小鼠胰岛的分离与纯化

目的 探讨稳定、高效的小鼠胰岛细胞的分离纯化方法.方法 采用胆总管内灌注不同浓度胶原酶(分别为0.5、1.0、1.5 g/L)消化胰腺的方法分离BALB/C小鼠胰岛,Ficoll-400不连续密度梯度离心法纯化胰岛.双硫腙(DTZ)对胰岛进行特异性染色计算胰岛产量及纯度,葡萄糖刺激释放试验体外测胰岛功能.结果 不同浓度的胶原酶在不同时间内消化胰腺后收获的胰岛数量有较大的差异,其中采用0.5 g/L胶原酶V、38 ℃水浴消化20 min组收获量最大为(230±20)个胰岛细胞团,纯度约为90%.DTZ染色后胰岛呈腥红色,形态完整.葡萄糖刺激释放实验示高糖刺激后胰岛素释放量为低糖刺激后的2.3倍.结论 胶原酶浓度、消化时间和温度是影响小鼠胰岛分离结果的重要因素,当胶原酶浓度为0.5 g/L,消化时间20 min时可获得数量较多,纯度较好的胰岛细胞.

Abstract：

Objective To investigate the stable and efficient method of isolation and purification from mice pancreas. Methods BALB/C mouse islets were isolated by different concentrations of collagenase digestion (0. 5, 1. 0, 1. 5 g/L respectively) and purificated by Ficoll density gradient centrifugation.The number, purity and vitality of the islets were analyzed. The production and purity of the islets were checked by Dithizone immunofluorescence staining. The glucose-induced insulin secretion was detected by enzyme linked immunosorbent assay (ELISA) for islet function in vitro. Results Different number of islets was obtained by mice pancreas digestion with different concentrations of collagenase and for different digestion durations.After the mouse pancreata were digested in 38 C and with 0. 5 g/L collagenase V for 20rmin, maximum number of islets was obtained, and the purity of the final preparation was ＞ 95%. After culture in vitro, insulin release of islets under high glucose stimulation was 2. 3 times of that under low glucose stimulation. Conclusion Concentration of collagenase, temperature, and digestion duration were important factors of islet isolation and purification from mice pancreas. More production and higher purity of islets were obtained under the concentration of 0. 5 g/L collagenase Ⅴ for 20 min.     

Comparison of Celsior and UW solution in experimental pancreas preservation

BACKGROUND: The University of Wisconsin solution (UW) is the gold standard for pancreas preservation. Celsior (CEL) was formulated specifically for heart preservation. Recently, experimental and clinical experience has been reported on the application of CEL to abdominal organs. In this animal study, pancreas preservation with CEL was compared with that in UW solution. PATIENTS AND MATERIALS: Heterotopic, allogeneic pancreaticoduodenal transplantation was performed in female G?ttingen Minipigs (n = 12 donors, n = 12 recipients). The grafts were flushed and stored for 6 h at 4 degrees C in UW or CEL. The recipients were randomized into two groups receiving either UW (n = 6)- or CEL (n = 6)-preserved grafts with a follow-up of 5 days. Blood flow (laser Doppler), partial oxygen tension, histological changes, endothelin-1 (plasma, immunohistochemistry), lipase, amylase, trypsinogen activation peptide, and C-reactive protein (CRP) were measured. RESULTS: Partial oxygen tension was lower in the CEL group (P < 0.05). However, blood flow did not differ between UW- and CEL-preserved organs. The histomorphologic analysis of the pancreatic grafts revealed significantly less edema in the UW-preserved organs. Serum levels of amylase, lipase, CRP, and TAP taken from the central venous blood were comparable in the two groups, except for higher amylase values 36 h after reperfusion in the CEL group compared to the UW group (P < 0.05). Likewise, TAP taken from the portal venous effluent of the graft was found to be higher in the CEL group than in UW (P < 0.05). Endothelin-1 serum levels rose significantly during reperfusion without differences between the two groups. ET-1 immunohistochemistry revealed increased local ET-1 during reperfusion in all grafts. However, the ET-1 immunostaining in the CEL group was more pronounced than that in the UW group (P < 0.05). CONCLUSIONS: Our results suggest that CEL solution is not as effective in preventing pancreatic ischemia/reperfusion damage as the standard UW solution in experimental pancreas transplantation. Increased ET-1 immunostaining and reduced p(ti)O(2) in the CEL group indicate increased microcirculatory damage in the CEL group.     

