摘要
目的
胰岛移植虽然发展前景良好,但仍面临着需要供胰量大,存活率较低,自身免疫性疾病复发所致移植物失功等问题。在临床经门静脉胰岛移植中,移植后大量胰岛丢失是主要的问题之一。在移植过程中,注入门静脉的胰岛与血液接触,发生一种凝血/炎症反应过程,我们称之为立即经血液介导的炎症反应(IBMIR)。IBMIR以凝血和补体系统的快速激活,胰岛内白细胞的募集和渗入,血小板的快速粘合和激活为特征。组织因子是IBMIR的主要启动因子。本实验旨在观察经胰岛—内皮细胞复合体的方法预处理的胰岛对IBMIR的影响,为临床胰岛移植胰岛预处理提供一种有效的新方法。
材料与方法
1、实验动物及分组
本实验采用雄性Wistar大鼠(中国医科大学实验动物中心提供),体重250～300g。实验分为三组:一组将分离、纯化的胰岛和大鼠主动脉内皮细胞混合培养,形成胰岛—内皮细胞复合体,称为实验组;一组将分离、纯化的胰岛直接置于CMRL1066培养液中培养,称为对照组;一组为PBS液,称为阴性对照组。
2、胰岛、内皮细胞的获取,胰岛—内皮细胞复合体的制备,胰岛功能的测定和模拟体内循环模型的建立
大鼠胰腺原位灌注消化液、不连续密度梯度离心法获得纯化胰岛。胶原酶消化贴片法获取大鼠主动脉内皮细胞,并传代培养。胰岛、内皮细胞混合培养5天,获得内皮细胞包被的胰岛,即胰岛—内皮细胞复合体。培养过程中进行葡萄糖刺激试验以测定胰岛素释放指数。实验组、对照组及PBS阴性对照组分别置于模拟体内循环模型的3个管中反应,分别于灌注前,5分钟、15分钟、30分钟、60分钟留取血浆进行分析。
3、观察指标
对不同时间阴性对照组、对照组和实验组血浆中的TAT、C3a、血小板的数量进行测量和统计。通过HE染色和免疫组化染色进行形态学观察。
4、统计学分析
将所得数据用均值士标准差表示,采取t检验和方差分析,P＜0.05认为有统计学意义,所有数据用SPSS11.5软件处理。
结果
实验组和对照组血浆中的TAT、C3a、血小板的数量在灌注后60分钟有明显差别(P＜0.05)。免疫组化染色提示胰岛—内皮细胞复合体组较未包被组中性粒细胞侵润明显减少。
结论
1、胰岛—内皮细胞复合体并不影响胰岛的功能。
2、胰岛—内皮细胞复合体能够显著减少胰岛移植初期的凝血激活、补体激活、血小板消耗和炎细胞浸润,从而显著减轻IBMIR的反应。
3、胰岛—内皮细胞复合体的方法有可能成为临床胰岛移植中胰岛预处理的一种新方法。
Preface
Although islet transplantation are good prospects for development,it is still faced with the need for pancreatic mass,a lower survival rate,recurrence of autoimmune diseases caused by graft failure and so on.In clinical islet transplantation through portal vein,the substantial islet loss after transplantation is one of the main problem.At transplantation,the portal vein injection of insulin and blood contact,the occurrence of a coagulation/inflammatory response process,we call the instant blood mediated inflammatory reaction.(IBMIR) IBMIR are characterized with coagulation and complement system activation,the collection and infiltration of leukocytes and platelets adhesion and activation.Tissue factor is the main initiation factor IBMIR.This experiment is to observe effect of composite islet-endothelial cell grafts to instant blood mediated inflammatory reaction in islet transplantation in rat.
Materials and Methods
Animal:SD rat of either sex with weigh ranging from 250-300g as the donor, provided by animal center of China medical university.Purified islets are obtain by perfusion of rat pancreatic digestive juice in situ and discontinuous density gradient centrifugation.The endothelial cells are obtained by collagenase digestion method from patch rat aortic,and cultured randomly divided into 8 groups Islet and endothelial cells co-cultured 5 days,accessing to islet coated endothelial cells,that is,composite islet-endothelial cell grafts.In the process of cultivate,we obtain insulin release index with glucose-stimulated test.Coated group,uncoated group and PBS control group were placed in simulated blood circulation system in the reaction tube 3,respectively, before reperfusion,5 minutes,15 minutes,30 minutes,60 minutes of collecting plasma for analysis.TAT,C3a and platelet are measured.Through HE staining and immunohistochemical staining we have a morphological observation.Statistical analysis:all the data expressed in meanlSD.The significant of difference was evaluated by t test or ANOVA.P＜0.05 was considered having statistical significance.
