人胰岛在分离、纯化过程中的细胞凋亡及氧化损伤

目的 探讨胰岛在获取、分离及纯化等过程中出现细胞凋亡的相关因素及应对策略.方法 经供者腹主动脉灌注4℃的UW液后,切取供者胰腺,并进行胶原酶灌注、消化及梯度离心,分离、提取胰岛.分别于胶原酶灌注前、灌注后、消化后及胰岛分离后等时点,采用原位末端标记法(TUNEL法)检测胰岛细胞凋亡情况,采用比色法检测超氧化物歧化酶(SOD)与丙二醛(MDA)水平,采用HE染色和双硫腙染色观察胰岛的形态学改变.结果 在分离、纯化胰岛的各个过程中,均出现了胰岛细胞凋亡的现象,其中在胶原酶灌注后和消化后最为明显,并伴随MDA的高表达,表达水平分别为(6.18±2.38)nmol/mgprot和(9.21±2.75)nmol/mgprot,均明显高于灌注前的(4.21±1.83)nmol/mgprot(P＜0.05和P＜0.01);而此时SOD水平则显著下降,至胰岛分离后仍处于较低水平,与灌注前相比,差异均有统计学意义(P＜0.05和P＜0.01).胶原酶灌注前,胰岛结构完整;胶原酶灌注后,胰岛周围组织结构膨胀;消化后,胰岛膜性结构被破坏,但能勉强维持岛状结构;胰岛分离后结构基本完整.结论 在提取胰岛的过程中,胶原酶灌注及消化可引起胰岛细胞凋亡,其机制可能与氧化损伤有关,抗氧化措施可作为移植前保护胰岛的手段.

Abstract：

Objective To observe the changes of islet cell apoptosis and oxidation-antioxidation before the transplantation, and to explore the pathways of islet protection. Methods Fifteen human pancreases were perfused with the Hanks solution containing collagenase, then digested and isolated. During the procedure, islet cell apoptosis was detected by TUNEL, SOD and MDA in the pancreas were measured by colorimetric method, and the morphologic changes were observed by H-E staining and dithizone staining. Results In the procedure of human islet isolation, especially in the stage of digestion, the apoptosis of human islet cells occurred. In the stages of perfusion and digestion, the MDA contents reached the high levels (6. 18 ± 2. 38 and 9. 21 ± 2. 75 umol/mg protein respectively),and the structures of the islets and tissues around the islets were damaged. Conclusion In the stages of perfusion and digestion, apoptosis of islet cells can be caused by oxidation. It suggests that antioxidation is a pathway for protection of islets before transplantation.     

骨髓间充质干细胞对同种异体胰岛的保护作用

目的 研究小鼠骨髓间充质干细胞(mesenehymal stem cells,MSCs)对同种异体胰岛的保护作用.方法 将C57BL/6小鼠骨髓间充质干细胞按照3×104/孔的密度预先接种至24孔培养板,次日分离纯化BALB/c小鼠胰岛,将其分为链脲菌素(streptozotocin,STZ)处理(诱导化学损伤)、混合淋巴细胞反应(mixed lymphocyte reaction,MLR)处理(诱导免疫损伤)、空白处理三组,与预先接种的MSCs进行共培养.作为实验对照,另设三组胰岛在不加MSCs的条件下进行体外培养.5 d后,用吖啶橙(acridine orange,AO)/碘化丙啶(propidium iodide,PI)荧光染色评估胰岛活力,葡萄糖刺激的胰岛素分泌实验检测其功能,比较MSCs共培养组与对照组胰岛在低糖、高糖刺激下的胰岛素分泌量及刺激指数(即高糖刺激胰岛素分泌量/低糖刺激胰岛素分泌量比值).结果 AO/PI染色显示,STZ或MLR处理的胰岛中有大量红色荧光的死细胞,而与MSCs共培养的胰岛中死细胞明显减少,绿色荧光的活细胞明显增加;STZ或MLR处理组胰岛在低糖、高糖刺激下的胰岛素分泌水平及刺激指数均显著下降,而与MSCs共培养者较相应对照组胰岛功能明显改善(P＜0.05).结论 小鼠骨髓间充质干细胞可减轻同种异体胰岛的化学及免疫损伤,保护游离胰岛的活力与功能.