Comparison of modified Celsior solution and M-kyoto solution for pancreas preservation in human islet isolation

Since the successful demonstration of the Edmonton protocol, islet transplantation has advanced significantly on several fronts, including improved pancreas preservation systems. In this study, we evaluated two different types of organ preservation solutions for human islet isolation. Modified Celsior (Celsior solution with hydroxyethyl starch and nafamostat mesilate; HNC) solution and modified Kyoto (MK) solution were compared for pancreas preservation prior to islet isolation. Islet yield after purification was significantly higher in the MK group than in the HNC group (MK = 6186 ± 985 IE/g; HNC = 3091 ± 344 IE/g). The HNC group had a longer phase I period (digestion time), a higher volume of undigested tissue, and a higher percentage of embedded islets, suggesting that the solution may inhibit collagenase. However, there was no significant difference in ATP content in the pancreata or in the attainability of posttransplant normoglycemia in diabetic nude mice between the two groups, suggesting that the quality of islets was similar among the two groups. In conclusion, MK solution is better for pancreas preservation before islet isolation than HNC solution due to the higher percentage of islets that can be isolated from the donor pancreas. MK solution should be the solution of choice among the commercially available solutions for pancreatic islet isolation leading to transplantation.     

Function of transplanted human pancreatic allografts after preservation in cold storage for 6 to 26 hours

Preservation of cadaveric pancreas allografts has been a difficult problem in clinical pancreas transplantation; most institutions use Collins solution and limit preservation time to less than 6 hr. Longer preservation times have been used at the University of Minnesota. Between August 1983, and December 1985, 47 human cadaveric pancreas grafts were transplanted into Type I diabetic recipients after cold storage at 4 degrees C in a modified, hyperosmolar silica-gel filtered plasma (SGFP), a solution previously found to allow dog pancreas grafts to be successfully preserved for up to 48 hr. Ten grafts were preserved for 2-5 hr (group 1); 20 for 6-11 hr (group 2; 17 for 12-26 hr (group 3). Graft function and late outcome were compared between these groups and another group of 7 cadaveric grafts (group 4), which were transplanted immediately and without any preservation. Analysis of exocrine pancreatic function early after transplantation showed a maximum mean serum amylase (IU/L) of 557, 440, 429, and 307 in groups 1, 2, 3, and 4, respectively. Primary preservation failure rates of 0, 5%, 5.8%, and 0%, and endocrine graft function rates at 1 month of 80%, 80%, 76%, and 86% were obtained for groups 1, 2, 3, and 4, respectively (P = NS). Only patients who were insulin-independent were counted as having functioning grafts. Detailed functional studies at 1 month showed that mean plasma glucose levels during 24-hr metabolic profiles were in the normal range in 71%, 68%, 72%, and 50%, while oral glucose tolerance test results were within the normal range in 38%, 81%, 76%, and 66% of groups 1, 2, 3, and 4, respectively (P = NS). At 1 year, patient survival rates were 57%, 88%, 75%, and 100% (P = NS), and the graft functional survival rates were 0, 25%, 33%, and 29% (P = NS) in the respective groups. Five patients in group 2, and 6 in group 3 have currently functioning grafts at 4 to 37 months after transplantation. We conclude that cadaver pancreas grafts can be safely preserved for 12-24 hr in modified SGFP solution, thus making the sharing of these organs between different centers practical and the transplant operation less of an emergency procedure.     

Iloprost to improve surfactant function in porcine pulmonary grafts stored for twenty-four hours in low-potassium dextran solution