Results
The plasma TAT,C3a and the number of platelets in uncoated group and the composite islet-endothelial cell grafts group at 60 minutes after reperfusion have a significant difference(P＜0.05).The neutrophil filtration decreased significantly in uncoatedgroup than in composite islet-endothelial cell grafts group from immunohistochemical staining of pancreatic islet tips.
Conclusions
1.The composite islet-endothelial cell grafts does not effect the function of islet.
2.The composite islet-endothelial cell grafts can significantly reduce the initial islet transplantation activation of coagulation,complement activation,platelet consumption and inflammatory cell infiltration,which significantly reduced the response IBMIR.
3.The way of composite islet-endothelial cell grafts provides a new method for clinical pancreatic transplantation.
引文
1 Obesity and Diabetes in the Developing World - A Growing ChallengeParvez Hossain, M.D., Bisher Kawar, M.D., and Meguid El Nahas, M.D., Ph.D.
2 Lee Goldman M.D.,Dennis Ausiello M.D.,Cecil Textbook of Medicine ,23rd ,893.
3 Obesity and Diabetes in the Developing World - A Growing ChallengeParvez Hossain, M.D., Bisher Kawar, M.D., and Meguid El Nahas, M.D., Ph.D.
4 Puestow CB, Jillesby WJ, et al. Retrograde surgical drainage of pancres for chronic relapsing pancreatitis. Arch Surg 1958;76: 898-906.
5 Zollinger RM, Keith LM, Ellison EH. Pancreatitis. N Engl J Med 1954;251:497-502.
6 Hering B J, Ricordi C. Islet transplantation for patients with type 1 diabetes: Results, research priorities and reasons for optimism. Graft 1999; 2: 12-27.
7 Soria B, Roche E, Berna G, Leon-Quinto T, Reig JA, Martin F. Insulin-secreting cells derived from embryonic stem cells normalize glycemia in streptozocin-induced diabetic mice. Diabetes 2000; 49:157-162.
8 Bonner-Weir S, Sharma A. Pancreatic stem cells. J Pathol 2002; 197:519-526.
9 Jonathan RT, Lakey TJ, Anderson RV, et al. Novel approaches to cryopreservation of human pancreatic islets .Transplantation. 2001, 72 :1005-1011.
10 Lee TC, Barshes NR, Brunicardi FC,et al. Procurement of the human pancreas for pancreatic islet transplantation. Transplantation 2004; 78(3): 481-483.
11 Bennet W, Sundberg B, Groth CG et al. Incompatibility between human blood and isolated islets of Langerhans: a finding with implications for clinical intraportal islet transplantation? Diabetes 1999; 48(10): 1907-1914) (Sever CE, Demetris AJ, Zeng Y et al. Islet cell allotransplantation in diabetic patients. Histologic findings in four adults simultaneously receiving kidney or liver transplants. Am J Pathol 1992; 140(5): 1255-1260
12 Ups J Med Sci. 2005;110(1):17-55. The role of the innate immunity in islet transplantation.Moberg L.
13 Xenotransplantation. 2007 Jul;14(4):288-97 Rapid loss of intraportally transplanted islets: an overview of pathophysiology and preventive strategies.van der Windt DJ, Bottino R, Casu A, Campanile N, Cooper DK.
14 Diabetes. 2007 Aug;56(8):2008-15. Epub 2007 May 31. Islet surface heparinization prevents the instant blood-mediated inflammatory reaction in islet transplantation.Cabric S, Sanchez J, Lundgren T, Foss A, Felldin M, Kallen R, Salmela K, Tibell A, Tufveson G, Larsson R, Korsgren O, Nilsson B.
15 Am J Transplant. 2006 Feb;6(2):305-12. Am J Transplant. 2006 Jun;6(6):1500. Dufrane, D [added]; Gianello, P [added]. Low molecular weight dextran sulfate: a strong candidate drug to block IBMIR in clinical islet transplantation.Johansson H, Goto M, Dufrane D, Siegbahn A, Elgue G, Gianello P, Korsgren O, Nilsson B.
16 Transplantation. 2007 Aug 15;84(3):308-15. Interference with tissue factor prolongs intrahepatic islet allograft survival in a nonhuman primate marginal mass model.Berman DM, Cabrera O, Kenyon NM, Miller J, Tarn SH, Khandekar VS, Picha KM, Soderman AR, Jordan RE, Bugelski PJ, Horninger D, Lark M, Davis JE, Alejandro R, Berggren PO, Zimmerman M, O'Neil JJ, Ricordi C, Kenyon NS.
