猪胰肾联合移植模型的建立及门静脉回流肠道引流和体静脉回流肠道引流术式对比的实验研究
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摘要
第一部分:猪胰肾联合移植模型的建立
[目的]建立猪胰肾联合移植内分泌门脉回流外分泌肠道引流(portal-emeric drainage,PE)和内分泌体静脉回流外分泌肠道引流(systemic-enteric drainage,SE)术式的模型。
[方法]四川当地杂交第一代长白猪48例,随机分成PE和SE组,每组24例。各组根据血型相容的原理进行配对,分别分成供受体。整块切取供体胰十二指肠及右肾,保留带腹腔动脉、肠系膜前动脉(相当于人肠系膜上动脉)和右肾动脉的腹主动脉段。手术切除受体胰腺制成Ⅰ型糖尿病模型,并完整切除受体右肾以备供体肾原位植入,保留左肾。PE组采用供体的门静脉和受体的肠系膜前静脉(相当于人肠系膜上静脉)或门静脉行端侧吻合,SE组将供体的门静脉和受体的肝后后腔静脉(相当于人肝下下腔静脉)行端侧吻合。供体的腹主动脉段与受体的腹主动脉段端侧吻合;右肾静脉和受体的右肾静脉端端吻合;移植胰腺胰管和移植肾脏输尿管分别置引流管,将胰液和尿液引出腹腔。供体十二指肠和受体空肠行端侧吻合。于术前1d、切胰后30min、关腹前、术后1～7d
Introduction: Simultanous pancreas-kidney (SPK) transplantation is regarded as definitive therapy for patients with type I diabetes and renal failure. But now, there is no definite surgical procedure .The controversy of the procedure lies on the difference of the endocrine of the graft pancreas. Compared with Systemic venous drainage (SVD), portal venous drainage(PVD) accords with physiology and can avoid hyperinsulinemia and hyperglucagonemia. The blood flow of the graft passes across the liver in PVD. Whether PVD has an immunologic advantage and what it's mechanism may be have remained unclear. Through the comparative research of PVD versus SVD, the immunologic benefits of PVD will be further confirmed and the impact of liver on the induce tolerance of PVD-SPK and mechanism of them can be delineated.Study 1 Establishment of the model of simultaneous pancreas-kidney transplantation in pigs
Objectives To establish the model of simultaneous pancreas-kidney transplantation with portal-enteric drainage or systemic drainage in pigs. Methods 48 first hybrid landraces were divided into PE group and SE group randomly and the donors and the recipients were matched according to the principle of blood type compatibility in each group. The grafts including duodenum, pancreas and right kidney were harvested en bloc and the abdominal aorta segment including the coeliac artery, anterior mesenteric artery (equal to the superior mesenteric artery in human) and right renal artery were keep intact. The graft pancreatic duct was catheterized for draining the pancreatic fluid. The grafts were perfused in situ with ice-cold Ringer's lactate. IDDM was induced surgically by total pancreatectomy. The right-sided nephrectomy was performed in all recipients in order to implant the graft kidney orthotopically and the left kidney was keep intact. By end-to-side fashion,the portal vein of the graft was anastomosed on the anterior mesenteric vein (equal to the superior mesenteric vein in human) or the portal vein of the recipicent in PE group, the portal vein on the posterior hepatic vena cava(equal to the inferior hepatic vena cava in human) in SE group and the abdominal aorta segmental of the graft was anastomosed on the abdominal aorta segmental of the recipient. By end-to-end fashion , the right renal vein was anastomosed to the same one in the recipient. The graft ureter was catheterized and the drainage tube was drained out of the peritoneal cavity. The drainage tube of the graft pancreatic duct was also drained out of the peritoneal cavity. By end-to-side fashion, the graft duodenum was anastomosed on the jejunum of the recipient. The level of fasting plasma glucose, insulin and glucagon was monitored on 1 day before the operation
and from 1st day to 7th day after transplantation. The volume of the graft pancreatic fluid and the urine creatinine of the graft renal were measured from the 1st day to the 7th day after transplantation in order to monitor the functions of the grafts.Results 12 cases of simultaneous pancreas-kidney transplantation with PE or SE were performed respectively. 1 case died from anesthetic accident in each group respectively. Phlebothrombosis occurred on one pig in PE group. 