OBJECTIVES: The optimal strategy for pulmonary graft preservation remains elusive. Experimental work and initial clinical experience support low-potassium dextran solutions as lung perfusates. We have previously shown a protective effect of prostaglandin E 1 on ischemia-reperfusion injury in lung transplantation by a shift from proinflammatory to anti-inflammatory cytokines in a rat lung transplantation model. In this study, we tested the hypothesis that the addition of a prostacyclin analog (iloprost) to low-potassium dextran might lead to improved surfactant and ultimately graft function. METHODS: In a randomized, blinded study with a porcine left single-lung transplantation model, donor lungs were flushed with 1 L of either low-potassium dextran solution or low-potassium dextran solution modified by the addition of 250 microg iloprost (n = 6 in each group). Grafts were stored at 4 degrees C for 24 hours. After transplantation, the right bronchus and pulmonary artery were clamped, and the animals remained dependent on the graft. Posttransplantation graft function was assessed throughout a 7-hour observation period by measuring oxygenation (30-minute intervals), different pulmonary and systemic hemodynamic parameters, and wet/dry lung weight ratios. Bronchoalveolar lavage fluid was obtained before and 2 hours after reperfusion. Surfactant function was measured from bronchoalveolar lavage fluid with a pulsating bubble surfactometer. Neutrophil sequestration was assessed by a myeloperoxidase assay performed on lung tissue specimens taken at the end of the observation period. RESULTS: Pulmonary vascular resistance remained lower in the iloprost group than in the control group (P < .05). Tissue water content after 7 hours of reperfusion remained lower in the iloprost group (P < .05). In addition, significantly reduced myeloperoxidase tissue activity was observed in the iloprost group (P < .05). Although there was no difference in degradation of surface active surfactant large aggregates to small aggregates, the surface tension measured at minimal bubble diameter was lower in the iloprost group (P < .05). CONCLUSIONS: Modification of low-potassium dextran solution with the prostacyclin analog iloprost resulted in a significant amelioration of ischemia-reperfusion injury and improved preservation of surfactant function in transplanted lungs. This intriguing approach merits further evaluation with respect to the mechanisms involved and, ultimately, potential introduction into clinical lung transplantation.     

Islet isolation and transplantation outcomes of pancreas preserved with University of Wisconsin solution versus two-layer method using preoxygenated perfluorocarbon

BACKGROUND: Previous small clinical trials indicate that the two-layer method (TLM) for pancreas preservation improves islet isolation outcome. However, the effect of TLM has not been evaluated in large-scale study. In addition, a direct benefit of TLM on islet transplantation outcome has not been addressed in the setting of any randomized controlled trials. METHODS: Between April 2003 and October 2005, human pancreata from brain-dead donors were preserved by TLM using preoxygenated perfluorocarbon (n = 75) or in University of Wisconsin (UW) solution (n = 91) prior to islet isolation. Islet isolation and transplantation outcomes were compared between the two groups. RESULTS: We did not find any significant differences in adenosine triphosphate content in pancreatic tissue after preservation, pre and postpurification islet yields, in vitro insulin secretory function, or utilization ratio of transplantation between the two groups. Transplanted mass and functional viability of islet isolated from TLM-preserved pancreas were similar to those from UW-preserved pancreas. Patients receiving the TLM-islet or the UW-islet showed a marked decrease in insulin requirement after transplantation. However, no significant difference was observed in a decrease in insulin requirement between patients receiving the TLM-islet and the UW-islet. CONCLUSIONS: No beneficial effect of TLM on islet isolation and transplantation outcomes was observed. Our findings bring into question the true merit of routine use of TLM prior to islet isolation.     

A comparison of some simplified lactobionate preservation solutions with standard UW solution and Eurocollins solution for pancreas preservation.

Fifty-two rat pancreas transplants were performed to investigate which components of the UW solution were essential for successful pancreas preservation. LEW rats were used and the pancreata stored at 4 degrees C for 48 hr after flushing with commercial UW solution (ViaSpan, DuPont Pharmaceuticals) or a number of simplified solutions. Following storage the pancreata were transplanted into syngeneic recipient animals with streptozotocin-induced diabetes mellitus. Graft function was assessed by regular postoperative blood sugar measurements and a glucose tolerance test on the 14th postoperative day. With commercial UW solution, 4 of 9 recipients (44%) showed satisfactory graft function, while only one of 5 pancreata preserved using Eurocollins solution demonstrated satisfactory function. With solution A, in which hydroxyethyl starch and insulin were omitted from the standard UW solution, 3 of 7 recipients (43%) showed satisfactory function. Omission of glutathione, allopurinol, and adenosine from this solution (solution B) gave satisfactory function in 4 of 8 cases (50%). Substitution of raffinose in solution B with an equimolar concentration of glucose (solution C) resulted in acceptable function in 5 of 8 cases (62%). Increasing the raffinose concentration in solution B to 100 mM/L resulted in only 2 of 8 grafts (25%) with adequate function. By contrast, reversing the Na/K concentrations in solution A resulted in 100% (7/7) satisfactory graft function. We conclude that the rat pancreas can be successfully transplanted following 48-hr cold preservation using UW solution and some simplified versions, and that a substantially simplified lactobionate-based solution with a reversed sodium/potassium ratio improved survival.     

