Impact of donor-related variables on islet isolation outcome in dogs

Summary Clinical human islet transplantation programmes are considerably hampered by the variability of islet isolation outcome. The effects of the islet content of the pancreas and other donor-related variables on isolation outcome have not been evaluated systematically so far — either in large animals, or in man. We studied the impact of interindividual differences in age, body weight and pancreatic islet content on the outcome of collagenase isolation of islets from the splenic pancreas of beagle dogs (n=31). The islet volume of the splenic pancreas amounted to a mean (± SEM) 15.7±0.9 l per gramme pancreas, and varied three-fold (from 8.4 to 27.3 l). Isolated islet yield was 7.6±0.7 l/g and varied nine-fold (1.8–16.3 l). Animals also varied in age eight-fold (867 months) and body weight two-fold (8.6–18.3 kg). Differences in body weight and age explained 60% of variance in the fractional islet volume of the pancreas and 50% of the variance in islet yield (p<0.001). Fractional islet volume of the splenic pancreas also explained 50% of the variance in islet yield (p<0.001). We conclude that the outcome of islet isolation may be predictable after controlling for the variable islet content of pancreases, and other donor-related variables, and suggest that similar studies should be done in man.     

Increased islet viability by addition of beraprost sodium to collagenase solution.

The digestion of pancreatic tissue with collagenase is an essential part of the islet isolation procedure. However, the process exposes islets to various types of harmful factors, including collagenase contaminants, enzymes released from the acinar cells, warm ischemia, and mechanical agitation. Nitrogen oxide production and cytokine release may also contribute to islet cell damage. Protection of islets from such damage would improve the islet yield, survival, and function. Beraprost sodium (BPS) is a prostaglandin I2 analogue, is stable in aqueous solution, and has a cytoprotective effect on various types of cells. BPS has been shown to improve the yield and function of cryopreserved and/or cultured islets. These findings prompted us to examine its cytoprotective effect on islets during the islet isolation process. Canine islets were isolated by means of a two-step digestion method and purified on Euro-Ficoll density gradient solutions (the procedure used for human islets). BPS at a concentration of 100 nM was added to the collagenase solution. After purification, the islet yield was 434,561 +/- 35.691 islet number expressed as 150 microm equivalent size (IEQ)/pancreas or 8,799 +/- 345 IEQ/g of pancreas in the BPS group and 349,987 +/- 52,887 IEQ/pancreas or 7,998 +/-1610 IEQ/g of pancreas in the control group (n = 8, each). The percent viability was 88.5 +/- 0.7% in the BPS group and 82.0 +/-0.9% in the control group (P < 0.01). Therefore, the recovery of viable islets (calculated by islet number x % viability) was 384,586 +/- 46,804 IEQ/pancreas (7,743 IEQ/g) in the BPS group and 286,989 +/- 43,367 IEQ/pancreas (6,558 IEQ/g) in the control group (P < 0.02). After culture, significantly higher numbers of islets were also recovered in the BPS group than in the control group. The islet insulin content was significantly higher in the BPS group than controls (237.8 +/- 38.5 versus 92.3 +/- 25.6 microU/IEQ; P < 0.02), although islets of both groups responded with high stimulation indices (>6). These results indicate that the addition of BPS to the collagenase solution increases the recovery of viable islets, and improves beta cell function.     

A method for large-scale, high-yield isolation of canine pancreatic islets of Langerhans

A modified collagenase digestion method is described for the isolation of large numbers of islets from the canine pancreas (approximately 3,500 islets/g). The islets isolated by this technique remained viable and released insulin in response to secretagogues after one week of maintenance in tissue culture. Islets isolated from a single donor pancreas were re-implanted into the spleen of the same animal made diabetic by subtotal pancreatectomy and two injections fo streptozotocin. Hyperglycemia was corrected in two dogs and decreased in a third dog followed for 30 days. The islet isolation method is described, therefore, provides a sufficiently large yield of viable islets from one donor pancreas to correct or improve diabetes in a recipient animal.     