Abstract：

Objective To study the protection of mouse bone marrow-derived mesenchymal stem cells ( MSCs) for allogenic islets. Method MSCs from C57BL/6 mice were preceded to a 24-well culture plate with the density of 3 × 104/well. On the second day, islets were isolated, purified and divided to undergo streptozotocin (STZ) induced chemical injury and mixed lymphocyte reaction ( MLR) respectively. Then, treated and control islets were respectively divided into the following groups: islets + MSCs, STZ-islets + MSCs, and MLR-islets + MSCs. As control groups for their counterparts, treated or non-treated islets were also cultured without MSCs. On the 5th day incubation, glucose-stimulated insulin secretion test was performed to assess the function of islets in different groups, comparing their insulin-secretion amount stimulated by low or high glucose and the stimulation index determined by the ratio of (insulin amount secreted under high-glucose stimulation)/(insulin amount secreted under low-glucose stimulation ). Islet viability was evaluated by acridine orange (AO)/propidium iodide (PI) fluorescence staining. Result As shown by AO/PI staining, large numbers of dead cell with red fluorescence could be observed in STZ- or MLR- treated islets without MSCs, while the number of dead cells obviously reduced in MSC-cocultured islets with increased viable cells of green fluorescence. STZ- or MLR- treated islets exhibited apparently decreased insulin-secretion amount either under low- or high-glucose stimulation, as well as the stimulation index. The insulin-secretion function was significantly improved in islets cocultured with allogenic MSCs (P ＜ 0. 05 ). Conclusions Bone marrow-derived MSCs can protect isolated allogenic islets against chemical and immunological injury.     

CD103分子介导CD8+T淋巴细胞对同种胰岛移植物的损伤

目的 检验CD103分子是否介导了CD8+T淋巴细胞对同种移植胰岛的免疫损伤.方法 用流式细胞仪检测野生型C57BL/6小鼠外周血CD8+T淋巴细胞表达CD103的情况.以Balb/c小鼠为供者,C57BL/6小鼠为受者,制作同种胰岛移植模型.受者分为3组:M290-SAP组小鼠注射CD103免疫毒素M290-SAP;M290组小鼠注射抗CD103单克隆抗体M290;另以仅接受胰岛移植、不注射任何药物的小鼠为未处理组.检测移植胰岛CD3、CD8、CD44和CD103阳性细胞的表达,检测肠系膜淋巴结中CD3、CD8和CD103阳性细胞的表达.移植物功能丧失或观察期结束时获取移植胰岛,行HE染色和免疫组织化学染色.结果 野生型C57BL/6小鼠外周血的CD8+T淋巴细胞中有44.06%表达CD103.未处理组移植胰岛浸润的细胞成分中有29%的CD8+T淋巴细胞表达CD103.M290-SAP组小鼠淋巴细胞不仅丧失了CD103的表达,而且CD8+T淋巴细胞的绝对数量也减少,该组小鼠血糖稳定时间超过100 d(未处理组为13 d,P＜0.05),移植胰岛组织学形态良好.结论 CD8+T淋巴细胞免疫损伤同种移植胰岛必须表达CD103,CD103有可能成为胰岛移植抗排斥反应治疗的新靶点.

Abstract：

Objective To test whether the CD103 molecule mediates CD8+ T lymphocytes on allogeneic islet graft immune injury. Methods By using flow cytometry, the expression of CD103 in peripheral CD8+ T lymphocytes in wild-type C57BL/6 mice was detected. Allogenic islet transplantation models were made using Balb/c donor mice and C57BL/6 recipient mice. Recipients were divided into 3 groups: M290-SAP-treated mice were injected with CD103 immunotoxin M290-SAP; M290-treated mice were injected with CD103 monoclonal antibody M290; untreated mice were only transplanted islet without any drug treatment. CD3, CD8, CD44 and CD103 positive cells were counted in islet allograft infiltrative lymphocytes. CD3, CD8, and CD103 positive cells were measured in the mesenteric lymph node. The islet allografts were removed and subjected to HE staining and immunohistochemical staining at the time of graft loss or the end of the observation period. Results 44. 06% peripheral CD8+ T cells expressed CD103 in wild-type C57BL/6 mice. 29 % CD8+ T cells expressed CD103 in the infiltrative lyrnphocytes of islet allografts in the untreated mice. In M290-SAP-treated mice, the lymphocytes had no CD103 expression and the absolute number of CD8+ lymphocytes was decreased as well The blood glucose was maintained stable for more than 100 days (13 days in untreated group, P＜0.05) in the M290-SAP-treated mice. Moreover, the transplanted islets retained intact. Conclusion CD103 expression is required for destruction of pancreatic islet allograft by CD8+ T cells. CD103 might provide a novel target for therapeutic intervention in islet allograft rejection.     