Preservation of the canine liver for 24-48 hours using simple cold storage with UW solution

The results of a series of 29 orthotopic liver transplants in the dog are described. The livers were preserved in a new cold storage fluid, UW solution, and were successfully transplanted after periods of storage of 24, 30, 36, and 48 hr. All six animals transplanted after 24 hr survived beyond 5 days after transplantation and had excellent graft function. Four of six survived for at least 5 days after 30 hr of cold storage, and five of five after 36 hr. Five of six consecutive dogs that received transplants that had been cold-stored for 48 hr survived for 5 or more days. This solution represents a substantial advance over all existing cold storage solutions for liver preservation.     

Development of a method for isolation of islets from human fetal pancreas

Three methods for the preparation of islets from human fetal pancreas (17.4 +/- 1.2 wk gestational age) were compared. In each method, the pancreases were minced and followed by 1) no collagenase digestion, 2) 5 min of collagenase digestion, or 3) 14 min of collagenase digestion. The culture conditions prevented adherence of the fragments. Culture for 6-7 wk of minced fetal pancreas without collagenase digestion resulted in fragments that were a mixture of cells positive for insulin or glucagon, ducts, necrotic debris, and other unidentified cells with complete degeneration of the acinar cells. Culture of minced pancreas digested for 5 min with collagenase resulted in fragments that superficially appeared to be islets but did not have the size characteristics of human fetal islets and contained fibrous and duct elements not seen in islets. Culture of minced pancreas digested for 14 min with collagenase resulted in islets that were released into the medium and harvested by picking. These islets were morphologically similar to islets of the intact human fetal pancreas and isolated islets from rat neonatal pancreas. These islets and fragments were viable for at least 7-8 wk in culture.     

A method for isolation of islets of Langerhans from the human pancreas

A method has been developed for the isolation of islets of Langerhans from the human pancreas. The average number of islets isolated was 1011 islets per gram of pancreas (SD 475, range 752-2111), and the purity of the preparation as defined by histologic examination and specific staining for insulin varied from 10% to 40%. Islet structure was well preserved and the islets were shown to be viable by supravital staining, demonstration of insulin response to glucose, and by transplantation of isolated islets beneath the renal capsule of nude mice. The essential features of this technique for isolation of human islets include injection of a high concentration of collagenase (6 mg/ml) into the pancreatic duct under pressure, followed by a short incubation (23 min) at 39 degrees C. The gland is then dispersed by a process of teasing and shaking, and the islets are separated by a two-stage process of filtration on a nylon mesh to remove the larger islets and centrifugation on a preformed Ficoll density gradient to separate the small islets.     

Ameliorating injury during preservation and isolation of human islets using the two-layer method with perfluorocarbon and UW solution

This study assessed the effects of a two-layer method (TLM), using perfluorocarbon and UW solution, on the quality of human pancreata following storage and islet yield/function after isolation. In part A, TLM was applied immediately after procurement and the energetic profile was compared to a group treated with UW solution only (control) throughout 24-h storage. In part B, cadaveric human pancreata were procured and subjected to a TLM after cold storage in UW solution (TLM group) or UW solution (control group). Energetics, lipid peroxidation, and islet recovery/function were assessed after preservation at 4 degrees C. In part A, after 9-h storage, the energetic profile (ATP, ATP/ADP, energy charge) for the TLM group was superior to controls. In part B, TLM treatment resulted in consistently greater ATP, ATP/ADP, and energy charge values than with storage in UW solution alone (p < 0.05). UW treatment resulted in 40% greater peroxidative damage than in the TLM group (p < 0.05). Islet recovery and functional viability were 30-40% higher following TLM treatment (p < 0.05). These data support the hypothesis that islet viability and yields can be significantly improved using a brief period of TLM treatment following conventional UW storage; reduced energetic and oxidative stress are implicated as potential mechanisms.