胰岛移植治疗糖尿病的现状和进展

胰岛移植是治疗糖尿病尤其是1型糖尿病的一种简单有效的方法,相较与胰腺移植,它较为简单和方便,但存在组织来源匮乏和免疫移植排斥等障碍。新的胰岛分离纯化方法提高了供移植的胰岛的纯度和活性。成体干细胞研究、异种移植研究,有望解决移植的供源问题。Edmonton方案在胰岛移植的临床应用中具有里程碑意义。新型的免疫抑制剂和免疫诱导剂的研究可以提高临床胰岛移植的成功率。     

Clinical islet cell transplantation

Summary Diabetes mellitus is perhaps the most devastating chronic disease of all time. A brief history of the evolution of treatment modalities is provided, culminating in the rationale for the physiologic replacement of a functioning β-cell mass by transplantation. Vascularized pancreas transplantation is discussed briefly as an introduction to the transplantation of the isolated islet. A detailed review of the current state of human islet transplantation for the cure of diabetes is then described. Finally, areas for future development are highlighted.     

The journey of islet cell transplantation and future development

Intraportal islet transplantation has proven to be efficacious in preventing severe hypoglycemia and restoring insulin independence in selected patients with type 1 diabetes. Multiple islet infusions are often required to achieve and maintain insulin independence. Many challenges remain in clinical islet transplantation, including substantial islet cell loss early and late after islet infusion. Contributions to graft loss include the instant blood-mediated inflammatory reaction, potent host auto- and alloimmune responses, and beta cell toxicity from immunosuppressive agents. Protective strategies are being tested to circumvent several of these events including exploration of alternative transplantation sites, stem cell-derived insulin producing cell therapies, co-transplantation with mesenchymal stem cells or exploration of novel immune protective agents. Herein, we provide a brief introduction and history of islet cell transplantation, limitations associated with this procedure and methods to alleviate islet cell loss as a means to improve engraftment outcomes.     

Anti‐diabetic and neuroprotective effects of pancreatic islet transplantation into the central nervous system

During the last decades, the central nervous system (CNS) was intensively tested as a site for islet transplantation in different animal models of diabetes. Immunoprivilege properties of intracranial and intrathecal sites were found to delay and reduce rejection of transplanted allo‐islets and xeno‐islets, especially in the form of dispersed single cells. Insulin released from islets grafted in CNS was shown to cross the blood–brain barrier and to act as a regulator of peripheral glucose metabolism. In diabetic animals, sufficient nutrition and oxygen supply to islets grafted in the CNS provide adequate insulin response to increase glucose level resulting in rapid normoglycemia. In addition to insulin, pancreatic islets produce and secrete several other hormones, as well as neurotrophic and angiogenic factors with potential neuroprotective properties. Recent experimental studies and clinical trials provide a strong support for delivery of islet‐derived macromolecules to CNS as a promising strategy to treat various brain disorders. This review article focuses mainly on analysis of current status of intracranial and intrathecal islet transplantations for treatment of experimental diabetes and discusses the possible neuroprotective properties of grafted islets into CNS as a novel therapeutic approach to brain disorders with cognitive dysfunctions characterized by impaired brain insulin signalling. Copyright © 2015 John Wiley & Sons, Ltd.     

Autologous islet cell transplantation to prevent surgical diabetes

Autologous islet transplantation (AIT) is performed to prevent surgical diabetes after total or semi-total pancreatectomy for the treatment of chronic pancreatitis with severe abdominal pain. In addition, AIT is used in cases of benign pancreatic tumors and pancreatic trauma. It has been shown that AIT results in better outcomes in terms of glycemic control compared with allogeneic islet transplantation. The reasons for the favorable outcomes of AIT are thought to be: (i) patients have no autoimmune diseases; (ii) the transplanted islets do not suffer allogeneic rejection; (iii) diabetogenic antirejection drugs are not required; (iv) pancreata do not undergo a cytokine storm as a result of periods of brain death; (v) the period of cold preservation of retrieved pancreata is short; (vi) the isolated islets are immediately transplanted without culture; and (vii) pancreata with pancreatitis may contain more progenitor cells. Further research into AIT would help improve the results of allogeneic islet transplantation. Conversely, the technical difficulties associated with islet isolation appear to be the largest hurdle for AIT; therefore, remote center islet isolation may prove to be key in the promotion of this treatment.     