1 case died from postbiopsy on day 5 after transplantation. Arterial thrombus occurred in 1 case in SE group on the 5th day after transplantation. There were no leak of intestinal anastomoses and infections of peritoneal cavity in the study. The pancreatic fluid could be seen in 10-60minutes after reperfusion of blood flow of the grafts and theurine could be seen in 5-30minutes.The normal level of fasting plasma glucose was recovered on the first day after transpantation. Pigs with SE had a fasting hyperglucagonemia and hyperinsulinemia compared with pigs before transplantation and pigs with PE. They were normal in pigs with PE. We didn't find significant changes in the level of fasting plasma glucose, insulin, glucagons and the volume of pancreatic fluid and urine of the grafts within each group from the 1st day to the 7th day after surgery. With the exception of level of insulin and glucagons, there were no differences in the others indexes between two groups.Conclusions The model of simultaneous pancreas kidney transplantationin pigs was established successfully and was stable and it can be applied to the further study. The primary experience showed that the two groups were comparable in technique. The systemic drainage of the endocrine is
associated to hyperinsulinemia and hyperglucagonemia. Study 2 The influence of differential vein drainage of simultaneouspancreas kidney transplantation on acute rejection in pigs Objectives To investigate the influence of systemic venous drainage and portal venous drainage of the endocrine on acute rejection of simultaneous pancreas kidney transplantation in pigs.Methods 58 first hybrid landraces were divided into three groups randomly, 12 pigs in group I (control group), 24 pigs in group II (group SE) and group III (group PE) respectively and they were match according to the method in dissertation 1. Pigs in the group 1 underwent laparotomy and the pancreas and the right kidney were isolated and the pancreatic duct was catheterized to drain the pancreatic fluid. Pigs in the group II and group III underwent the procedure as the dissertation 1 to establish the PE and SE model. The blood sample was collected to monitor fasting plasma glucose from day 1 to day 7 and insulin , glucagons, amylase and the volume of graft renal urine and the pancreatic fluid and the urine creatinine were measured on day 1 before transplantation and dayl, day3, day5 and day 7 after transplantation. The grafts tissue biopsies obtained by surgery for pathological studies on day 3 and day 7 after transplantation.Results 1. There was no difference of statistic in fasting plasma glucose and the volume of the graft pancreatic fluid and the urine creatinine after operation in three groups (P>0.05). 2.The level of insulin and glucose in
group SE was higher than group control and group PE (P < 0.05) . There was no difference of statistics between group control and group PE (P> 0.05). 3. Compared with group PE, the incidence and the density of acute rejection in group SE were more (P < 0.05).Conclusions The technique and the incidence of the surgical complications are comparable between PE-SPK and SE-SPK. Portal drainage in SPK can reduce the incidence of acute rejection and the density of acute rejection. Study 3 The investigation of the possible mechanism and the role of liver in the induction of tolerance in simultaneous pancreas kidney transplantationObjective To investigate the possible mechanism and the role of liver in the induction of tolerance in simultaneous pancreas kidney transplantation. Methods 58 first hybrid landraces were divided into three groups randomly as the dissertation 2. The models were established as study 1. The tissues of the graft pancreas and kidney were gained by surgery for pathologic studies and the expression of mRNA and protein of local IFN- Y and IL4 was examined by RT-PCR and ELISA on day 3, 7 after transplantation. Results Compared with pigs with SE , the incidence of acute rejection and the density of rejection were all less in pigs with PE(P < 0.05). The local expression of mRNA and protein of IFN in both group SE and group PE was significantly up-regulated.The local expression of mRNA and protein of IL-4 was significantly up-regulated in PE group and down-regulated, even no
expression in group SE.Conclusions Liver plays a key role in the induction of tolerance of PE-SPK. The induction of tolerance may be associated with the mechanism in which liver modulates a Thl to Th2 immune shift.
