The Experimental Study Of Cold Ischemia Injury On Graft In Pancreas Transplantation

Objective 1.To establish a stable model of miniature swine pancreas transplantation; 2.To observe the effects of the function and blood perfusion of pancreas graft with the different period of cold ischemia; 3.To investigate the relationship of the pancreas graft cell apoptosis and the cold ischemia injury and the effect of the expression of Bcl-2 and Bax protein.Method1. To establish the minature swine model of whole pancreaticoduodenal transplantation: a total of 30 miniature swines were divided into two groups (donors and recipients ) randomly. Cooling of the grafts was accomplished with flush in situ with 4℃UW preservation solution via a aortic cannula. Animal model was established with pancreas transplantation. The total pancreas with a short segment of duodenum was transplanted to a recipient by anastomosis of vessels to the recipient abdominal aorta and vena and small bowel segment to the jejunum with enteric drainage. During recipient operation, mean arterial pressure(MAP) and central venous pressure(CVP) were monitored. Liquid supplement, anti-coagulation and anti-infection were conducted postoperatively. Meanwhile, the recipients were observed closely in postoperative period.2. The experimental study of cold ischemia injury on pancreas graft: a total of 40 miniature swine were divided into donors and recipients randomly and animal model was established . According to the time of cold ischemia injury ,recipients were divided into four groups: cold ischemia time 0h, cold ischemia time 3h ,cold ischemia time 6h ,cold ischemia time 9h.TypeⅠdiabetes mellitus model was established by resecting pancreas of the recipient. The blood perfusion was detected and the pancreas graft tissue was taked at 30 minutes after pancreas transplantation. The tissue was divided 3 parts which was used for electron microscope observation, detecting cell apoptosis (TUNEL) and the expression of Bcl-2 and Bax protein respectively. In addition, the blood was taked consecutively for measuring the level of blood glucose, serum amylase and insulin in serum.Results11.Of the 15 miniature swine recipients studied, there were 14 cases survived exceeding 1 day. The average operation time of donor and recipient was 69.3 and 75.5 minutes. One recipient died at 2 hour after operation because of circulatory failure due to too much blood loss during operation, another died at 3 day after operation because of anastomotic stoma fistula due to infection.2.The blood glucose started to lower at 2 hour after reperfusion and stabilized at 24 hour after reperfusion. The level of the blood glucose and serum amylase, artery resistance index (RI) of the transplanted pancreas at 2h and 8h after operation in the group of CIT 9h was higher than the rest three groups (P<0.05).The level of the blood glucose and serum amylase,artery resistance index at 2h and 8h after operation in the group of CIT 3h, 6h was higher than the group of CIT 0h (P<0.05).But it hasn't significant difference between the group of CIT 3h and 6h (P>0.05). The level of the artery maximum velocity(Vmax) of the transplanted pancreas in the group of CIT9h was lower than the rest three groups(P<0.05), while at 2h and 8h after operation in the group of CIT 3h, 6h was lower than the group of CIT 0h (P<0.05). But it hasn't significant difference between the group of CIT 3h and 6h(P>0.05).3.At 0.5h after reperfusion apoptosis cells were found in the pancreas graft tissue in each group and the number of apoptosis cells was more along with prolonging of cold storage time. Apoptosis index has significant difference among each group(P<0.05). The expression of bcl-2 protein and bcl-2/bax ratio were the highest in the group of CIT 0h(P<0.05), while the expression of bcl-2 protein and bcl-2/bax ratio in the group of CIT 3h , 6h were higher than the group of CIT 9h (P<0.05),but it hasn't significant difference between the group of CIT 3h and 6h(P>0.05). The expression of bax protein was the lowest in the group of CIT 0h, while the expression of bax protein in the group of CIT 3h , 6h was lower than the group of CIT 9h (P<0.05),but it hasn't significant difference between the group of CIT 3h and 6h(P>0.05).Conclusion1. The animal model of pancreas transplantation is easy, stable and reliable by anastomosis of vessels to the recipient abdominal aorta and vena cava and small bowel segment to the jejunum with enteric drainage in miniature swine, so it can be a ideal model for studying ischemia reperfusion injury of pancreas graft.2. Cold ischemia injury is one of the important reasons of pancreas graft functional defect after transplantation.3. Apopotsis as a kind of cell death form participates the process of pancreas cold ischemia injury and the degree of cell apoptosis aggravates along with prolonging of cold ischemia time.4. Bcl-2 participates the process of pancreas ischemia injury and the mechanism might be that the expression of Bcl-2 protein inhibit apoptosis , the expression of Bax protein induce apoptosis , while Bcl-2/Bax ratio has a negative correlation with apoptosis.