Ex Vivo Evaluation in Normal Dogs of Insulin Released by a Bioartificial Pancreas Containing Isolated Rat Islets of Langerhans

In bioartificial pancreatic systems, isolated islets of Langerhans are protected against immune rejection by an artificial membrane, permeable to glucose and insulin, but not to immunoglobulins and lymphocytes. Some of these devices, referred to as vascular systems, are set up to be connected to a vascular site in the recipient, with blood circulating in contact with one side of the membrane, and the islets on the other side. Such a bioartificial pancreas, containing isolated rat islets of Langerhans, was connected to an arteriovenous shunt of a normal anesthetized dog. The aim of this experiment was to investigate the kinetics of the insulin secretory response of the system to a glucose load. Glucose was infused upstream of the system, increasing the glucose concentration inside the bioartificial pancreas from 7 to 14 mmol/l, without altering the blood glucose concentration of the dog. Insulin concentration was determined simultaneously upstream and downstream of the bioartificial pancreas. Insulin production was calculated by multiplying the difference between these values by the blood flow rate. Blood flow rate (Q) was estimated from the change in the glucose concentration produced by the glucose infusion using a mass transfer analysis derived from Fick's principle. Insulin production increased from 20 +/- 8 to 59 +/- 15 microU/100 islets/min within 15 min following the beginning of the stimulation (n = 6, p less than 0.05). Five min after the end of the stimulation, insulin production decreased from 75 +/- 13 to 50 +/- 9 microU/100 islets/min (p less than 0.05) to reach the basal level (21 +/- 3 microU/100 islets/min) 30 min after the end of the glucose stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

非糖皮质激素的免疫抑制方案对同种异体移植胰岛的保护作用

目的　评价非糖皮质激素的免疫抑制方案对防止大鼠同种异体胰岛移植排斥反应的效果。方法　大鼠同种异体胰岛移植后 ,分别应用他克莫司 (FK5 0 6 ) 霉酚酸酯 (MMF)和FK5 0 6 MMF 泼尼松 (Pred)行免疫抑制治疗两周 ,并设对照组 ,动态观察受者血糖、胰岛素及C肽变化 ,并作移植部位的形态学检测。结果　FK5 0 6 MMF组和FK5 0 6 MMF Pred组与对照组相比 ,移植胰岛存活时间明显延长 ,移植后 2个月在受者肝汇管区可见较多形态完整的胰岛细胞。FK5 0 6 MMF组维持术后正常血糖、胰岛素及C肽的时间超过 6 0d ,而FK5 0 6 MMF Pred组与前者比较 ,分泌C肽较少 (P <0 .0 5 ) ,血糖维持在较高水平 (P <0 .0 1) ,胰岛素水平两组差异无显著性。FK5 0 6 MMF Pred组停药两周以后的血糖水平较用药期间、停药两周内有明显降低 (P <0 .0 5 ) ,胰岛素和C肽分泌有所增多 ,但差异无显著性。结论　应用FK5 0 6 MMF和FK5 0 6 MMF Pred均有很强的免疫抑制效应 ,但糖皮质激素对胰岛细胞有毒性作用。小剂量FK5 0 6与MMF联用对移植胰岛细胞有较强的保护作用。     

包裹胰岛微囊的物理性能研究

用３％海藻酸钠及２．２％ＣａＣｌ＿２所制备的微囊在高流速及高浓度的蔗糖溶液冲击下未见破裂，且微囊包裹活细胞经过二小时的剧烈搅拌，亦无损漏。改进法所制备的微囊具有良好的机械强度，其物理性能已达到临床进行包膜胰岛移植治疗糖尿病的要求。     

A One–Step, Operator–Independent Method for Isolating Islets of Langerhans from the Porcine Pancreas