Ghrelin对离体大鼠胰岛分泌胰岛素的影响及其机制

目的 观察外源性Ghrelin对胰岛素分泌和胰岛内向整流钾通道(Kir6.2)表达的影响.方法 Wistar大鼠按10 nmoL/kg的剂量予Ghrelin腹腔注射2周后,分离胰岛进行胰岛素释放实验,分离β细胞检测静息膜电位;检测胰岛Kir6.2 mRNA和蛋白表达变化.结果 在11.1、16.7mmol/L葡萄糖刺激下,对照组的胰岛素释放分别为22.5、43.5 uIU/胰岛/h,Ghrelin组为15.2、30.1uIU/胰岛/h,较对照组显著降低(P＜0.05).对照组β细胞的静息膜电位为(-73.2±24.8)mV,Ghrelin组为(-95.4±33.7)mV,较对照组显著降低(P＜0.05).而Ghrelin组Kir6.2表达显著高于对照组(P＜0.05).结论 Ghrelin与其受体结合后抑制胰岛素分泌,其机制可能与上调Kir6.2表达,使β细胞兴奋性降低有关.

Abstract：

Objective To investigate the influence of ghrelin administration on the insulin secretion, and the expression of Kir6. 2 channels in islets. Methods Ghrelin was intraperitoneally administrated in Wistar rats at the doses 10 nmol/kg every day for 2 weeks. Islets were isolated for insulin release experiments. Single β cells were isolated for electrophysiological experiments. Meanwhile, the expression levels of Kir6. 2 mRNA and protein in islets were detected. Results At 11. 1 and 16. 7 mmol/L glucose,the levels of insulin release in control group were 22. 5 and 43.5 uIU/islet per h, and those in ghrelin-treated group were 15. 2 and 30. 1 uIU/islet per h (P ＜0. 05 ). In control group, the resting membrane potential reached ( - 73.2 ± 24. 8 ) mV, but in ghrelin-treated group, it was hyperpolarized to ( - 95.4 ± 33.7 )mV ( P ＜ 0. 05 ). The Kir6. 2 expression levels were significantly up-regulated by ghrelin ( P ＜ 0. 05 ).Conclusion Ghrelin via pancreatic GHSR up-regulates the Kir6. 2 expression in islets, hyperpolarizing the resting membrane potential, and resulting in the inhibition of insulin release.     

腺病毒载体介导蛋白激酶B基因转染大鼠胰岛的研究

Objective To confirm in vitro gene delivery to rat islets by adenovirus vectors. Methods The adenovirus vector, AdS-Aktl, was constructed following the standard molecular methods. The purified islets were infected with either Ad5-EGFP or Ad5-Akt1 adenovirus the multiplicity of infection (MOI) was 500). The expression of Aktl in Ad5-Akt1 islet transfectants was confirmed by using Western blot. Results Adenovirus vector Ad5-Akt1 was constructed and the the titer of the virus was 5.3 × 109 pfu/ml. The transfection efficiency of Ad5-EGFP was 60% -70%, and Akt1 protein was detected in Ad5-Aktl islet transfectants. Conclusion Adenovirus is efficient to deliver exogenous gene into rat islets in vitro.     

腺病毒载体介导蛋白激酶B基因转染大鼠胰岛的研究

Objective To confirm in vitro gene delivery to rat islets by adenovirus vectors. Methods The adenovirus vector, AdS-Aktl, was constructed following the standard molecular methods. The purified islets were infected with either Ad5-EGFP or Ad5-Akt1 adenovirus the multiplicity of infection (MOI) was 500). The expression of Aktl in Ad5-Akt1 islet transfectants was confirmed by using Western blot. Results Adenovirus vector Ad5-Akt1 was constructed and the the titer of the virus was 5.3 × 109 pfu/ml. The transfection efficiency of Ad5-EGFP was 60% -70%, and Akt1 protein was detected in Ad5-Aktl islet transfectants. Conclusion Adenovirus is efficient to deliver exogenous gene into rat islets in vitro.     

