Small intestinal submucosa improves islet survival and function during in vitro culture

AIM: To evaluate the recovery and function of isolated rat pancreatic islets during in vitro culture with small intestinal submucosa (SIS). METHODS: Pancreatic islets were isolated from Wistar rats by standard surgical procurement followed by intraductal collagenase distension, mechanical dissociation and Euroficoll purification. Purified islets were cultured in plates coated with multilayer SIS (SIS-treated group) or without multilayer SIS (standard cultured group) for 7 and 14 d in standard islet culture media of RPMI 1640. After isolation and culture, islets from both experimental groups were stained with dithizone and counted. Recovery of islets was determined by the ratio of counts after the culture to the yield of islets immediately following islet isolation. Viability of islets after the culture was assessed by the glucose challenge test with tow (2.7 mmol/L) and high glucose (16.7 mmol/L) solution supplemented with 50 mmol/L 3-isobutyl-1-methylxanthine (IBMX) solution. Apoptosis of islet cells after the culture was measured by relative quantification of histone-complexed DNA fragments using ELISA. RESULTS: After 7 or 14 d of in vitro tissue culture, the recovery of islets in SIS-treated group was significantly higher than that cultured in plates without SIS coating. The recovery of islets in SIS-treated group was about twice more than that of in the control group. In SIS-treated group, there was no significant difference in the recovery of islets between short- and long-term periods of culture (95.8±1.0% vs 90.8±1.5%, P>0.05). When incubated with high glucose (16.7 mmol/L) solution, insulin secretion in SIS-treated group showed a higher increase than that in control group after 14 d of culture (20.7±1.1 mU/L vs 11.8±1.1 mU/L, P<0,05). When islets were placed in high glucose solution containing IBMX, stimulated insulin secretion was higher in SIS-treated group than in control group. Calculated stimulation index of SIS-treated group was about 23 times of control group. In addition, the stimulation index of SIS-treated group remained constant regardless of short- and long-term periods of culture (9.5±0.2 vs 10.2±1.2, P>0.05). Much less apoptosis of islet cells occurred in SIS-treated group than in control group after the culture. CONCLUSION: Co-culture of isolated rat islets with native sheet-like SIS might build an extracellular matrix for islets and provide possible biotrophic and growth factors that promote the recovery and subsequent function of islets.     

Pancreatic islet isolation variables in non-human primates (rhesus macaques)

BACKGROUND: Non-human primates (NHPs) are important preclinical models for pancreatic islet transplantation (PIT) because of their close phylogenetic and immunological relationship with humans. However, low availability of NHP tissue, long learning curves and prohibitive expenses constrain the consistency of isolated NHP islets for PIT studies. To advance preclinical studies, we attempted to identify key variables that consistently influence the quantity and quality of NHP islets. METHODS: Seventy-two consecutive pancreatic islet isolations from rhesus macaques were reviewed retrospectively. A scaled down, semi-automated islet isolation method was used, and monkeys with streptozotocin-induced diabetes, weighing 3-7 kg, served as recipients for allotransplantation. We analysed the effects of 22 independent variables grouped as donor factors, surgical factors and isolation technique factors. Islet yields, success of isolation and transplantation results were used as quantitative and qualitative outcomes. RESULTS: In the multivariate analysis, variables that significantly affected islet yield were the type of monkey, pancreas preservation, enzyme lot and volume of enzyme delivered. The variables associated with successful isolation were the enzyme lot and volume delivered. The transplant result was correlated with pancreas preservation, enzyme lot, endotoxin levels and COBE collection method. CONCLUSIONS: Islet quantity and quality are highly variable between isolations. The data reviewed suggest that future NHP isolations should use bilayer preservation, infuse more than 80 ml of Liberase into the pancreas, collect non-fractioned tissue from the COBE, and strictly monitor for infection.     