9a Techniques of pancreas and islet transplantation

Techniques for vascularized pancreas transplantation are relatively standardized, whereas those for human islet isolation and transplantation are rapidly changing and evolving. The commonest method for transplanting the vascularized pancreas is to use the entire pancreas together with a segment of donor duodenum, and to anastomose this to the recipient bladder. This technique offers the advantages of technical ease, the maximum β-cell mass is transplanted and graft function can be monitored by measuring urinary amylase levels. Human islet isolation requires that the pancreas is dissociated, the islets purified and then transplanted to a wellvascularized location. The pancreas is dispersed by a combination of the intraductal injection of collagenase and gentle mechanical agitation, and the islets separated from contaminating exocrine tissue by density-gradient centrifugation. Once purified, the islets can be placed into tissue culture or cryopreserved. The commonest site for human islet transplantation is intraportal injection so that the islets are embolized throughout the liver. Alternatives include transplantation to the renal subcapsular space or the spleen.     

Improvement of collagenase distribution with the ductal preservation for human islet isolation

A delivery of collagenase at the islet-exocrine interface is crucial for successful human islet isolation. In this study, we investigated how the ductal preservation method at the procurement site affected collagenase distribution. At first, we analyzed human islet isolation data among groups using Serva collagenase with or without ductal injection (DI) or using new Liberase MTF with DI. Then, to assess the distribution of collagenase, human pancreata were classified into two groups: without DI (no DI, n = 5) and with DI at the procurement site (DI, n = 5). Collagenase with 1% marking dye was perfused in the same manner as in our clinical isolation. The distension of the pancreas and the microscopic distribution of the dyed collagenase in pancreas sections were examined. For microscopic analysis, islets were counted and classified into three criteria: unreached, dye didn't reach the islet surface; surface, dye resided on the surface of the islet but not inside; and inside, dye was found inside the islet. As a result, DI groups substantially improved islet yields. In addition, Liberase MTF with DI significantly improved efficacy of pancreas digestion. All pancreata were well distended macroscopically. However, microscopically, the majority of islets in the no DI group were untouched by the dyed collagenase. Ductal preservation substantially improved dyed collagenase delivery on the surface of islets. In conclusion, delivery of collagenase on the surface of islets was unexpectedly insufficient without DI, which was substantially improved by DI. Thus, ductal preservation is a potent method to improve collagenase delivery and islet yields.     

Improvement of collagenase distribution with the ductal preservation for human islet isolation

A delivery of collagenase at the islet-exocrine interface is crucial for successful human islet isolation. In this study, we investigated how the ductal preservation method at the procurement site affected collagenase distribution. At first, we analyzed human islet isolation data among groups using Serva collagenase with or without ductal injection (DI) or using new Liberase MTF with DI. Then, to assess the distribution of collagenase, human pancreata were classified into two groups: without DI (no DI, n = 5) and with DI at the procurement site (DI, n = 5). Collagenase with 1% marking dye was perfused in the same manner as in our clinical isolation. The distension of the pancreas and the microscopic distribution of the dyed collagenase in pancreas sections were examined. For microscopic analysis, islets were counted and classified into three criteria: unreached, dye didn’t reach the islet surface; surface, dye resided on the surface of the islet but not inside; and inside, dye was found inside the islet. As a result, DI groups substantially improved islet yields. In addition, Liberase MTF with DI significantly improved efficacy of pancreas digestion. All pancreata were well distended macroscopically. However, microscopically, the majority of islets in the no DI group were untouched by the dyed collagenase. Ductal preservation substantially improved dyed collagenase delivery on the surface of islets. In conclusion, delivery of collagenase on the surface of islets was unexpectedly insufficient without DI, which was substantially improved by DI. Thus, ductal preservation is a potent method to improve collagenase delivery and islet yields.     