17 Transplantation. 2003 Nov 15;76(9): 1285-8. Nicotinamide inhibits tissue factor expression in isolated human pancreatic islets: implications for clinical islet transplantation.Moberg L, Olsson A, Berne C, Felldin M, Foss A, Kallen R, Salmela K, Tibell A, Tufveson G, Nilsson B, Korsgren O.
18 Diabetes. 2002 Jun;51(6): 1779-84. Inhibition of thrombin abrogates the instant blood-mediated inflammatory reaction triggered by isolated human islets: possible application of the thrombin inhibitor melagatran in clinical islet transplantation.Ozmen L, Ekdahl KN, Elgue G, Larsson R, Korsgren O, Nilsson B.
19 Evert de Jonge, Pascale E. P. Dekkers, Abla A. Creasey, C. Erik Hack, Susan K. Paulson, Aziz Karim, Jozef Kesecioglu, Marcel Levi, Sander J. H. van Deventer, and Tom van der Poll Blood, Vol. 95 No. 4 (February 15), 2000: pp. 1124-1129 FOCUS ON HEMATOLOGY
20 Drake TA, Morrissey JH, Edgington TS. Selective cellular expression of tissue factor in human tissues. Implications for disorders of hemostasis and thromb
21 osis. Am J Pathol 1989;134(5):1087-97.
1 Lee Goldman M.D.,Dennis Ausiello M.D.,Cecil Textbook of Medicine ,23~(rd),893 .
2 Diabetes Care, January 2002, supplement 1
3 Obesity and Diabetes in the Developing World - A Growing ChallengeParvez Hossain, M.D., Bisher Kawar, M.D., and Meguid El Nahas, M.D., Ph.D.
4 Diabetes Control and Complications Trial Research Group.Diabetes Control and Complacations Trial(DCCT):Results of feasibility study.Diabetes Care 1992;10:1-19.
5 Puestow CB, Jillesby WJ, et al. Retrograde surgical drainage of pancreas for chronic relapsing pancreatitis. Arch Surg 1958;76: 898-906.
6 Zollinger RM, Keith LM, Ellison EH. Pancreatitis. N Engl J Med 1954;251:497-502.
7 La key J R , Wa r nock GL , Shapi ra AM , et al . Int raductal colla2genase delivery into the human pancreas using syringe loading oncontrolled perfusion [ J ] . Cell Transplantant, 1999 , 8(3):285-292.1 14
8 Ryan EA , Lakey J R , Paty BW, et al . Successf ul islet t ransplantation : continued insulin reserve provides longterm glycemic cont rol[J]. Diabetes , 2002 ,51(7): 2148 - 2157.
9 Jonathan RT, Lakey TJ, Anderson RV, et al. Novel approaches to cryopreservation of human pancreatic islets .Transplantation. 2001, 72 :1005-1011 .
10 Shaplm AM,Lakey JR,Ryan EA,et al .Islet transplantation in seven patients with typeⅠ diabetes mellitus using a glucocorticoid free immunosuppessive regimen.N Engl J Med,2000,343:230-238
11 Gustafsson D, Antonsson T, Bylund R, Eriksson U, Gyzander E, Nilsson I,Elg M, Mattsson C, Deinum J, Pehrsson S, Karlsson O, Nilsson A, SorensenH: Effects of Melagatran, a new low-molecular-weight thrombin inhibitor,on thrombin and fibrinolytic enzymes. Thromb Haemost 79:110-118, 1998。
12 Bennet W, Sundberg B, Groth CG et al. Incompatibility between human blood and isolated islets of Langerhans: a finding with implications for clinical intraportal islet transplantation? Diabetes 1999; 48(10): 1907-1914) (Sever CE, Demetris AJ, Zeng Y et al. Islet cell allotransplantation in diabetic patients. Histologic findings in four adults simultaneously receiving kidney or liver transplants. Am J Pathol 1992; 140(5): 1255-1260
13 Ups J Med Sci. 2005;110(1):17-55. The role of the innate immunity in islet transplantation.Moberg L
14 Moberg L, Johansson H, Lukinius A et al. Production of tissue factor by pancreatic islet cells as a trigger of detrimental thrombotic reactions in clinical islet transplantation. Lancet 2002; 360(9350):2039-2045.
15 Diabetes. 2005 Jun;54(6):1755-62. Tissue factor produced by the endocrine cells of the islets of Langerhans is associated with a negative outcome of clinical islet transplantation. Johansson H, Lukinius A, Moberg L, Lundgren T, Berne C, Foss A, Felldin M, Kallen R, Salmela K, Tibell A, Tufveson G, Ekdahl KN, Elgue G, Korsgren O, Nilsson B. 16 Monroe D, Key N. The tissue factor: FVIIa complex-procoagulant activity, regulation,and multitasking. J Thromb Haemost 2007;(In Press)
17 Drake TA, Morrissey JH, Edgington TS. Selective cellular expression of tissue factor in human tissues. Implications for disorders of hemostasis and thrombosis. Am J Pathol 1989;134(5):1087-97.