Risks and benefits of transplantation in the cure of type 1 diabetes: whole pancreas versus islet transplantation. A single center study

BACKGROUND: Pancreas and islet transplantation are the only available options to replace beta-cell function in patients with type 1 diabetes. Great variability in terms of rate of success for both approaches is reported in the literature and it is difficult to compare the respective risks and benefits. OBJECTIVES: The aim of this study was to analyze risks and benefits of pancreas transplantation alone (PTA) and islet transplantation alone (ITA) by making use of the long-term experience of a single center where both transplantations are performed. We focused on the risks and benefits of both procedures, with the objective of better defining indications and providing evidence to support the decision-making process. The outcomes of 33 PTA and 33 ITA were analyzed, and pancreas and islet function (i.e., insulin independence), perioperative events, and long-term adverse events were recorded. RESULTS: We observed a higher rate of insulin independence in PTA (75%) versus ITA (59%), with the longer insulin independence among PTA patients receiving tacrolimus. The occurrence of adverse events was higher for PTA patients in terms of hospitalization length and frequency, re-intervention for surgical and immunological acute complications, CMV reactivation, and other infections. CONCLUSIONS: In conclusion, these results support the practice of listing patients for PTA when the metabolic control and the progression of chronic complications require a rapid normalization of glucose levels, with the exception of patients with cardiovascular disease, because of the high surgical risks. ITA is indicated when replacement of beta-cell mass is needed in patients with a high surgical risk.     

Clinical islet transplantation in Japan

Introduction

The results of clinical islet transplantation in Japan are, here in, reported and discussed its efficacy and problems.

Methods

Since the first islet transplantation was performed in 2004, 65 islet isolations and 34 islet transplantations to 18 type 1 diabetic patients have been performed in Japan.

Results

Following islet transplantation, patients experienced decreased insulin requirements and lower hemoglobin A1C levels, and positive serum C-peptide levels. All patients achieved stabilized blood glucose levels and the disappearance of hypoglycemic unawareness. Although three patients achieved insulin independency for a limited period, persistent islet graft function was difficult to maintain. Overall islet graft survival was 86.5% at 6 months, 78.7% at 1 year, and 62.9% at 2 years after the first islet transplantation. In our institution, we carried out 23 islet isolations and six islet transplantations to four patients. Although insulin independency was not achieved, all patients showed a disappearance of hypoglycemic unawareness.

Conclusions

Using data from the Japanese Trial of Islet Transplantation, the effectiveness of islet transplantation was shown even when using the pancreata from non-heart-beating donors. Although there are a number of problems to be solved and further improvement is needed, we can state that the introduction of clinical islet transplantation offers hope for type 1 diabetic patients.     

Current status of islet cell transplantation

Despite substantial advances in islet isolation methods and immunosuppressive protocol, pancreatic islet cell transplantation remains an experimental procedure currently limited to the most severe cases of type 1 diabetes mellitus. The objectives of this treatment are to prevent severe hypoglycemic episodes in patients with hypoglycemia unawareness and to achieve a more physiological metabolic control. Insulin independence and long term-graft function with improvement of quality of life have been obtained in several international islet transplant centers. However, experimental trials of islet transplantation clearly highlighted several obstacles that remain to be overcome before the procedure could be proposed to a much larger patient population. This review provides a brief historical perspective of islet transplantation, islet isolation techniques, the transplant procedure, immunosuppressive therapy, and outlines current challenges and future directions in clinical islet transplantation.     

Partial hepatectomy improves the outcome of intraportal islet transplantation by promoting revascularization

Revascularization of grafts is one of the important key factors for the success of islet transplantation. After partial hepatectomy, many growth factors such as hepatocyte growth factor and vascular endothelial growth factor are increased in the remnant liver. These growth factors have properties that promote angiogenesis. This might be an optimal environment for revascularization of islets transplanted intraportally. To verify this hypothesis, syngeneic islets (330 per recipient) were transplanted into the right hepatic lobes of streptozotocin-induced diabetic Balb/c mice with (hepatectomy group) or without (control group) left liver resection. Blood glucose was monitored for 28 d after transplantation. Glucose tolerance test was performed on post-operative day (POD) 30, and histological assessments were performed on POD 7 and 30 respectively. Analysis revealed that 36.7% of the control and 90.0% of the hepatectomy mice attained normoglycemia during the observation period (*p = 0.0142). Glucose tolerance was improved in the hepatectomy group (Area under the curve of intraperitoneal glucose tolerance tests on POD 30, Control; 47,700 ± 5,890 min*mg/dl, Hepatectomy; 26,000 ± 2,060 min*mg/dl: **p = 0.00314). Revascularization of grafted islets was more pronounced in the hepatectomy group (Vessel number per islet area on POD 7, Control; 3.20 ± 0.463 × 10^?4/µm², Hepatectomy; 7.08 ± 0.513 × 10^?4/µm²: **p < 0.01). In the present study, partial hepatectomy (30%) improved the outcome of intraportal islet transplantation. Revascularization of islets transplanted into the liver may have been promoted by the induction of liver regeneration.     