腺病毒载体介导蛋白激酶B基因转染大鼠胰岛的研究

Objective To confirm in vitro gene delivery to rat islets by adenovirus vectors. Methods The adenovirus vector, AdS-Aktl, was constructed following the standard molecular methods. The purified islets were infected with either Ad5-EGFP or Ad5-Akt1 adenovirus the multiplicity of infection (MOI) was 500). The expression of Aktl in Ad5-Akt1 islet transfectants was confirmed by using Western blot. Results Adenovirus vector Ad5-Akt1 was constructed and the the titer of the virus was 5.3 × 109 pfu/ml. The transfection efficiency of Ad5-EGFP was 60% -70%, and Akt1 protein was detected in Ad5-Aktl islet transfectants. Conclusion Adenovirus is efficient to deliver exogenous gene into rat islets in vitro.     

腺病毒载体介导蛋白激酶B基因转染大鼠胰岛的研究

Objective To confirm in vitro gene delivery to rat islets by adenovirus vectors. Methods The adenovirus vector, AdS-Aktl, was constructed following the standard molecular methods. The purified islets were infected with either Ad5-EGFP or Ad5-Akt1 adenovirus the multiplicity of infection (MOI) was 500). The expression of Aktl in Ad5-Akt1 islet transfectants was confirmed by using Western blot. Results Adenovirus vector Ad5-Akt1 was constructed and the the titer of the virus was 5.3 × 109 pfu/ml. The transfection efficiency of Ad5-EGFP was 60% -70%, and Akt1 protein was detected in Ad5-Aktl islet transfectants. Conclusion Adenovirus is efficient to deliver exogenous gene into rat islets in vitro.     

腺病毒载体介导蛋白激酶B基因转染大鼠胰岛的研究

Objective To confirm in vitro gene delivery to rat islets by adenovirus vectors. Methods The adenovirus vector, AdS-Aktl, was constructed following the standard molecular methods. The purified islets were infected with either Ad5-EGFP or Ad5-Akt1 adenovirus the multiplicity of infection (MOI) was 500). The expression of Aktl in Ad5-Akt1 islet transfectants was confirmed by using Western blot. Results Adenovirus vector Ad5-Akt1 was constructed and the the titer of the virus was 5.3 × 109 pfu/ml. The transfection efficiency of Ad5-EGFP was 60% -70%, and Akt1 protein was detected in Ad5-Aktl islet transfectants. Conclusion Adenovirus is efficient to deliver exogenous gene into rat islets in vitro.     

腺病毒载体介导蛋白激酶B基因转染大鼠胰岛的研究

Objective To confirm in vitro gene delivery to rat islets by adenovirus vectors. Methods The adenovirus vector, AdS-Aktl, was constructed following the standard molecular methods. The purified islets were infected with either Ad5-EGFP or Ad5-Akt1 adenovirus the multiplicity of infection (MOI) was 500). The expression of Aktl in Ad5-Akt1 islet transfectants was confirmed by using Western blot. Results Adenovirus vector Ad5-Akt1 was constructed and the the titer of the virus was 5.3 × 109 pfu/ml. The transfection efficiency of Ad5-EGFP was 60% -70%, and Akt1 protein was detected in Ad5-Aktl islet transfectants. Conclusion Adenovirus is efficient to deliver exogenous gene into rat islets in vitro.     

