and immunological change after pancreas transplantation.Part 2:Bone marrow(BM)-derived DCs modified with NF-κB decoy oligodeoxynucleotidesObjective To obtain Lewis rats bone marrow-derived modified DCs by using NF-κB decoy oligodeoxynucleotidesMethods DCs were propagated from Lewis rats bone marrow with GM-CSFand IL4; their maturation was arrested by treatment with ligodeoxyribonucleotides(ODN) specifically against nuclear factor NF-κB;and the modified DC were stimulated by LPS. DC phenotype(MHC-Ⅱ,CD80,CD86) was examined by flow cytometry.Results Compared with mature DC,NF-κB decoy ODN treated DCs exhibited features of immature DCs,with low level of surface molecule and costimulatory molecule (MHC-Ⅱ,CD80,CD86) expression.After being stimulated by LPS,the NF-κB decoy ODN treated DCs still had the features of immature DCs.Conclusions The modified DCs,with NF-κB decoy ODN treated,exhibite features of immature DCs steadly.Part 3:Transfection of NF-κB decoy oligodeoxynucleotides using ultrasound-mediated gene transfer into donor PancreaseObjective To obtain the optimization of ultrasound-SonoVue-mediated gene transfection procedure into pancrease tissue.Methods SD rats were used as experimental animals.5OD NF-κB-decoy ODN(FITC labeled) was dissolved in 0.1ml saline containing an echocardiographic contrast agent, SonoVue,at three different concentrations(0%,10%,or 25%each concentration),and infused into the pancreas through the portal vein.The pancreas was removed and exposed to ultrasound using an ultrasound transducer(Misonix XL2020) at 2 MHz carrier frequency in a water bath;signal intensity- was 2.5 W/cm2 and the exposure duration was varied from 0.5 to 2min.The pancreas transfected with NF-κB decoy ODN(FITC labeled) was then transplanted.The grafts were harvested on day 2,and luciferase activity was evaluated by luciferase assa.Amylase in recipients serum were detected.Results Ultrasound application significantly enhanced transfection of FITC labeled NF-κB decoy ODN in proportion to its duration up to 1 min.2 min of ultrasound exposure caused marked tissue injury(Amylase enhanced significantly).The addition of SonoVue significantly enhanced NF-κB decoy ODN transfection when SonoVue was added at a concentration of 10%or 25%.SonoVue caused marked tissue injury at 25%(Amylase enhanced significantly)Conclusions Ultrasound(2MHz) with 1min exposure and SonoVue at a concentration of 10%,efficiently transfects NF-κB decoy ODN into pancreas tissue without marked tissue injury.Administration of recipient BM-derived DCs treated with NF-κB decoy oligodeoxynucleotides,transfection of NF-κB decoy oligodeoxynucleotides into donor pancreas by applying ultrasound-SonoVue mediator prevents acute rejection in rats pancreas transplantation.Part 4 Experimental study on preventing rejection in rats pancreaticoduodenal transplantation using imDC treated with NF-κB decoy oligodeoxynucleotidesObjective To investigate the mechanism and effect of recipient bone marrow derived DCs treated with NF-κB-decoy oligodeoxynucleotides,transfection of NF-κB decoy oligodeoxynucleotides into donor pancreas by applying ultrasound-SonoVue mediator and combination of two approaches on preventing acute rejection in rats pancreas transplantationMethods DA and Lewis rats served as donor and recipient,respectively.Pancreas transplantations in rats were performed.Recipients were divided into 4 groups according to different treatments.Group1(n=12):no treatment and as controls;Group2(n=12):treated with recipient BM-derived DCs treated with NF-κB-decoy oligodeoxynucleotides; Group3(n=12):treated with transfection of NF-κB decoy oligodeoxynucleotides into donor pancreas by applying ultrasound-SonoVue mediator;Group4(n=12):treated with combination of the two approaches.The graft pancreas functions were detected and the concentrations of IFN-γ,IL-4,IL-10 in recipients serum were measured by ELISA on day 1,4,7,I0 after pancreas transplantation respectively.Acute rejection in graft tissue was investigated by pathological detection on day 4 after pancreas transplantation.The mRNA expression of preceding cytokins in grafts were determined with RT-PCR,and apoptosis in pancreas,peripheral lymphocytes were evaluated by TUNEL method.Results Survival time was prolonged in Group2,Group3 and Group4,and Group4 has the most prolongation.Graft pancreas functions in Group3 and Group4 were better than those in Group1 and Group2.Serum concentrations and the mRNA expression in grafts of IFN-γ,IL-4,IL-10 all increased in Group1(controls),but the mRNA expression in grafts of IL-4,IL-10 increased in Group2,Group3 and Group4.The acute rejection of gtaft tissue in Group4 was lighter than it in Group1.The lympocytes apoptosis was observed in the graft and spleen in Group2 and Group4.Conclusions Administration of recipient BM-derived DCs treated with NF-κB decoy oligodeoxynucleotides,transfection of NF-κB decoy oligodeoxynucleotides into donor pancreas by applying ultrasound-SonoVue mediator prevents acute rejection in rats pancreas transplantation.Combination of the two approaches can obviously enhance the effect of preventing acute rejection on pancreas transplantation in rats;the mechanism of which might be related to preventing ischemia-reperfusion injury and the apoptosis of lymphocytes in grafts.