Human pancreatic islet function at the onset of Type 1 (insulin-dependent) diabetes mellitus

Summary Viable human pancreatic islets isolated from a recent-onset Type 1 (insulin-dependent) diabetic patient were used to perform in vitro studies. Pre-proinsulin mRNA and insulin content, as well as insulin response were analysed. Insulin response to glucose and forskolin was completely absent in diabetic islets, as compared to control islets. Insulin content was reduced to only one-third of control values (395.0±3.5 vs 989.0±46.3 U/islet) and 20.7±3.9% of islets from the diabetic pancreas contained insulin-positive cells in immunofluorescence studies. Northern blot analysis revealed a severe reduction in the content of pre-proinsulin mRNA in diabetic pancreatic tissue. Our results indicate that although markedly decreased, beta cells in human pancreatic islets at the onset of Type 1 diabetes are still present. Never-theless, pancreatic islet function is disproportionately impaired with a complete absence of an insulin response.     

Effect of pharmacological agents and fasting on pancreatic islet norepinephrine in the golden hamster

Summary Using a specific and sensitive radioenzymatic assay that utilizes the partially purified enzyme phenylethanolamine-N-methyltransferase, studies were done to determine if pharmacological agents and/or fasting alter the norepinephrine concentration of collagenase-isolated golden hamster pancreatic islets. The norepinephrine concentration (42.1±8.07 mol/kg net weight, mean±SEM) and the monoamine oxidase activity (5,407±530 pmol product /mg tissue/min) of hamster pancreatic islets was at least five times higher than acinar pancreas, kidney, heart, median eminence or cerebral cortex. The catechol-o-methyltransferase activity of hamster islets (7±2.3 pmol product/mg tissue/min) was one half or less than the other tissues. Islet norepinephrine was not increased by two days administration of the monoamine oxidase inhibitor tranylcypromine. Islet norepinephrine concentration was increased 2-fold by administration of the norepinephrine precursor DL-threo-dihydroxyphenylserine.This increase was enhanced by prior administration of tranylcypromine (3.5-fold) and prevented by prior administration of the decarboxylase inhibitor N¹-(DL-seryl)-N²-(2,3,4-trihydroxybenzyl) hydrazine (RO-4-4602). There was a good correlation between the islet norepinephrine concentration and the plasma glucose concentration after pharmacological agents. Reserpine administration markedly depleted the islet norepinephrine concentra tion. Fasting of 24, 48 and 72 h did not alter the norepinephrine concentration in islets and heart. It is concluded that the pancreatic islets of the hamster have an active noradrenergic system, but that islet norepinephrine does not appear to play an important role in the impaired insulin secretion of fasting hamsters.     

Insulin secretory characteristics of monkey pancreatic islets: a simple method of islet isolation and the effect of various density gradients on separation

We describe a simple stationary digestion method of islet isolation and separation by various density gradients from monkey pancreas (Macaca radiata radiata). Effective method, different types and concentrations of collagenase were standardized. Sigma type XI collagenase yielded >1000 islets/gram pancreas at the concentration of 4 mg/ml and 3 ml Hank's/gram pancreas. Slow digestion with less concentration of collagenase was suitable for monkey islet isolation. Discontinuous density gradients of bovine serum albumin (BSA) and dextran were compared with standard Ficoll for separation of islets. Islet yield (1038 +/- 81), insulin secretory response (stimulation index, S.I.11) and histological examination revealed dextran gradients were more appropriate for monkey islets when compared to BSA and Ficoll. Insulin secretory characteristics of monkey islets were studied by exposing them to low and high concentrations glucose (S.I.11.5), arginine (S.I.4.2), leucine (S.I.2.3) and tolbutamide (S.I.1.7). The results indicated that the magnitude of glucose induced insulin secretion of monkey islet is about half as that of rat and mouse islets. However, it is higher than that of porcine and bovine islets. In conclusion, the knowledge of insulin secretory ability of Indian bonnet monkey islets together with the techniques of isolation and separation are useful tool for diabetic research especially islet transplantation.     