猪胰岛的分离与纯化

目的 探索大规模猪胰岛细胞分离纯化的方法.方法 联合器官切取,胶原酶P主胰管灌注,COBE2991细胞分离机及HCA-Ficoil纯化猪胰岛细胞.通过双硫腙(DTZ)染色,倒置显微镜下计数胰岛细胞的数量和纯度,胰岛素释放试验检测胰岛细胞的分泌功能.结果 消化后平均每条胰腺可平均获得(275 000±20 895)胰岛细胞当量(IEQ),纯化后平均为(230 350±26 679)IEQ,平均每克胰腺组织可获得(2710±229)IEQ,纯化后胰岛细胞平均纯度为(50.2±1.95)%.纯化后的胰岛细胞对胰岛素释放刺激反应良好,高糖(16.7 mmol/L)时胰岛素的释放量为低糖(3.3 mmol/L)时的4.74倍(P≤0.001).结论 成功建立了猪胰岛细胞分离、纯化的方法,纯化的猪胰岛细胞具有良好的生物活性.     

一种经济高效的SD大鼠胰岛细胞分离技术

目的 建立一种经济高效的大鼠胰岛细胞分离纯化方法,为胰腺的修复重建奠定实验基础.方法 成年雄性SD大鼠25只,体重230～380g,共进行5次实验,每5只大鼠一组进行消化和分离.采用医用复方氯化钠注射液(compound sodium chloride injection, CSCI)经胰总管灌注大鼠胰腺,0.5mg/mL V型胶原酶消化后,分别采用浓度为27.0%、23.0%、20.5%和11.0%的Ficoll 400形成不连续密度梯度介质,离心纯化胰岛细胞.双硫腙(dithizon, DTZ)染色行纯化前后胰岛细胞计数和纯度检测;荧光染料碘化丙啶(propidium iodide, PI)和二乙酸荧光素(fluorescein diacetate, FDA)储存液双染色鉴定胰岛细胞活性;RPMIl640培养基培养3d后,分别用浓度为2.8mmol/L的低糖和25.0mmol/L的高糖行葡萄糖刺激胰岛素释放实验检测胰岛细胞功能.结果 5次实验胰岛细胞消化时间为(13.8±1.6)min.DTZ染色鉴定纯化前胰岛细胞数为(5626±422)个,纯化后为(2914±485)个,纯化后的胰岛细胞数较纯化前明显减少(P<0.01),回收率51.6%±6.0%,每个胰腺收获胰岛细胞数为(583±97)个/只.5次分离获得的胰岛细胞纯度为90.2%±3.4%,活性为81.6%±7.0%.培养3d后,葡萄糖刺激胰岛素释放实验显示:低糖环境下胰岛素水平为(39.7±7.5)EU/L,高糖环境为(116.1±17.4)EU/L,比较差异有统计学意义(P<0.01)刺激指数为3.0±0.4.结论 采用CSCI作为大鼠胰岛细胞分离纯化的主要液体试剂,并采用低浓度V型胶原酶消化,不仅可降低实验成本,同时可获得高质量的胰岛细胞.     

三种消化酶在大鼠胰岛分离纯化中的效果比较

目的　分别用 3种酶消化大鼠胰腺 ,比较分离纯化后胰岛细胞的收获量、纯度、活性和功能。方法　将 2 1只SD大鼠按应用释放酶、胶原酶P、胶原酶V消化胰腺随机均分为A、B、C组。结果　A组的胰岛细胞收获量比B组或C组多 ( 93 0± 78比 692± 68或 70 6± 70 ,P <0 .0 5 ) ;纯度比B组或C组高 ( 93 .8± 2 .3比 88.4± 2 .0或 89.2± 2 .1,P <0 .0 5 ) ;成活率比B组或C组高( 97.2± 1.4比 92 .2± 1.9或 92 .9± 2 .1,P <0 .0 5 ) ;A组培养后胰岛素分泌量和葡萄糖刺激时胰岛素分泌量比B组或C组多 (P <0 .0 5 ) ;A组逆转糖尿病大鼠的高血糖状态时间比B组或C组长( 7.5± 1.3比 5 .8± 1.6或 6.3± 1.2 ,P <0 .0 5 )。结论　与胶原酶P和胶原酶V比较 ,释放酶消化大鼠胰腺可提高胰岛细胞的收获量、纯度、活性和功能。     