[目的]建立猪胰肾联合移植内分泌门脉回流外分泌肠道引流(portal-emeric drainage,PE)和内分泌体静脉回流外分泌肠道引流(systemic-enteric drainage,SE)术式的模型。
[方法]四川当地杂交第一代长白猪48例,随机分成PE和SE组,每组24例。各组根据血型相容的原理进行配对,分别分成供受体。整块切取供体胰十二指肠及右肾,保留带腹腔动脉、肠系膜前动脉(相当于人肠系膜上动脉)和右肾动脉的腹主动脉段。手术切除受体胰腺制成Ⅰ型糖尿病模型,并完整切除受体右肾以备供体肾原位植入,保留左肾。PE组采用供体的门静脉和受体的肠系膜前静脉(相当于人肠系膜上静脉)或门静脉行端侧吻合,SE组将供体的门静脉和受体的肝后后腔静脉(相当于人肝下下腔静脉)行端侧吻合。供体的腹主动脉段与受体的腹主动脉段端侧吻合;右肾静脉和受体的右肾静脉端端吻合;移植胰腺胰管和移植肾脏输尿管分别置引流管,将胰液和尿液引出腹腔。供体十二指肠和受体空肠行端侧吻合。于术前1d、切胰后30min、关腹前、术后1～7d
Introduction: Simultanous pancreas-kidney (SPK) transplantation is regarded as definitive therapy for patients with type I diabetes and renal failure. But now, there is no definite surgical procedure .The controversy of the procedure lies on the difference of the endocrine of the graft pancreas. Compared with Systemic venous drainage (SVD), portal venous drainage(PVD) accords with physiology and can avoid hyperinsulinemia and hyperglucagonemia. The blood flow of the graft passes across the liver in PVD. Whether PVD has an immunologic advantage and what it's mechanism may be have remained unclear. Through the comparative research of PVD versus SVD, the immunologic benefits of PVD will be further confirmed and the impact of liver on the induce tolerance of PVD-SPK and mechanism of them can be delineated.Study 1 Establishment of the model of simultaneous pancreas-kidney transplantation in pigs
Objectives To establish the model of simultaneous pancreas-kidney transplantation with portal-enteric drainage or systemic drainage in pigs. Methods 48 first hybrid landraces were divided into PE group and SE group randomly and the donors and the recipients were matched according to the principle of blood type compatibility in each group. The grafts including duodenum, pancreas and right kidney were harvested en bloc and the abdominal aorta segment including the coeliac artery, anterior mesenteric artery (equal to the superior mesenteric artery in human) and right renal artery were keep intact. The graft pancreatic duct was catheterized for draining the pancreatic fluid. The grafts were perfused in situ with ice-cold Ringer's lactate. IDDM was induced surgically by total pancreatectomy. The right-sided nephrectomy was performed in all recipients in order to implant the graft kidney orthotopically and the left kidney was keep intact. By end-to-side fashion,the portal vein of the graft was anastomosed on the anterior mesenteric vein (equal to the superior mesenteric vein in human) or the portal vein of the recipicent in PE group, the portal vein on the posterior hepatic vena cava(equal to the inferior hepatic vena cava in human) in SE group and the abdominal aorta segmental of the graft was anastomosed on the abdominal aorta segmental of the recipient. By end-to-end fashion , the right renal vein was anastomosed to the same one in the recipient. The graft ureter was catheterized and the drainage tube was drained out of the peritoneal cavity. The drainage tube of the graft pancreatic duct was also drained out of the peritoneal cavity. By end-to-side fashion, the graft duodenum was anastomosed on the jejunum of the recipient. The level of fasting plasma glucose, insulin and glucagon was monitored on 1 day before the operation
and from 1st day to 7th day after transplantation. The volume of the graft pancreatic fluid and the urine creatinine of the graft renal were measured from the 1st day to the 7th day after transplantation in order to monitor the functions of the grafts.Results 12 cases of simultaneous pancreas-kidney transplantation with PE or SE were performed respectively. 1 case died from anesthetic accident in each group respectively. Phlebothrombosis occurred on one pig in PE group. 