Abstract: Large–scale isolation of islets of Langerhans is one of the major obstacles in islet transplantation. Until now, isolation methods relied on enzymatic digestion, the duration of which relies on a decision dictated by the operator's experience. This approach has always hindered development of an automated method. The aim of this study was to develop a one–step method based on complete digestion of the pancreas. The original aspect of the technique (derived from the Ricordi method) is use of the University of Wisconsin (UW) solution in the digestion medium and a continuous flow collagenase processing circuit with local cooling and rewarming to allow tissue digestion to proceed at 37°C while settling of the cell suspension takes place at 4°C. A stopcock system permits the alternate use of two settling chambers so that while one is in the circuit, the other can be removed for cen–trifugation, resuspension of the crude islet preparation in collagenase in free UW solution, and further purification in a density gradient system. Ten experiments were performed, and 545, 750 ± 48, 670 purified pig islets were obtained per totally digested pancreas. Histological studies showed cell integrity. Insulin secretion in response to double glucose stimulation under perfusion conditions demonstrated the functional viability of the isolated islets. In conclusion, this one–step method makes it possible to obtain a high number of viable islets of Langerhans in the absence of any decision by an operator, and it should therefore provide basis for an automated method.     

IL-1β、TNF-α和IFN-γ引起的大鼠胰岛细胞凋亡及牛磺酸的影响

目的： 观察白细胞介素-1β(IL-1β)、肿瘤坏死因子-α（TNF-α）和γ-干扰素（IFN-γ）引起胰岛细胞凋亡、胰岛素分泌、Bcl-xL和Bax蛋白表达变化及其牛磺酸的影响。方法： 应用体外单层培养Wistar大鼠胰岛细胞，分别检测IL-1β、TNF-α和 IFN-γ对胰岛细胞凋亡细胞百分率、DNA片段、培养液中NO^-₂/ NO^-₃含量及NOS活性、胰岛素分泌、Bcl-xL和Bax表达的影响，并进一步观察牛磺酸的作用。结果： IL-1β、TNF-α和 IFN-γ联合可诱导胰岛细胞凋亡率明显增加，DNA明显片段化，同时NO^-₂/ NO^-₃含量和NOS活性亦明显升高，胰岛素分泌明显降低, Bcl-xL表达下降和Bax表达增强（P<0.01）；牛磺酸能阻断上述细胞因子的作用（P<0.01），并有一定的剂量依赖性。结论：牛磺酸能够改善IL-1β、TNF-α和 IFN-γ诱导的胰岛细胞凋亡，其机制可能与抑制NOS活性从而减少NO的生成以及下调Bax/Bcl-xL比例有关。     

Effects of Na+, K+ and Mg2+ on45Ca2+ uptake by pancreatic islets

Microdissected pancreatic islets of noninbredob/ob-mice were used to study ionic effects on the lanthanum-nondisplaceable⁴⁵Ca²⁺ uptake by islet cells. Omission of Mg²⁺ from the incubation medium had no effect, but the⁴⁵Ca²⁺ uptake was increased by omission of Na⁺ and decreased by omission of K⁺. Excess Mg²⁺ (1.2–15 mM) inhibited and excess K⁺ (4.7–25 mM) stimulated the⁴⁵Ca²⁺ uptake in a concentration-dependent manner. Stimulation of⁴⁵Ca²⁺ uptake in Na⁺-deficient islets was associated with an enhancement of the basal insulin release. Total abolishment of glucose-stimulated⁴⁵Ca²⁺ uptake in K⁺-deficient islets did not preclude a significant secretory response to glucose. It is concluded that the lanthanum-nondisplaceable⁴⁵Ca²⁺ uptake shows a partial correlation to insulin release.     

人胰岛微血管和A细胞的微机三维重建

采用微机辅助三维重建的方法重建了人胰岛的微血管的A细胞，结果表明，所使用的软件重建速度快，图像速真，清晰度高，视觉效果好，可对重建物体进行多角度，多轴旋转，也可进行任何部位的切割，以便能进一步了解胰岛内各结构的详细情况，为进一步研究胰岛内微血管和细胞的立体位置关系提供方法学基础。     

The microanatomy of canine islets of Langerhans: implications for intra-islet regulation