Acute insulin response of donors is correlated with pancreatic islet isolation outcome in the pig

Aims/hypothesis Unpredictability of islet isolation outcome remains a frustrating and costly issue in the clinical implementation of islet transplantation. The aim of this experimental study was to test the hypothesis that the donors insulin secretory reserve, an in vivo surrogate of functional pancreatic mass, is correlated with the outcome of islet isolation.Methods Insulin secretory reserve was evaluated in 28 healthy adult minipigs prior to pancreatectomy and islet isolation. Blood glucose and insulinaemia were measured before and 1, 3, 5, 10, 15, 30, 60 and 90 min after glucose infusion. Following total pancreatectomy, islet isolation was performed according to Ricordis semi-automated method, and the total number of islets obtained was determined. Fasting blood glucose, insulinaemia, acute insulin response (AIR), maximal insulinaemia and the glucose decay constant (K_G) were calculated, and possible associations with the outcome of islet isolation were assessed.Results AIR and maximal insulinaemia after glucose injection were correlated with the outcome of islet isolation (p<0.01). Mean values for AIR and maximal insulinaemia were significantly different between animals in which islet isolation was successful (n=11) vs those in which it was unsuccessful (n=17) (77.6±13.7 U/ml vs 42.3±7.8 U/ml, p<0.05; 144.7±21.6 U/ml vs 71.9±10.4 U/ml, p<0.05, respectively).Conclusions/interpretation This study suggests that the donors pancreatic endocrine mass, as estimated by AIR, is a major determinant of the outcome of islet isolation in large mammals. Our results may explain the frustrating variability of human islet isolation outcome and could lead to a new approach for optimising the selection of brain-dead and/or living pancreas donors.     

Human pancreatic tissue dissociation enzymes for islet isolation: Advances and clinical perspectives

Background and aimsSuccessful clinical human allo or auto-islet transplantation requires the recovery of a sufficient number of functional islets from either brain-dead or chronic pancreatitis pancreases respectively.MethodsIn the last two decades (2000–2019), significant progress has been made in improving the human islet isolation procedures and in standardizing the use of different tissue dissociation enzyme (TDE; a mixture of collagenase and protease enzymes) blends to recover higher islet yields.Results and ConclusionsThis review presents information focusing on properties and role of TDE blends during the islet isolation process, particularly emphasizing on the current developments, associated challenges and future perspectives within the field.     

Can We Re-Engineer the Endocrine Pancreas?

Purpose of Review

Engineering endocrine pancreatic tissue is an emerging topic in type 1 diabetes with the intent to overcome the current limitation of β cell transplantation. During islet isolation, the vascularized structure and surrounding extracellular matrix (ECM) are completely disrupted. Once implanted, islets slowly engraft and mostly are lost for the initial avascular phase. This review discusses the main building blocks required to engineer the endocrine pancreas: (i) islet niche ECM, (ii) islet niche vascular network, and (iii) new available sources of endocrine cells.

Recent Findings

Current approaches include the following: tissue engineering of endocrine grafts by seeding of native or synthetic ECM scaffolds with human islets, vascularization of native or synthetic ECM prior to implantation, vascular functionalization of ECM structures to enhance angiogenesis after implantation, generation of engineered animals as human organ donors, and embryonic and pluripotent stem cell-derived endocrine cells that may be encapsulated or genetically engineered to be immunotolerated.

Summary

Substantial technological improvements have been made to regenerate or engineer endocrine pancreatic tissue; however, significant hurdles remain, and more research is needed to develop a technology to integrate all components of viable endocrine tissue for clinical application.

