The Establish Of Model Of Pancreas Transplantation In Rabbit And The Experimental Study Of Relations Of Th1/Th2 And Fas-mRNA In Acute Rejection

PartⅠ:The establish of model of partial pancreas transplantation and whole pancreas transplantation[Objective]To establish the model of partial pancreas transplantation(PPT) and whole pancreas transplantation(WPT) in rabbit,and to explore the feasibility and value of different surgical model of pancreas transplantation for experimental study.[Method]Japanese rabbit(JR)were selected for donors,and New Zealand White rabbits (NZR) for receptors.A total of 40 NZRs were induced by alloxan to establish the experimental diabetes model.Group B,the number of 14 NZRs underwent the PPT opertation(JR→NZR,as experimental group;Group C,the number of 13 NZRs underwent the WPT opertation(JR→NZR),and treated with cyclosporine,as a immunosuppression group;Group D,the number of 13 NZR were accepted the WPT operation(JR→NZR),as experimental group.The steps of PPT operation were the body and tail of pancreas,and the spleen were taken out in the donor rabbit by splenic artery perfusion in donor,the splenic vein was anastomosed with recipient's splenic vein while the splenic artery was anastomosed with recipient's splenic artery in recipient operation,the pancreatic juice was drainaged by tube in vitro.The steps of WPT operation were the whole pancreas were dissociation and taken out in donor,to identify the main pancreatic duct,to retain a length of aorta abdominalis,inclued the celiac artery and the superior mesenteric artery;to retain a length of superior mesenteric vein to portal vein,the far stump of abdominal aortic was enclosed.The portal vein was performed the end-to-side anastomosed with recipient's inferior vena cava,while the aorta abdominalis was anastomosed with recipient's aorta abdominalis,a tube was fixed into the pancreatic duct,and performed the purse string suture with bladder.The fasting plasma bloods were sampled via peripheral vein 1 day before the operation and on the 1^st,3^rd,5^th,7^thand10^th after the operation.All the samples were preservation to detect the level of blood glucose,insulin,and serum amylase.The situation of the pancreatic juice drainaged in PPT group was observed everyday after the operation.The urine samples were collected in each group on the 1^st,3^rd,5^th,7^thand 10^th after the operation,to detect the level of urine amylase.[Results]The criterion of successful operations was survival time of receptor animals was over three days.A total of 14 operations in PPT group,the successful rate of the receptor was 85.7%(12/14),the survival rate of first week was 83.3%(10/13).One case underwent the failure of anastomose.Three cases were dead in one week as for the formation of thrombus in splenic vein and infection of abdomino.There were 26 cases in WPT group,the successful rate of the receptor was 92.3%(24/26),two cases were dead within 2d,the reason was the formation of thrombus in arterial anastomotic stoma.The survival rate of first week was 80.8%(21/26),three cases were dead from 3d to 1 week,the following reasons were the formation of thrombus in arterial and venous anastomotic stoma,infection of abdomino,acute rejection reaction.After reperfusion,the secretion of pancreatic juice can be seen in drainage tube within 5-20 minutes.The level of fasting plasma blood glucose were unstable from 1d to 3d in postoperative.If the receptor animals can survive over 3d,the level of blood glucose were close to the normal range usually.From 5d to 10d,the level of blood glucose and insulin were maintaining stability.On the 1d after operation,the level of serum amylase rised in each group.It were gradually return to normal range from 7d to 10d. The level of urine amylase rised in group B on the 3d after operation,were also gradually return to normal range from 7d to 10d.The level of urine amylase significantly rised in group C and D,compared wtin group B.It started to decline on the 7d in group C,5d in group D,and also return to normal range on the 7d later.There were little differences of the level of blood glucose within groups.The level of blood glucose in group B were higher than group C and D.The level of insulin and urine amylase in group B were significantly lower than other two groups.