Influence of donor age on bovine pancreatic islet isolation

BACKGROUND: Pancreatic islets from pigs are largely used for experimental studies. However, pancreas harvesting requires modification of conventional slaughtering to reduce ischemia time. It has been shown that bovine pancreatic islets can be more easily obtained and they show satisfactory in vitro and in vivo function. To improve the isolation procedure we compared the effect of bovine donor age on islet isolation. METHODS: Islets were isolated by collagenase digestion and sequential sieving from calves (6 months of age) and from adult bovine (> 16 months of age). After isolation the number of islet equivalents was calculated and histological and immunohistochemical studies performed. The purity and viability of islet for each preparation was also estimated. In vitro function of islets was evaluated by static insulin secretion assay, and alginate encapsulated islets were transplanted in streptozotocin-induced diabetic rats for in vivo functional evaluation. RESULTS: A significantly higher number of islets were obtained from calf pancreas, compared with adult bovine pancreas. Hystological examination showed intact morphologic features of islets. The purity of islet preparations was higher from calf pancreas than from adult pancreas. Cell viability, and insulin production in presence of high glucose concentration, were not affected by donor age. All animals receiving microencapsulated islets from calves showed normoglycemia for prolonged periods (17-40 days). CONCLUSIONS: These results indicate that pancreatic islet isolation is more efficient from juvenile bovine than from adult. Calf pancreas is a good and convenient source of tissue for massive islet isolation for experimental studies.     

Isolation and Purification of Islet Cells From Adult Pigs

We used in situ perfusion and a multiple-organ harvesting technique to collect islets from adult pig pancreata. The tissues were digested with collagenase P followed by purification in a lympholyte discontinuous gradient using a COBE2991 cell separator. The yield and purity of isolated islets were evaluated with a light microscope after dithizone (DTZ) staining. Islet function was assessed using an in vitro insulin release assay. The results showed that before purification 275,000 ± 20,895 islet equivalents (IEQ) were obtained from 1 digested pancreas. After purification with gradient centrifugation, the islet yield was 230,350 ± 26,679 IEQ/pancreas. Each gram of the purified pancreatic tissues yielded 2710 ± 229 IEQ with an average purity of 50.2 ± 2.0%. The purified islet cells responded to stimulation with high glucose concentrations (16.7 mmol/L), namely, 4.74-fold greater than the insulin secretion with exposure to the basal level of glucose (3.3 mmol/L; P < .001). These results suggested that the established isolation method can be applied to large-scale purification of fully functional islets from pig pancreata.     

Automated method for isolation of human pancreatic islets

We describe an automated method for the isolation of human pancreatic islets. The procedure meets the following requirements: 1) minimal traumatic action on the islets, 2) continuous digestion in which the islets that are progressively liberated can be saved from further enzymatic action, 3) minimal human intervention in the digestion process, and 4) high yield and purity of the isolated islets. After purification on Ficoll gradients, an average of 164,600 islets/pancreas was obtained (2279 islets/g), with an average purity of 78.5% islets. The average volume and average insulin content of the final islet preparation were 348 mm3 and 93.4 U, respectively. The islets were morphologically intact with a normal degree of beta-granulation and responded to glucose stimulation with a fivefold increase of insulin secretion over basal levels. The procedure is now being used for the initiation of the second phase of clinical trials on human islet transplants.     

Studies of the isolation, viability, and preservation of purified islets after surgical pancreatectomy in large pigs

Abstract: Previous studies have shown separation of mass quantities of islets from slaughterhouse pig pancreases. In these studies, we examined the isolation, viability, and preservation of pig islets obtained by surgical pancreatectomy. Pigs aged 6 to 30 months weighing 100 to 200 kg were subjected to laparotomy under general anesthesia. The splenic lobe of pancreas was mobilized without warm ischemia. Islets were immediately isolated by collagenase perfusion through the duct, automated dissociation, and Ficoll purification. Yields of islets (150 μm size) were 8500 to 9300/g for two different collagenases before purification. Purification yielded 90% pure islets, but yields decreased to 400 to 600/g. Perifusate insulin release was biphasic after glucose/theophylline with 3 to 5-fold stimulation. Following culture/cryopreservation marked islet losses occurred but viability was preserved. Quantities of 1,500 islet equivalents resulted in euglycemia in nude mice. These data show that mass quantities of viable islets can be isolated after pancreatectomy, but there is marked islet loss during purification and subsequent preservation due to inherent islet fragility.     