     

Effect of increased pancreatic islet norepinephrine,dopamine and serotonin concentration on insulin secretion in the Golden hamster

Summary The purpose of this study was to determine if increased concentrations of pancreatic islet norepinephrine, dopamine, or serotonin alter insulin secretion. Golden hamsters received intraperitoneal injections of the norepinephrine precursor DL-threo-dihydroxyphenylserine, the dopamine precursor L-3,4-dihydroxyphenylalanine, or the serotonin precursor 5-hydroxytryptophan with and without pretreatment of the hamsters with the monoamine oxidase inhibitor tranylcypromine. Administration of the monoamine precursors to animals pretreated with tranylcypromine resulted in a mean increase in plasma glucose of 192% and a mean decrease in plasma insulin of 58%. Using a collagenase isolation technique, islets from control and treated animals were evaluated for monoamine content and insulin secretory capacity. The monoamine concentrations in control islets, in mol/kg wet weight, were: norepinephrine 42±8; dopamine 8±2; and serotonin 26±9. Administration of the appropriate precursor to control hamsters resulted in a 1.9-fold (norepinephrine), 6-fold (dopamine), and 22-fold (serotonin) increase in monoamines. There was no alteration in the glucose (16.3 mmol/l)-stimulated in vitro insulin secretion from islets obtained from these hamsters. Administration of the precursors to hamsters pretreated with tranylcypromine resulted in a 3.5-fold (norepinephrine), 22-fold (dopamine), and 59-fold (serotonin) increase in monoamines. Glucose-stimulated in vitro insulin secretion from islets obtained from these hamsters was completely blocked. This study suggests that high concentrations of norepinephrine, dopamine, and serotonin in the pancreatic islets can decrease glucose-stimulated insulin secretion.     

Improved quality and yield of islets isolated from human pancreata using a two-step digestion method

A new approach, involving a two-step digestion process and Los Angeles preservation solution #1 (LAP-1), a cold storage solution, was developed for isolation of high-quality islets from human pancreata for transplantation. This approach markedly improves the islet yield, purity and viability, and the isolation success rate. In this method, the pancreas was digested first in warm collagenase solution for up to 20 minutes. After decanting the enzyme solution, partially digested tissue was dissociated by gentle agitation in cold LAP-1 solution without additional collagenase. The digested tissues were stored in cold LAP-1 solution until islet purification on Euro-Ficoll. Forty-six islet isolations were performed consecutively by the new method (group 1). These results were compared to those obtained earlier with 46 consecutive isolations, using our previous method that had been used before development of the new method (group 2). Our old method was a modification of Ricordi's method involving only warm collagenase digestion and the storage of digested tissues in cold Hanks balanced salt solution. All pancreata were partial, containing the body and tail. There were no significant differences in both groups with regard to the donor age, cold ischemic time, harvesting conditions, and pancreatic weight. Pancreas digestion was completed in approximately 1 hour in both groups. The isolation success rate as determined by viable islets after 2 days in culture was 93.5% (43 of 46 cases) in group 1, and 56.5% (26 of the 46) in group 2. Immediately after isolation, the new method yielded a total of 335,739 +/- 36,244 islets equivalent to 150 microm (IEQ) and 6,233 +/- 681 IEQ/g of pancreas with 83 +/- 2.5% purity, whereas the old method yielded a total of 195,587 +/- 25,242 IEQ and 3,763 +/- 5,509 IEQ/g with 69.2 +/- 4.7% purity. Isolated islets in group 1 maintained a good three-dimensional structure, displayed normal insulin release to high glucose stimulation in vitro, and restored euglycemia after transplantation into streptozotocin-diabetic athymic mice. The two-step digestion method provides a sufficient number of islets for transplantation from a single pancreas.     