Review of experimental attempts of islet allotransplantation in rodents:Parameters involved and viability of the procedure

The purpose of the present study was to organize the parameters involved in experimental allotransplantation in rodents to elaborate the most suitable model to supply the scarcity of islet donors. We used the PubMed database to systematically search for published articles containing the keywords "rodent islet transplantation" to review. We included studies that involved allotransplantation experiments with rodents’ islets, and we reviewed the reference lists from the eligible publications that were retrieved. We excluded articles related to isotransplantation, autotransplantation and xenotransplantation, i.e., transplantation in other species. A total of 25 studies related to allotransplantation were selected for systematic review based on their relevance and updated data. Allotransplantation in rodents is promising and continues to develop. Survival rates of allografts have increased with the discovery of new immunosuppressive drugs and the use of different graft sites. These successes suggest that islet transplantation is a promising method to overcome the scarcity of isletdonors and advance the treatment options for type 1 diabetes.     

不同血型成人胰腺用于ICA检测的比较研究

目的应用免疫组化法(ABC法)检测ICA,比较研究A型、B型、AB型与O型4种不同血型成人胰腺抗原底物对ICA检测的特异性和敏感性的影响。方法取24例1型糖尿病病人的ICA阳性不同JDF单位的血清标本和23例正常人ICA阴性血清标本,以A型、B型、AB型与O型4种不同血型成人胰腺的冰冻切片为抗原底物,应用ABC免疫组化法检测ICA。结果以O型血成人胰腺为对照,应用3例A型和B型血胰腺和2例AB型血胰腺为抗原底物进行ICA检测,24例ICA阳性血清和23例ICA阴性血清检测结果的特异性和敏感性基本一致,未见假阳性或假阴性结果。结论本文研究结果证明A型、B型或AB型血成人胰腺与O型血成人胰腺一样,可用于ICA免疫组化(ABC法)检测,不同血型人胰腺作为抗原底物不影响ICA免疫组化法检测的特异性和敏感性。     

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.     

移植胰岛功能保护的研究进展

移植胰岛的坏死和凋亡严重影响胰岛移植的效果。近年来通过改善供体胰腺组织的保存、胰岛分离和纯化方法,抑制非特异性炎症反应,减轻经血液介导的瞬时炎症反应,诱导免疫耐受和基因修饰胰岛移植等措施,移植胰岛的功能保护研究取得了一定进展。     

大囊包被胰岛非低温长期保存的研究

目的　研究一种理想的、能维持胰岛活性和功能的长期保存方法。方法　作者采用琼脂糖和胶原包被大鼠胰岛后于３７ ℃、５ ％ Ｃ Ｏ２ 孵箱中培养，并在体内外条件下研究存放后胰岛的活性和功能。结果　此方法能保存胰岛达４０ 周之久，并能维持胰岛的正常形态、活性及胰岛素分泌功能。接受保存１ ～２７ 周的大囊包被胰岛移植后，９２ ．８ ％ 的糖尿病小鼠血糖恢复正常，大部分宿主的正常血糖持续到大囊包被胰岛取出为止。结论　采用琼脂糖和胶原包被胰岛并培养于３７ ℃、５ ％ Ｃ Ｏ２ 孵箱中是移植前长期保存胰岛的理想方法。     

Influence of heme oxygenase-1 gene transfer on the viability and function of rat islets in in vitro culture