Comparison of automated and manual methods for islet isolation

The authors used the principal features of a collagenase perfusion technique and an automated dissociation technique to determine if islets could be isolated from the large mammal pancreas and to compare the effects of the two methods on isolated islets. The pancreases of 16 dogs were cannulated and perfused with collagenase at 4 degrees C, then warmed to 37 degrees C. Group 1 (eight) pancreases were perfused at 37 degrees C until digested, then dissociated manually by teasing and trituration. Group 2 (eight) pancreases were transferred to a closed chamber for continued collagenase digestion and dissociation at 37 degrees C. Islets were purified using identical Ficoll gradients. Aliquots were stained with dithizone and evaluated for number, size and purity. Total islet volume was calculated. Group 2 pancreases were thoroughly digested leaving only a few residual ducts, but undigested fragments persisted in group 1 pancreases. Islet size was similar in both groups. There was a greater islet volume before and after Ficoll purification in group 2, but the difference was not significant. Purity was greater than 90% in both groups. Perifusion with 28 mM glucose elicited a biphasic insulin release from islets in both groups. The data show that the combined protocol enables mass isolation of purified islets from the canine pancreas. Compared with the manual technique, the automated protocol for pancreas dissociation tends to improve the yield of islets without compromising islet size and viability. It provides the advantages of a closed system with increased control over the extent of collagenase digestion.     

Improvement of porcine islet isolation by inhibition of trypsin activity during pancreas preservation and digestion using α1-antitrypsin

Porcine islets are considered to be a promising resource for xenotransplantation. However, it is difficult to isolate porcine islets because of the marked fragility and rapid dissociation. Endogenous trypsin is one of the main factors to damage islets during the isolation procedure. Recent studies have suggested that trypsin inhibitors during the preservation of pancreas or the collagenase digestion can improve the result of islet isolation. In this study, we examined whether α1-antitrypsin (Aralast?), which inhibits several endogenous proteases and has immunomodulatory properties, can protect islets from the proteases and improve the results of porcine islet isolation. Twelve porcine pancreata were divided into three groups: without Aralast group (standard, n = 5), preserved with Aralast using the ductal injection (DI) method (DI, n = 3), and with Aralast using the DI method and in the collagenase solution (DI+C, n = 4). Efficacy of islet isolation was assessed by islet yields, purity, and viability. The trypsin activity of the preservation and the digestion solution during the isolation procedure was measured. During islet isolation, the trypsin activity in DI+C group was significantly inhibited compared to the standard group, whereas DI group showed less effect than DI+C group. The average of postpurification islet equivalents (IEQ) per pancreas weight in the DI+C group was significantly higher than the standard group (standard: 3516 ± 497 IEQ/g, DI: 4607 ± 1090 IEQ/g, DI+C: 7097 ± 995 IEQ/g; p = 0.017 between standard and DI+C). In the DI+C group, stimulation index was higher than in other groups, although there was no significant difference. The presence of Aralast in both DI solution and collagenase solution markedly inhibited trypsin activity during pancreas digestion procedure and improved the porcine islet isolation. Inhibition of trypsin activity by Aralast could improve porcine islet isolation.     

国产胶原酶在小鼠胰岛分离中的效果研究#br#

目的 研究一种国产胶原酶在小鼠胰岛分离中的分离效果,探索该胶原酶在胰岛分离中应用的 可行性。方法 将国产胶原酶分别配成不同浓度的胶原酶溶液和含中性蛋白酶的胶原酶的溶液,经胰管逆行灌注胶原酶的方法进行小鼠胰岛分离,采用Ficoll液对胰岛进行纯化,计数所得的胰岛细胞团,培养 6 h后检测活性,对最佳分离结果的胰岛做同系糖尿病小鼠的肾被膜下移植,监测术后血糖和进行组织学 检查,以进口Sigma胶原酶V为对照。结果 国产胶原酶在小鼠胰腺消化时间、所得胰岛数量和活性效果 上跟进口胶原酶V有一定差距（P＜0.01）,但含中性蛋白酶的国产胶原酶组在小鼠胰岛分离数量和当量上 与进口胶原酶组无差异（P＞0.05）,分离的胰岛在体内移植后降血糖效果与进口胶原酶组无差异（P＞0.05）。 结论 国产胶原酶结合中性蛋白酶可用于小鼠胰岛的分离。