1 case died from postbiopsy on day 5 after transplantation. Arterial thrombus occurred in 1 case in SE group on the 5th day after transplantation. There were no leak of intestinal anastomoses and infections of peritoneal cavity in the study. The pancreatic fluid could be seen in 10-60minutes after reperfusion of blood flow of the grafts and theurine could be seen in 5-30minutes.The normal level of fasting plasma glucose was recovered on the first day after transpantation. Pigs with SE had a fasting hyperglucagonemia and hyperinsulinemia compared with pigs before transplantation and pigs with PE. They were normal in pigs with PE. We didn't find significant changes in the level of fasting plasma glucose, insulin, glucagons and the volume of pancreatic fluid and urine of the grafts within each group from the 1st day to the 7th day after surgery. With the exception of level of insulin and glucagons, there were no differences in the others indexes between two groups.Conclusions The model of simultaneous pancreas kidney transplantationin pigs was established successfully and was stable and it can be applied to the further study. The primary experience showed that the two groups were comparable in technique. The systemic drainage of the endocrine is
associated to hyperinsulinemia and hyperglucagonemia. Study 2 The influence of differential vein drainage of simultaneouspancreas kidney transplantation on acute rejection in pigs Objectives To investigate the influence of systemic venous drainage and portal venous drainage of the endocrine on acute rejection of simultaneous pancreas kidney transplantation in pigs.Methods 58 first hybrid landraces were divided into three groups randomly, 12 pigs in group I (control group), 24 pigs in group II (group SE) and group III (group PE) respectively and they were match according to the method in dissertation 1. Pigs in the group 1 underwent laparotomy and the pancreas and the right kidney were isolated and the pancreatic duct was catheterized to drain the pancreatic fluid. Pigs in the group II and group III underwent the procedure as the dissertation 1 to establish the PE and SE model. The blood sample was collected to monitor fasting plasma glucose from day 1 to day 7 and insulin , glucagons, amylase and the volume of graft renal urine and the pancreatic fluid and the urine creatinine were measured on day 1 before transplantation and dayl, day3, day5 and day 7 after transplantation. The grafts tissue biopsies obtained by surgery for pathological studies on day 3 and day 7 after transplantation.Results 1. There was no difference of statistic in fasting plasma glucose and the volume of the graft pancreatic fluid and the urine creatinine after operation in three groups (P>0.05). 2.The level of insulin and glucose in
group SE was higher than group control and group PE (P < 0.05) . There was no difference of statistics between group control and group PE (P> 0.05). 3. Compared with group PE, the incidence and the density of acute rejection in group SE were more (P < 0.05).Conclusions The technique and the incidence of the surgical complications are comparable between PE-SPK and SE-SPK. Portal drainage in SPK can reduce the incidence of acute rejection and the density of acute rejection. Study 3 The investigation of the possible mechanism and the role of liver in the induction of tolerance in simultaneous pancreas kidney transplantationObjective To investigate the possible mechanism and the role of liver in the induction of tolerance in simultaneous pancreas kidney transplantation. Methods 58 first hybrid landraces were divided into three groups randomly as the dissertation 2. The models were established as study 1. The tissues of the graft pancreas and kidney were gained by surgery for pathologic studies and the expression of mRNA and protein of local IFN- Y and IL4 was examined by RT-PCR and ELISA on day 3, 7 after transplantation. Results Compared with pigs with SE , the incidence of acute rejection and the density of rejection were all less in pigs with PE(P < 0.05). The local expression of mRNA and protein of IFN in both group SE and group PE was significantly up-regulated.The local expression of mRNA and protein of IL-4 was significantly up-regulated in PE group and down-regulated, even no
expression in group SE.Conclusions Liver plays a key role in the induction of tolerance of PE-SPK. The induction of tolerance may be associated with the mechanism in which liver modulates a Thl to Th2 immune shift.