AIM: To investigate the influence of heme oxygenase-1 (HO-1) gene transfer on the viability and function of cultured rat islets in vitro. METHODS: Islets were isolated from the pancreata of Sprague-Dawley rats by intraductal collagenase digestion, and purified by discontinuous Ficoll density gradient centrifugation. Purified rat islets were transfected with adenoviral vectors containing human HO-1 gene (Ad-HO-1) or enhanced green fluorescent protein gene (Ad-EGFP), and then cultured for seven days. Transfection was confirmed by fluorescence microscopy and Western blot. Islet viability was evaluated by acridine orange/propidium iodide fluorescent staining. Glucose-stimulated insulin release was detected using insulin radioimmunoassay kits and was used to assess the function of islets. Stimulation index (SI) was calculated by dividing the insulin release upon high glucose stimulation by the insulin release upon low glucose stimulation. RESULTS: After seven days culture, the viability of cultured rat islets decreased significantly (92% +/- 6% vs 52% +/- 13%, P < 0.05), and glucose-stimulated insulin release also decreased significantly (6.47 +/- 0.55 mIU/L/30IEQ vs 4.57 +/- 0.40 mIU/L/30IEQ, 14.93 +/- 1.17 mIU/L/30IEQ vs 9.63 +/- 0.71 mIU/L/30IEQ, P < 0.05). Transfection of rat islets with adenoviral vectors at an MOI of 20 was efficient, and did not impair islet function. At 7 d post-transfection, the viability of Ad-HO-1 transfected islets was higher than that of control islets (71% +/- 15% vs 52% +/- 13%, P < 0.05). There was no significant difference in insulin release upon low glucose stimulation (2.8 mmol/L) among Ad-HO-1 transfected group, Ad-EGFP transfected group, and control group (P > 0.05), while when stimulated by high glucose (16.7 mmol/L) solution, insulin release in Ad-HO-1 transfected group was significantly higher than that in Ad-EGFP transfected group and control group, respectively (12.50 +/- 2.17 mIU/L/30IEQ vs 8.87 +/- 0.65 mIU/L/30IEQ; 12.50 +/- 2.17 mIU/L/30IEQ vs 9.63 +/- 0.71 mIU/L/30IEQ, P < 0.05). The SI of Ad-HO-1 transfected group was also significantly higher than that of Ad-EGFP transfected group and control group, respectively (2.21 +/- 0.02 vs 2.08 +/- 0.05; 2.21 +/- 0.02 vs 2.11 +/- 0.03, P < 0.05). CONCLUSION: The viability and function of rat islets decrease over time in in vitro culture, and heme oxygenase-1 gene transfer could improve the viability and function of cultured rat islets.     

抗体预处理对胰岛移植物的影响

目的：研究抗猪B淋巴细胞抗血清预处理胰岛制备物对其免疫原性和功能的影响。方法：用混合成年猪B淋巴细胞为抗原制备抗血清,预处理猪ICCs。以胰岛－淋巴细胞混合培养和胰岛内SLA－DR阳性细胞数检测胰岛的免疫原性;以胰岛素释放试验检测胰岛功能。结果：经抗猪B淋巴细胞抗血清预处理组胰岛－淋巴细胞混合培养的cpm值,MHCI类抗原和SLA－DR抗原阳性细胞数较对照组均明显降低,有非常显著性差异（P＜0．001）,胰岛素释放值无明显差异,结论：应用抗猪B淋巴细胞血清预处理能降低胰岛制备物的免疫原性,对胰岛素释放功能无明显影响。     

年龄对胰腺部分切除的小鼠胰岛细胞再生和凋亡的影响

目的 探讨年龄对胰岛细胞再生的影响,初步揭示胰岛再生的机制以及与老龄性糖尿病的关系.方法 对清洁级3月龄[n=12,体重（16.7±1.9）g]、6月龄[n=12,体重（25.4±2.3）g]、12月龄[n=12,体重（28.5±3.0）g]近交品系C57B6小鼠胰腺切除65%～70%,在饮用水中加入BrdU以标记增殖细胞,各组动物分别于1、2、4周处死取材,通过测量空腹血糖、静脉糖耐量并应用免疫组织化学的方法对胰岛细胞的再生以及胰岛细胞的凋亡进行全面的研究.所得数据通过Graphpad Prism 5.0软件的2-way ANOVA进行统计学处理.结果 在3月龄的年轻小鼠部分胰腺切除后,术后1、2、4周胰岛β细胞再生率分别为15.1%±10.0%、25.5%±8.1%、45.7%±14.6%;6月龄小鼠胰岛再生率分别为23.9%±8.7%、28.3%±7.6%和27.2%±5.7%;而对于12月龄老龄的小鼠,胰岛细胞再生率分别为4.4%±6.9%、6.1%±5.8%和5.1%±7.0%,用GraphPad Prism 5.0软件进行2-way ANOVA分析表明时间、年龄对胰岛再生均有影响（P＜0.0001,P＜0.05）,各组间差异有统计学意义.3月龄的小鼠1、2、4周胰岛β细胞凋亡率分别为2.7%±2.4%、3.7%±3.4%、3.1%±3.6%,12月龄的小鼠1、2、4周胰岛β细胞凋亡率分别为5.6%±1.4%、5.8%±4.3%、6.7%±2.6%,3月龄小鼠凋亡的β细胞数量明显少于12月龄的小鼠（P＜0.05）.结论 随着年龄的增长,小鼠胰岛β细胞的再生能力明显降低,并且更易发生凋亡.这可能是导致老龄人群糖尿病发病率升高的主要原因之一.     