Abnormal insulin secretion and glucose metabolism in pancreatic islets from the spontaneously diabetic GK rat

Summary Insulin secretion and islet glucose metabolism were compared in pancreatic islets isolated from GK/Wistar (GK) rats with spontaneous Type 2 (non-insulin-dependent) diabetes mellitus and control Wistar rats. Islet insulin content was 24.5±3.1 U/ng islet DNA in GK rats and 28.8±2.5 U/ng islet DNA in control rats, with a mean (±SEM) islet DNA content of 17.3±1.7 and 26.5±3.4 ng (p < 0.05), respectively. Basal insulin secretion at 3.3 mmol/l glucose was 0.19±0.03 · ng islet DNA^–1· h^–1 in GK rat islets and 0.40±0.07 in control islets. Glucose (16.7 mmol/l) stimulated insulin release in GK rat islets only two-fold while in control islets five-fold. Glucose utilization at 16.7 mmol/l glucose, as measured by the formation of ³H₂O from [5-³ H]glucose, was 2.4 times higher in GK rat islets (3.1±0.7 pmol · ng islet DNA^–1 · h^–1) than in control islets (1.3±0.1 pmol · ng islet DNA^–1 · h^–1; p<0.05). In contrast, glucose oxidation, estimated as the production of ¹⁴CO₂ from [U-¹⁴C]glucose, was similar in both types of islets and corresponded to 15±2 and 30±3 % (p<0.001) of total glucose phosphorylated in GK and control islets, respectively. Glucose cycling, i. e. the rate of dephosphorylation of the total amount of glucose phosphorylated, (determined as production of labelled glucose from islets incubated with ³H₂O) was 16.4±3.4% in GK rat and 6.4±1.0% in control islets, respectively (p<0.01). We conclude that insulin secretion stimulated by glucose is markedly impaired in GK rat islets. Glucose metabolism is also altered in GK rat islets, with diminished ratio between oxidation and utilization of glucose, and increased glucose cycling, suggesting links between impaired glucose-induced insulin release and abnormal glucose metabolism.     

A new collagenase blend increases the number of islets isolated from mouse pancreas

Diabetes is a predominant metabolic disorder in the industrialized nations. Since pancreatic islets play a key role in type I and type II diabetes, the isolation of islets from pancreatic tissues represents an important step in diabetes research. However, to date, only a small fraction of all islets, resident within any given pancreas, are harvested by using currently available enzyme blends. This holds true for islet isolation from both the mouse and the human pancreas. In the present study, the newly developed Liberase TL Research Grade was compared to the widely used Liberase RI to investigate the effect of increased collagenase purity on islet yield. The study shows that reducing the degradation products of collagenases during Liberase production significantly increases the number of islets isolated from the mouse pancreas by 28%, and, therefore, is expected to lower the numbers of mice and resulting costs for diabetes research accordingly. Furthermore, this study also points to a possibility to increase the number and mass of islets isolated from human pancreases, for which only a limited donor pool exists.     

A new collagenase blend increases the number of islets isolated from mouse pancreas

Diabetes is a predominant metabolic disorder in the industrialized nations. Since pancreatic islets play a key role in type I and type II diabetes, the isolation of islets from pancreatic tissues represents an important step in diabetes research. However, to date, only a small fraction of all islets, resident within any given pancreas, are harvested by using currently available enzyme blends. This holds true for islet isolation from both the mouse and the human pancreas. In the present study, the newly developed Liberase TL Research Grade was compared to the widely used Liberase RI to investigate the effect of increased collagenase purity on islet yield. The study shows that reducing the degradation products of collagenases during Liberase production significantly increases the number of islets isolated from the mouse pancreas by 28%, and, therefore, is expected to lower the numbers of mice and resulting costs for diabetes research accordingly. Furthermore, this study also points to a possibility to increase the number and mass of islets isolated from human pancreases, for which only a limited donor pool exists.     