引文
1. Robert J, Stratta M, Shokouh-Amiri HM. et al. A prospective comparision of simultaneous kidney-pancreas transplantation with systemic-enteric versus portal-enteric drainage. Ann Surg, 2001; 233(6): 740-751
2. American Diabetes Association. Pancreas transplantation for pantients with type 1 diabetes. Diabetes Care, 2000; 23(1): 117
3. Ojo AO, Meier-Kriesche Hu, Harson JA, et al. The impact of simultaneous pancreas-kidney transplantation on large-term patient survival. Transplantation, 2001(1): 82-89
4. Gruessner AC, Sutherland DE. Pancreas transplant outcomes for United States (US) and non-US cases as reported to the International Pancreas Transplant Registry (IPTR) as of May 2003. Clin Transpl 2003;:21-51
5. Stegall MD, Kim DY, Prieto M, et al. Thymoglobulin induction decreases rejection in solitary pancreas transplantation. Transplantation, 2001; 72(10): 1671-1675
6. Humar A, Ramcharan T, Kandaswamy R, et al. Technical failures after pancreas transplants: Why grafts fail and the risk factors- A multivariate analysis. Transplantation, 2004; 78(8): 1188-1192
7. Shokouh-Amiri MH, Rahimi-Saber S, Andersen HO. Segrnental pancreatic autotransplantation in the pig. Transplantation, 1989; 47(1): 42-44
8. Anthony Dorling, Kristian Riesbeck, Anthony Warrens, et al. Clinical xenotransplantation of solid organs. Lancet, 1997,349;867-71.
9.罗开,黎介寿,李为苏,等.异体猪全胰十二指肠移植实验研究.中华器官移植杂志,1997;18(4):196-197
10. Gruessner RW, Nakhleh R, Schechner J, et al. Differences in rejection grading after simultaneous pancreas and kidney transplantation in pigs. Transplantation, 1994; 57(7): 1021-1028
11.韩方海,张肇达,董高宏等.猪全胰十二指肠移植空肠引流及门静脉回流模型的建立.四川大学学报(医学版),2003:34(3):527—529
12.田志华.猪血型遗传多样及其在育种中的应用.西南民族学院学报(自 然科学版),1998;24(1):67-71
13.唐勇,张肇达,魏江涛,等.猪全胰十二指肠移植模型的改进.四川大学学报(医学版),2004;35(6):863-866
14. Gruessner AC, Sutherland DER. Pancreas transplants for the united states US and non-US cases as reported to the International Pancreas Transplant Registry (IPTR) and to United Network for Organ Sharing (UNOS) In: Terasaki PI, Cecka JM, eds. Clinical Tranaplants 1997. Los Angels, CA;UCLA Tissue Typing Laboratory; 1997
15. 15. Allen RD, Wilson TG, Grierson JM, et al. Percutaneous biopsy of bladder-drained pancreas transplants. Transplantation, 1991; 51:1213-1216
16. Sollinger HW, Odorico JS, Knechetle SJ, et al. Experience with 500 simultaneous pancreas-kidney transplants. Ann Surg, 1998; 228:284-296
17. White SA, Kinber R, Vertch PS, et al. Surgical treatment of diabetes mellitus by islet cell and pancreas transplantation. BMJ, 2001; 77(908): 383-387
18. Stratta RJ, Sollinger HW, Perlman SB. Early detection of rejection in pancreas transplantatioin. Diabetes, 1989; 38:63-67
19. Macmillan N, Fernandel L, Ricart NJ, et al. Venous graft thrombosis clinical pancreas transplantation: Options for a rescue treatment. Transplant Proc, 1998; 30:425-428