Improved vascular engraftment and function of autotransplanted pancreatic islets as a result of partial pancreatectomy in the mouse and rat

Aims/hypothesis The few patients subjected to autotransplantation of pancreatic islets after pancreatectomy usually become normoglycaemic after using islets from the resected organ only, whereas allogeneic recipients usually require at least two grafts to retain normoglycaemia. Previous experimental studies have demonstrated that islets transplanted to non-pancreatectomised recipients acquire a markedly decreased blood vessel density, which leads to a hypoxic microenvironment. The aim of the present study was to test the hypothesis that autotransplanted islets have better vascular engraftment and function as a result of the pancreatic surgery involved. Materials and methods In the present study, athymic mice and inbred rats were subjected to a 60% pancreatectomy and transplanted with human or rat islets, respectively, 4 days later. Control animals underwent sham surgery. Blood flow, oxygen tension, vascular density and endocrine volume in the islet grafts were measured 1 month after transplantation. Separate grafts were used for perfusion experiments and for assessment of beta cell proliferation and endocrine cellular apoptosis at different time periods after transplantation. Results Islet grafts in partially pancreatectomised recipients had an increased blood flow, oxygen tension, blood vessel density and endocrine mass 1 month post-transplantation compared with control animals. They also exhibited increased insulin release in perfusion experiments performed 1 month post-transplantation, and decreased cellular apoptosis early after transplantation. Conclusions/interpretation The present study shows that the pancreatectomy procedure itself has beneficial effects on the engraftment of transplanted human and rat islets. Our results provide an additional explanation, besides diminished immunological responses, of the much better outcome of islet autotransplantations compared with allogeneic transplantations in the clinic.     

介导胰岛新生的蛋白质研究进展

传统观念认为,胰岛是不可再增殖、分化的终末期细胞,但晚近的研究发现有一系列蛋白质可介导胰岛的新生,如甲状旁腺激素相关蛋白、肝细胞生长因子、胎盘催乳素、胰升糖素样肽1、β细胞调节素、胰岛素样生长因子1和2、Reg蛋白和胰岛新生相关蛋白等,这些蛋白可促进胰岛β细胞增殖、分化、新生并抑制其凋亡。这为胰岛新生疗法的临床应用带来了希望,如可以用于移植后胰岛的保护以根治1型糖尿病。     

Suitability of human juvenile pancreatic islets for clinical use

AIMS/HYPOTHESIS: The limited availability of deceased donor pancreases suitable for pancreas and islet transplantation calls for a broader utilisation of donor tissue for transplantation purposes. Young donors, representing, fortunately, a minor but significant pool of individuals, have been largely under-employed, mainly because of anatomical and functional incompatibilities with potential recipients. For islet transplantation, the isolation of pancreatic islets from young donors rarely occurs, because of technical problems. As a result of the peculiar characteristics of young donor pancreases, the standard isolation procedure does not allow efficient separation of the islets from the surrounding exocrine tissue, and favours the generation of mantled islets. Nonetheless, young donor islets offer high qualitative and clinically appealing characteristics. SUBJECTS AND METHODS: We standardised a modified methodology to obtain purified and mantle-free human islets from young donors. This method principally involves efficient delivery of isolation enzyme with reduced mechanical disruption of the pancreas combined with additional filtration steps. RESULTS: We were able to obtain purified and mantle-free human islets from donors as young as 6 months of age with good morphological and functional properties. The good qualitative characteristics of the islets, evidenced in vitro, were proven in vivo, as they were qualitatively superior to islets of older donors in transplantation studies. CONCLUSIONS/INTERPRETATION: This study justifies the utilisation of islets derived from young donors for islet transplantation.     