Summary In recent years models for the internal (intra-islet) regulation of hormone secretion have been proposed to explain how different islet cells might regulate each other by means of their respective secretory peptides. Models that emphasize the importance of a directed intra-islet blood flow and sequence of perfusion of islet cells rely on a certain type of islet microanatomy and vascular supply. The experimental studies underlying these models have partly been performed in dogs. To extend the incomplete morphological knowledge of the canine endocrine pancreas both canine islets of Langerhans and extrainsular cells have been analysed in immunostained serial semithin (0.5 m) sections. In addition to their occurrence within islets of Langerhans, all endocrine cell types are also found at extrainsular sites (about 9% of all endocrine cells) where they are distributed in different quantities among the epithelial lining of exocrine acini or excretory ducts and the connective tissue. There are continuous transitions from single extrainsular cells to small mono-and polycellular cell groups to islets. In a comprehensive analysis of whole islets, including computer-assisted three-dimensional reconstructions, the size, shape and vascularization of the islets as well as their cellular composition and the microtopology of islet cells have been studied. We have found marked intra-and inter-islet heterogeneities of the parameters investigated that are not compatible with concepts of a uniform and directed vascular perfusion of the various islet cell populations. Instead, their paracrine regulation may occur primarily via hormonal secretion into the intercellular spaces or vascular hormonal delivery to adjacent cells.     

Bcl-2对免疫抑制剂培养中胰岛细胞的保护作用

目的探讨免疫抑制剂对体外培养的大鼠胰岛细胞的毒性作用以及抗凋亡基因Bc l-2对胰岛细胞的保护作用。方法腺病毒介导的基因转染后表达Bc l-2的大鼠胰岛细胞和对照胰岛细胞分别加入不同浓度的他克莫司培养基中,48 h后检测胰岛细胞的凋亡率和分泌胰岛素的功能。结果低剂量和高剂量的他克莫司都能诱导胰岛细胞凋亡,减少胰岛素分泌,Bc l-2能抑制凋亡,改善胰岛素分泌。结论他克莫司对体外培养的大鼠胰岛细胞有直接的损害作用,而Bc l-2能防治这种损害作用。     

球形脂联素、葡萄糖和游离脂肪酸对胰岛β细胞单磷酸腺苷激活的蛋白激酶和乙酰辅酶A羧化酶磷酸化的影响

目的:探讨不同浓度的葡萄糖和游离脂肪酸对胰岛β细胞内单磷酸腺苷激活的蛋白激酶(adenosine-5'-monophosphate activated protein kinase,AMPK)和乙酰辅酶A羧化酶(acetyl CoA carboxylase,ACC)磷酸化的影响,以及球形脂联素对AMPK和ACC磷酸化的作用.方法:培养INS-1胰岛细胞系,用5 mmol/L葡萄糖和0.25 mmol/L游离脂肪酸处理,确定在不同时间对AMPK和ACC磷酸化的影响;观察不同浓度葡萄糖和游离脂肪酸对AMPK和ACC磷酸化的影响;在葡萄糖和游离脂肪酸存在的条件下观察AMPK的药理激活剂氨基咪唑-4-甲酰胺核苷酸(AICAR)和球形脂联素对AMPK和ACC磷酸化的影响.结果:不同浓度的葡萄糖和游离脂肪酸都能够在60 min时抑制AMPK和ACC的磷酸化水平,采用AMPK的药理激动剂AICAR能够明显升高AMPK和ACC的磷酸化水平.2.5 mg/L球形脂联素可以使基础状态的AMPK和ACC磷酸化水平分别增加23%(P＜0.05)和50%(P＜0.05).在5 mmol/L葡萄糖的基础上加用球形脂联素使AMPK和ACC磷酸化水平分别增加1.4倍(P＜0.05)和3倍(P＜0.01),在0.25 mmol/L游离脂肪酸的基础上加用球形脂联素使AMPK和ACC磷酸化水平分别增加3倍(P＜0.05)和5倍(P＜0.01).结论:在体外培养的胰岛INS-1细胞中,不同浓度的葡萄糖和游离脂肪酸能够降低AMPK和ACC的磷酸化水平,而AMPK的药理激动剂氨基咪唑-4-甲酰胺核苷酸(5'-aminoimidazole-4-carboxamide riboside,AICAR)和2.5 mg/L球形脂联素可以提高AMPK和ACC的磷酸化水平,此作用可以进一步增强胰岛β细胞内的脂肪酸氧化水平,减轻甘油三酯聚集,保护胰岛β细胞功能.