20. Humar A, Johnson EM, Gillingh KJ, et al. Venous thromboembolic complications after kidney and kidney-pancreas transplantation: a multivariate analysis. Transplantation, 1998; 65(2): 229—234
2. American Diabetes Association. Pancreas transplantation for pantients with type 1 diabetes. Diabetes Care, 2000; 23(1): 117
3. Ojo AO, Meier-Kriesche Hu, Harson JA, et al. The impact of simultaneous pancreas-kidney transplantation on large-term patient survival. Transplantation, 2001(1): 82-89
4. Gruessner AC, Sutherland DE. Pancreas transplant outcomes for United States (US) and non-US cases as reported to the International Pancreas Transplant Registry (IPTR) as of May 2003. Clin Transpl 2003;:21-51
5. Stegall MD, Kim DY, Prieto M, et al. Thymoglobulin induction decreases rejection in solitary pancreas transplantation. Transplantation, 2001; 72(10): 1671-1675
6. Humar A, Ramcharan T, Kandaswamy R, et al. Technical failures after pancreas transplants: Why grafts fail and the risk factors- A multivariate analysis. Transplantation, 2004; 78(8): 1188-1192
7. Shokouh-Amiri MH, Rahimi-Saber S, Andersen HO. Segrnental pancreatic autotransplantation in the pig. Transplantation, 1989; 47(1): 42-44
8. Anthony Dorling, Kristian Riesbeck, Anthony Warrens, et al. Clinical xenotransplantation of solid organs. Lancet, 1997,349;867-71.
9.罗开,黎介寿,李为苏,等.异体猪全胰十二指肠移植实验研究.中华器官移植杂志,1997;18(4):196-197
10. Gruessner RW, Nakhleh R, Schechner J, et al. Differences in rejection grading after simultaneous pancreas and kidney transplantation in pigs. Transplantation, 1994; 57(7): 1021-1028
11.韩方海,张肇达,董高宏等.猪全胰十二指肠移植空肠引流及门静脉回流模型的建立.四川大学学报(医学版),2003:34(3):527—529
12.田志华.猪血型遗传多样及其在育种中的应用.西南民族学院学报(自 然科学版),1998;24(1):67-71
13.唐勇,张肇达,魏江涛,等.猪全胰十二指肠移植模型的改进.四川大学学报(医学版),2004;35(6):863-866
14. Gruessner AC, Sutherland DER. Pancreas transplants for the united states US and non-US cases as reported to the International Pancreas Transplant Registry (IPTR) and to United Network for Organ Sharing (UNOS) In: Terasaki PI, Cecka JM, eds. Clinical Tranaplants 1997. Los Angels, CA;UCLA Tissue Typing Laboratory; 1997
15. 15. Allen RD, Wilson TG, Grierson JM, et al. Percutaneous biopsy of bladder-drained pancreas transplants. Transplantation, 1991; 51:1213-1216
16. Sollinger HW, Odorico JS, Knechetle SJ, et al. Experience with 500 simultaneous pancreas-kidney transplants. Ann Surg, 1998; 228:284-296
17. White SA, Kinber R, Vertch PS, et al. Surgical treatment of diabetes mellitus by islet cell and pancreas transplantation. BMJ, 2001; 77(908): 383-387
18. Stratta RJ, Sollinger HW, Perlman SB. Early detection of rejection in pancreas transplantatioin. Diabetes, 1989; 38:63-67
19. Macmillan N, Fernandel L, Ricart NJ, et al. Venous graft thrombosis clinical pancreas transplantation: Options for a rescue treatment. Transplant Proc, 1998; 30:425-428
20. Humar A, Johnson EM, Gillingh KJ, et al. Venous thromboembolic complications after kidney and kidney-pancreas transplantation: a multivariate analysis. Transplantation, 1998; 65(2): 229—234