[Conclusion](1) The model of partial pancreas transplantation(PPT) and whole pancreas transplantation(WPT) were established successfully in this experiment,and with a high successful rate of surgery.The endocrine and exocrine function of pancreas can gradually return to normal in successful surgerys.These models can be applied to the experimental research for pancreas transplantation in early phase.(2) There were significantly differences of the level of insulin between PPT and WPT groups.The possible reason for this represent were the venous return of insulin in PPT group was portal vein drainage(PVD);on the contrary,the WPT group was systemic venous drainage(SVD).(3) The different method of pancreatic exocrine drainage,to a certain degree,can supply a approch to observe the exocrine function of pancreas and to infer the survival of graft indirectly.(4) There were little changes of the level of blood glucose and insulin within each experimental groups.The level of urine amylase had a significant reduction in WPT group,probably due to acute rejection reaction. PartⅡ:The experimental study of relations of Th1/Th2 and Fas-mRNA in acute rejection.[Objective]To explore the relations between the dynamic changes of representation cytokines of Th1/Th2 in serum,the expression of Fas-mRNA in pancreatic tissue and the acute rejection reaction[Method]The number of 16 cases as the living donors were from the pre-test of PPT model (NZR→NZR),there were no death,as group A(normal control group);All the experimental groups were allograft,Japanese rabbits(JR) as a donor and New Zealand rabbits(NZR)as a receptor.Group B,included 14 cases of PPT surgerys(experimental group B);The total of 26 cases underwent the WPT surgerys,as allograft rejection group(JR→NZR).After operation,they were randomly divided into two groups;Group C,13 cases were treated with cyclosporine(immunosuppression Group);Group D,13 cases without any particular treatment(acute rejection,AR group).The bloods were sampled via peripheral vein 1 day before the operation and on the 1^st,3^rd,5^th,7^th and10^th after the operation.All the samples were preservation to detect the level of serum INF-γand IL-4 by ELISA assay.Four animals in group C and D were randomly selected to obtain the pancreatic tissue biopsies by surgery,one part of tissue treated with 10%neutral formalin to reserve,then HE staining,observed under the microscope;the other part used to decete Fas-mRNA expression by using RT-PCR method.[Results](1)In postoperative,the level of INF-γin control group was significantly lower than the experimental group in serum(P＜0.05).(2)In immunosuppression group,as a representative cytokines of Th1,the level of INF-γwas significantly lower than AR group(P＜0.05);As a representative cytokines of Th2,the level of IL-4 was stability,without significant difference within group(P＞0.05),but had significantly higher than the AR group(P＜0.05).(3) In AR group,the level of INF-γ,were significantly higher than immunosuppression group(P＜0.05).The level of IL-4 decreased on the 3d,and less than immunosuppression group(P＜0.05);On the 7d to10d,the level of IL-4 was significantly low or even can't be detected.(4) In two experimental groups,there were no expression of Fas-mRNA in pancreas tissu before surgery.On the 1d after operation,the expression of Fas-mRNA can be detected both in immunosuppression group and AR group;On the 3d,the expression of Fas-mRNA decreased in both groups,but the level of expression in AR group was significantly higher than immunosuppression group(P＜0.05).In AR group, the level of expression was consistent with the AR degree of pathology.(5) In AR group,the pathological changes was earlier than immunosuppression group,and at same time,the extent of rejection was more severe than the later group.[Conclusion](1) Using CsA,the level of INF-γhad a slow upward trend,and the level of IL-4 was stability basically(2)When acute rejection occured,the level of INF-γsignificantly increased,while the IL-4 had a low expression or even no expression;The significant changes of INF-γ/IL-4 were earlier than histopathological,and the extent of the INF-γ/IL-4 were consistent with the AR degree of pathology.(3) There were no expression of Fas-mRNA in normal tissues,the expression level of it in the AR group was higher than the immunesuppression group.(4) The time point of significantly expression of Fas-mRNA was earlier than the performance changes of histopathological.