肾上腺髓质素在胰岛素瘤患者体内的表达情况

目的研究胰岛细胞瘤患者体内肾上腺髓质素的表达与分布情况;并通过对比研究阐明其表达的特异性.方法收集9例手术治疗的胰岛素瘤患者手术前后的血浆及手术标本,分别用放免法检测血浆中肾上腺髓质素浓度;免疫组化ABC法测定肾上腺髓质素在肿瘤组织中的表达与分布.结果胰岛素瘤患者体内的肾上腺髓质素浓度(7.1±2.7 fmol/m1)比正常对照组(2.6±1.6 fmol/m1)显著增高.在手术前后监测的9例中,血浆肾上腺髓质素浓度从7.1±2.7fmol/ml下降到2.9±1.7 fmol/ml.免疫组化结果显示肾上腺髓质素分布于肿瘤细胞中,而在胰腺基质和主要的胰腺导管中无表达.结论胰岛素瘤患者血浆中肾上腺髓质素明显增高,且在肿瘤组织中明显表达.     

Expression of the electrogenic Na+–HCO3?-cotransporters NBCe1-A and NBCe1-B in rat pancreatic islet cells

It was recently proposed that, in rat pancreatic islets, the production of bicarbonate accounts for the major fraction of the carbon dioxide generated by the oxidative catabolism of nutrient insulin secretagogues. In search of the mechanism(s) supporting the membrane transport of bicarbonate, the possible role of the electrogenic Na⁺–HCO₃ ⁻-cotransporters NBCe1-A and NBCe1-B in rat pancreatic islet cells was investigated. Expression of NBCe1-A and NBCe1-B in rat pancreatic islet cells was documented by RT-PCR, western blotting, and immunocytochemistry. The latter procedure suggested a preferential localization of NBCe1-B in insulin-producing cells. Tenidap (3–100 μM), previously proposed as an inhibitor of NBCe1-A-mediated cotransport in proximal tubule kidney cells, caused a concentration-related inhibition of glucose-stimulated insulin secretion. It also inhibited 2-ketoisocaproate-induced insulin release and to a relatively lesser extent, the secretory response to l-leucine. Tenidap (50–100 μM) also inhibited the metabolism of d-glucose in isolated islets, increased ²²Na net uptake by dispersed islet cells, lowered intracellular pH and provoked hyperpolarization of plasma membrane in insulin-producing cells. This study thus reveals the expression of the electrogenic Na⁺–HCO₃ ⁻-cotransporters NBCe1-A and NBCe1-B in rat pancreatic islet cells, and is consistent with the participation of such transporters in the process of nutrient-stimulated insulin secretion.     

Remission of Digestive Insufficiency by Islet Transplantation to the Pancreas

Pancreatic digestive insufficiency is a common problem in both Type 1 and Type 2 diabetes and remains a serious consequence of diabetes in developing countries. The problem is not corrected by supportive therapies including exogenous insulin injections. It is our hypothesis that digestive insufficiency may be corrected or diminished by the transplantation of islets to the pancreas, thereby supplying islet hormones directly to acinar tissue analogous to the normal pancreas. Diabetic rats received 1000 syngeneic islets and dogs received 7600 autologous islets per kilogram as a transplant to the pancreas. Blood glucose and amylase concentrations were normalized in islet recipients in contrast with controls receiving no islets or islets transplanted to the renal capsule. These results suggest that diabetic digestive insufficiency may be corrected by intrapancreatic islet transplantation.     

实验性链脲佐菌素糖尿病动物模型的研究

采用腹腔内１次注射６０ｍｇ／ｋｇ体重ＳＴＺ方法，建立了速发型链脲佐菌素Ｗｉｓｔａｒ大鼠糖尿病模型。采用每周１次连续３周腹腔内注射（ＣＦＡ０．５ｍｌ和ＳＴＺ（２５ｍｇ／ｋｇ）方法，建立了迟发型Ｗｉｓｔａｒ大鼠糖尿病模型。结果提示：速发型模型建立的机制与ＳＴＺ直接损伤胰β细胞有关，迟发型大鼠糖尿病模型胰β细胞损伤可能与Ｔ淋巴细胞介导的免疫机制有关。