Human islet cell transplantation--future prospects.

BACKGROUND: Islet transplantation has the potential to cure diabetes mellitus. Nevertheless despite successful reversal of diabetes in many small animal models, the clinical situation has been far more challenging. The aim of this review is to discuss why insulin-independence after islet allotransplantation has been so difficult to achieve. METHODS: A literature review was undertaken using Medline from 1975 to July 2000. Results reported to the International Islet Transplant Registry (ITR) up to December 1998 were also analysed. RESULTS: Up to December 1998, 405 islet allotransplants have been reported the ITR. Of those accurately documented between 1990 and 1998 (n = 267) only 12% have achieved insulin-independence (greater than 7 days). However with refined peri-transplant protocols insulin independence at 1 year can reach 20%. CONCLUSIONS: There are many factors which can explain the failure of achieving insulin-independence after islet allotransplantation. These include the use of diabetogenic immunosuppressive agents to abrogate both islet allo-immunity and auto-immunity, the critical islet mass to achieve insulin-independence and the detrimental effects of transplanting islets in an ectopic site. However recent evidence most notably from the Edmonton group demonstrates that islet allotransplantation still has great potential to become an established treatment option for diabetic patients.     

Partial hepatectomy improves the outcome of intraportal islet transplantation by promoting revascularization

Revascularization of grafts is one of the important key factors for the success of islet transplantation. After partial hepatectomy, many growth factors such as hepatocyte growth factor and vascular endothelial growth factor are increased in the remnant liver. These growth factors have properties that promote angiogenesis. This might be an optimal environment for revascularization of islets transplanted intraportally. To verify this hypothesis, syngeneic islets (330 per recipient) were transplanted into the right hepatic lobes of streptozotocin-induced diabetic Balb/c mice with (hepatectomy group) or without (control group) left liver resection. Blood glucose was monitored for 28 d after transplantation. Glucose tolerance test was performed on post-operative day (POD) 30, and histological assessments were performed on POD 7 and 30 respectively. Analysis revealed that 36.7% of the control and 90.0% of the hepatectomy mice attained normoglycemia during the observation period (*p = 0.0142). Glucose tolerance was improved in the hepatectomy group (Area under the curve of intraperitoneal glucose tolerance tests on POD 30, Control; 47,700 ± 5,890 min*mg/dl, Hepatectomy; 26,000 ± 2,060 min*mg/dl: **p = 0.00314). Revascularization of grafted islets was more pronounced in the hepatectomy group (Vessel number per islet area on POD 7, Control; 3.20 ± 0.463 × 10 (-4) /μm ( 2) , Hepatectomy; 7.08 ± 0.513 × 10 (-4) /μm ( 2) : **p < 0.01). In the present study, partial hepatectomy (30%) improved the outcome of intraportal islet transplantation. Revascularization of islets transplanted into the liver may have been promoted by the induction of liver regeneration.     

Nerve growth factor is associated with islet graft failure following intraportal transplantation

Nerve growth factor (NGF) has recently been recognized as an angiogenic factor with an important regulatory role in pancreatic β-cell function. We previously showed that treatment of pancreatic islets with NGF improved their quality and viability. Revascularization and survival of islets transplanted under the kidney capsule were improved by NGF. However, the usefulness of NGF in intraportal islet transplantation was not previously tested. To resolve this problem, we transplanted syngeneic islets (360 islet equivalents per recipient) cultured with or without NGF into the portal vein of streptozotocin-induced diabetic BALB/c mice. Analysis revealed that 44.4% (4/9) of control and 12.5% (1/8) of NGF-treated mice attained normoglycemia (≤ 200 mg/dL) (p = 0.195). NGF-treated islets led to worse graft function (area under the curve of intraperitoneal glucose tolerance tests (IPGTT) on post-operative day (POD) 30, control; 35,800 ± 3,960 min*mg/dl, NGF-treated; 47,900 ± 3,220 min*mg/dl: *p = 0.0348). NGF treatment of islets was also associated with increased graft failure [the percentage of TdT-mediated dUTP-biotin nick-end labeling (TUNEL)-positive and necrotic transplanted islets on POD 5, control; 23.8% (5/21), NGF-treated; 52.9% (9/17): p = 0.0650] following intraportal islet transplantation. Nonviable (TUNEL-positive and necrotic) islets in both groups expressed vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α). On the other hand, viable (TUNEL-negative and not necrotic) islets in both groups did not express VEGF and HIF-1α. In the present study, pre-transplant NGF treatment was associated with impaired survival and angiogenesis of intraportal islet grafts. The effect of NGF on islet transplantation may significantly vary according to the transplant site.     