Islet isolation for allotransplantation: variables associated with successful islet yield and graft function

Aims/hypothesis Efficient islet isolation is an important prerequisite for successful clinical islet transplantation. Although progressively improved, islet yield and quality are, however, unpredictable and variable and require standardisation.Methods Since 1989 we have processed 437 pancreases using the automated method. The donor characteristics, pancreas procurement, and digestion and purification procedures including a wide enzyme characterisation of these pancreases were analysed and correlated with islet yield and transplant outcome.Results By univariate analysis, islet yield was significantly associated with donor age (r=0.16; p=0.0009), BMI (r=0.19; p=0.0004), good pancreas condition (p=0.0031) and weight (r=0.15; p=0.0056), total collagenase activity (r=0.22; p=0.0001), adjusted collagenase activity/mg (r=0.18; p=0.0002), collagenase activity/solution volume (r=0.18; p=0.0002) and neutral protease activity/solution volume (r=0.14; p=0.0029). A statistically significant contribution to the variability of islet yield in a multivariate analysis performed on donor variables was found for donor BMI (p=0.0008). In a multivariate analysis performed on pancreas variables a contribution was found for pancreas weight (p=0.0064), and for a multivariate analysis performed on digestion variables we found a contribution for digestion time (p=0.0048) and total collagenase activity (p=0.0001). Twenty-four patients with type 1 diabetes received single islet preparations from single donors. In these patients, multivariate analyses showed that the reduction in insulin requirement was significantly associated with morphological aspects of islets (p=0.0010) and that 1-month C-peptide values were associated with islet purity (p=0.0071).Conclusions/interpretation These data provide baseline donor, digestion and purification selection criteria for islet isolation using the automated method and indicate that the morphological aspect may be a clinically relevant measure of islets on which the decision for transplant can be based.     

恒河猴胰岛的半自动分离和纯化

目的探讨恒河猴胰岛分离和纯化的技术方法。方法采用自制的半自动胰腺消化分离装置,对3只成年恒河猴胰腺进行消化分离,用非连续密度梯度高渗枸橼酸盐嘌呤溶液（HCA液）-蔗聚糖液（Ficoll液）纯化恒河猴胰岛,通过双硫腙染色在倒置显微镜计数胰岛的数量和纯度,用胰岛素释放试验检测胰岛的分泌功能。分离和纯化结果采用配对t检验进行统计分析。结果消化后平均每个胰腺可获得（101420±12054）胰岛当量（IEQ）,纯化后平均每个胰腺可获得（71480±8054）IEQ,平均每克恒河猴胰腺组织可获得（2310±252）IEQ,纯化后胰岛平均纯度为（89．8±8．7）％,活率为（93．1±2．8）％。纯化后的胰岛对葡萄糖刺激反应良好,高糖（16．7mmol／L）时胰岛素的释放量为低糖（3．3mmol／L）时的5．9倍（t=45．2,P〈0．01）。分离胰岛在移植到糖尿病大鼠肾包膜下后能够影响降低血糖至支持水平。结论通过采用半自动胰腺消化分离装置,胶原酶P灌注消化及非连续密度梯度法纯化,能获取较高纯度及生物活性良好的恒河猴胰岛。     

The location of VIP in the pancreas of man and rat

Summary VIP has powerful stimulatory effects on both endocrine and exocrine pancreas but its localisation within the gland has not been established. In this study, human pancreas was obtained fresh at surgery (eleven) or within four hours of death (seven). The pancreas was also removed from rats (twenty-two). Immunocytochemical staining showed VIP to be present in fine nerve fibres in all areas of the pancreas. Many fibres were seen in the exocrine pancreas, running between the acini, and around ducts and blood vessels. In addition, dense networks of fibres were observed forming meshes around islets and occasional ganglia were found containing immunoreactive cell bodies. In general, there were fewer VIP fibres in the rat pancreas than in the human, but overall distribution was identical. The mean VIP content of whole human pancreatic tissue was 42±10 pmol/g wet weight (38±9 pmol/g in head, 49±6 pmol/g in body and 42±11 pmol/g in tail). Whole rat pancreatic tissue contained 28±7 pmol/g wet weight while preparations of isolated islets were found to contain 374±30 pmol/g. It is possible that the heavy VIP innervation of the islets described here indicates a role in the regulation of islet hormone release.