18 Moberg L, Johansson H, Lukinius A et al. Production of tissue factor by pancreatic islet cells as a trigger of detrimental thrombotic reactions in clinical islet transplantation. Lancet 2002; 360(9350): 2039-2045.
19 Lisa (?) zmen, Kristina Nilsson Ekdahl, Graciela Elgue, Rolf Larsson, Olle Korsgren, 1 and Bo Nilsson, Inhibition of Thrombin Abrogates the Instant Blood-Mediated Inflammatory Reaction Triggered by Isolated Human Islets, DIABETES, VOL. 51, JUNE 2002
20 Transplantation. 2008 Apr 27;85(8):1193-9. Acute antibody-mediated complement activation mediates lysis of pancreatic islets cells and may cause tissue loss in clinical islet transplantation. Tjernberg J, Ekdahl KN, Lambris JD, Korsgren O, Nilsson B.
21 Clin Exp Immunol. 2005 Oct;142(1):125-31. Neutrophilic granulocytes are the predominant cell type infiltrating pancreatic islets in contact with ABO-compatible blood.Moberg L, Korsgren O, Nilsson B
22 Xenotransplantation. 2004 Mar;11(2):184-94. Adult porcine islets produce MCP-1 and recruit human monocytes in vitro.Ehrnfelt C, Kumagai-Braesch M, Uzunel M, Holgersson J.
23 Xenotransplantation. 2007 Jul;14(4):288-97 Rapid loss of intraportally transplanted islets: an overview of pathophysiology and preventive strategies.van der Windt DJ, Bottino R, Casu A, Campanile N, Cooper DK.
24 Diabetes. 2007 Aug;56(8):2008-15. Epub 2007 May 31. Islet surface heparinization prevents the instant blood-mediated inflammatory reaction in islet transplantation.Cabric S, Sanchez J, Lundgren T, Foss A, Felldin M, Kallen R, Salmela K, Tibell A, Tufveson G, Larsson R, Korsgren O, Nilsson B.
25 Am J Transplant. 2006 Feb;6(2):305-12. Am J Transplant. 2006 Jun;6(6):1500. Dufrane, D [added]; Gianello, P [added]. Low molecular weight dextran sulfate: a strong candidate drug to block IBMIR in clinical islet transplantation.Johansson H, Goto M, Dufrane D, Siegbahn A, Elgue G, Gianello P, Korsgren O, Nilsson B.Transplantation. 2007 Aug 15;84(3):308-15. Interference with tissue factor prolongs intrahepatic islet allograft survival in a nonhuman primate marginal mass model.Berman DM, Cabrera O, Kenyon NM, Miller J, Tarn SH, Khandekar VS, Picha KM, Soderman AR, Jordan RE, Bugelski PJ, Horninger D, Lark M, Davis JE, Alejandro R, Berggren PO, Zimmerman M, O'Neil JJ, Ricordi C, Kenyon NS.
26 Transplantation. 2003 Nov 15;76(9):1285-8. Nicotinamide inhibits tissue factor expression in isolated human pancreatic islets: implications for clinical islet transplantation.Moberg L, Olsson A, Berne C, Felldin M, Foss A, Kallen R, Salmela K, Tibell A, TufBveson G, Nilsson B, Korsgren O.
27 Diabetes. 2002 Jun;51(6):1779-84. Inhibition of thrombin abrogates the instant blood-mediated inflammatory reaction triggered by isolated human islets: possible application of the thrombin inhibitor melagatran in clinical islet transplantation.Ozmen L, Ekdahl KN, Elgue G, Larsson R, Korsgren O, Nilsson B.
28 Evert de Jonge, Pascale E. P. Dekkers, Abla A. Creasey, C. Erik Hack, Susan K. Paulson, Aziz Karim, Jozef Kesecioglu, Marcel Levi, Sander J. H. van Deventer, and Tom van der Poll Blood, Vol. 95 No. 4 (February 15), 2000: pp. 1124-1129 FOCUS ON HEMATOLOGY.
29 Linn T, Schneider K, Hammes HP et al. Angiogenic capacity of endothelial cells in islets of Langerhans. Faseb J2003; 17(8): 881-883.
30 Sever CE , Demetris AJ , Zeng Y, et al. Islet cell allotransplantation in diabetic patients. Histologic findings in four adults simultaneously receiving kidney or liver transplants. AmJ Pathol, 1992,140:12552-1260.