Nerve growth factor is associated with islet graft failure following intraportal transplantation

Nerve growth factor (NGF) has recently been recognized as an angiogenic factor with an important regulatory role in pancreatic β-cell function. We previously showed that treatment of pancreatic islets with NGF improved their quality and viability. Revascularization and survival of islets transplanted under the kidney capsule were improved by NGF. However, the usefulness of NGF in intraportal islet transplantation was not previously tested. To resolve this problem, we transplanted syngeneic islets (360 islet equivalents per recipient) cultured with or without NGF into the portal vein of streptozotocin-induced diabetic BALB/c mice. Analysis revealed that 44.4% (4/9) of control and 12.5% (1/8) of NGF-treated mice attained normoglycemia (≤ 200 mg/dL) (p = 0.195). NGF-treated islets led to worse graft function (area under the curve of intraperitoneal glucose tolerance tests (IPGTT) on post-operative day (POD) 30, control; 35,800 ± 3,960 min*mg/dl, NGF-treated; 47,900 ± 3,220 min*mg/dl: *p = 0.0348). NGF treatment of islets was also associated with increased graft failure [the percentage of TdT-mediated dUTP-biotin nick-end labeling (TUNEL)-positive and necrotic transplanted islets on POD 5, control; 23.8% (5/21), NGF-treated; 52.9% (9/17): p = 0.0650] following intraportal islet transplantation. Nonviable (TUNEL-positive and necrotic) islets in both groups expressed vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α). On the other hand, viable (TUNEL-negative and not necrotic) islets in both groups did not express VEGF and HIF-1α. In the present study, pre-transplant NGF treatment was associated with impaired survival and angiogenesis of intraportal islet grafts. The effect of NGF on islet transplantation may significantly vary according to the transplant site.     

In vivo assessment of isolated pancreatic islet viability using the streptozotocin-induced diabetic nude rat

Summary In order to establish a model for the in vivo assessment of islet function we have used the Rowett nude rat with transplantation of allogeneic and xenogeneic (mouse) islets into the renal subcapsular space following a minimal period of diabetic induction. Thirty-one nude rats were given streptozotocin and 30 became diabetic with blood glucose levels of greater than 20 mmol/l at 48 h. Rat and mouse islets were prepared by intraductal collagenase and bovine serum albumin density gradient isolation. Eight rats received transplants of freshly prepared allogeneic islets and 8 rats received transplants of 48 h cultured allogeneic islets. Seven rats received transplants of 48 h cultured mouse islets. Diabetes was reversed in all animals and all remained normoglycaemic for 21 days. Graft removal by nephrectomy resulted in hyperglycaemia in 22 out of 23 animals. Histological examination of the grafts showed a band of endocrine tissue beneath the renal capsule which stained strongly positive for insulin and there was no evidence of lymphocytic infiltration/rejection. One rat remained normoglycaemic after graft removal, which may represent recovery of the animal's own islets from the streptozotocin-induced diabetes. Control rats remained diabetic until death. In conclusion, the athymic nude rat can be used for the assessment of allogeneic and xenogeneic islet function when a short (48 h) period of streptozotocin-induced diabetes is used. This model offers a potential method for assessing in vivo function of isolated human islets.     

Islet transplantation in IDDM patients

Summary This single-centre study investigated parameters that positively correlated with the success rate after islet allotransplantation in insulin-dependent diabetic (IDDM) patients. Twenty-one intrahepatic, fresh islet transplantations were performed in 20 IDDM patients (one patient had two transplants), after or simultaneous with kidney transplantation. The correlation between number and purity of transplanted islets and final outcome was investigated. One patient died of a cardiac arrest several hours after islet transplantation; this patient was not included in the follow-up analysis. Three patients (15 %) experienced acute, irreversible, early failure of islet function, which was considered as a ’presumed rejection'. Nine patients (45 %) achieved either complete insulin-independence (seven cases) or a reduction (> 50 %) of exogenous insulin requirement (two cases), with sustained serum C-peptide secretion (0.89 ± 0.04 nmol/l; duration: 21 ± 7 months, range 2–58 months). Liver biopsy, performed 3 years after transplantation in one successful case, showed normal islets within the hepatic parenchyma. Eight cases (40 %) did not show any metabolic effect of islet transplantation, with low serum C-peptide levels (’presumed function exhaustion'). Metabolic investigations performed in successful cases showed an early phase of insulin release after arginine, mild and reversible postprandial hyperglycaemia and normal HbA_1c levels. Success of islet transplantation positively correlates with the number (p < 0.05) of the transplanted islets. Islet transplantation is a safe procedure, with 45 % success rate, in terms of insulin-independence or relevant reduction of exogenous insulin requirement, although success can be transient. [Diabetologia (1997) 40: 225–231] Received: 20 June 1996 and in final revised form: 28 October 1996     

An effective purification method using large bottles for human pancreatic islet isolation

The purification process is one of the most difficult procedures in pancreatic islet isolation. It was demonstrated that the standard purification method using a COBE 2991 cell processor with Ficoll density gradient solution harmed islets mechanically by high shear force. We reported that purification using large bottles with a lower viscosity gradient solution could improve the efficacy of porcine islet purification. In this study, we examined whether the new bottle purification method could improve the purification of human islets. Nine human pancreata from brain-dead donors were used. After pancreas digestion, the digested tissue was divided into three groups. Each group was purified by continuous density gradient using ET-Kyoto and iodixanol gradient solution with either the standard COBE method (COBE group) or the top loading (top group) or bottom loading (bottom group) bottle purification methods. Islet yield, purity, recovery rate after purification, and in vitro and in vivo viability were compared. Islet yield per pancreas weight (IE/g) and the recovery rate in the top group were significantly higher than in the COBE and bottom groups. Furthermore, the average size of purified islets in the top group was significantly larger than in the COBE group, which indicated that the bottle method could reduce the shear force to the islets. In vivo viability was also significantly higher in the top group compared with the COBE group. In conclusion, the top-loading bottle method could improve the quality and quantity of human islets after purification.     

An effective purification method using large bottles for human pancreatic islet isolation

The purification process is one of the most difficult procedures in pancreatic islet isolation. It was demonstrated that the standard purification method using a COBE 2991 cell processor with Ficoll density gradient solution harmed islets mechanically by high shear force. We reported that purification using large bottles with a lower viscosity gradient solution could improve the efficacy of porcine islet purification. In this study, we examined whether the new bottle purification method could improve the purification of human islets. Nine human pancreata from brain-dead donors were used. After pancreas digestion, the digested tissue was divided into three groups. Each group was purified by continuous density gradient using ET-Kyoto and iodixanol gradient solution with either the standard COBE method (COBE group) or the top loading (top group) or bottom loading (bottom group) bottle purification methods. Islet yield, purity, recovery rate after purification, and in vitro and in vivo viability were compared. Islet yield per pancreas weight (IE/g) and the recovery rate in the top group were significantly higher than in the COBE and bottom groups. Furthermore, the average size of purified islets in the top group was significantly larger than in the COBE group, which indicated that the bottle method could reduce the shear force to the islets. In vivo viability was also significantly higher in the top group compared with the COBE group. In conclusion, the top-loading bottle method could improve the quality and quantity of human islets after purification.     

Islet-after-failed-pancreas and pancreas-after-failed islet transplantation: Two complementary rescue strategies to control diabetes

For selected patients with type 1 diabetes, β-cell replacement is the treatment of choice, either by islet transplantation (ITX) or whole pancreas transplantation (PTX). When either modality fails, current practice is to consider retransplantation, or return to exogenous insulin. We investigate outcomes with PTX after failed ITX (PAI), and ITX after failed PTX (IAP). All patients receiving PAI or IAP at a single institution were identified. Donor and recipient variables were documented, including transplant outcomes analyzed for insulin requirement and metabolic control. Five subjects were listed for PAI, and 2 received transplants. Of the 4 listed for IAP, 3 have received transplants. The mean waitlist time was 4.5 ± 4.1 y for PAI and 0.35 ±0 .4 y for IAP (p = 0.08). Metabolic control was excellent after PAI, with 2/2 insulin-independent. After IAP, 1/2 achieved insulin independence and good metabolic control after 2 islet infusions. The third could not receive 2^nd infusion and presented c-peptide levels < 0.1 nmol/L. Both strategies are feasible. The outcomes after PAI in our center must be offset by much longer waitlist time due to the sensitization status of these patients. Data from multicentre experience will allow more robust comparative outcomes to be made, the current observations being restricted to a limited patient set.