大鼠的不同纯度胰岛中干细胞标志物CK-19及PDX-1 mRNA的表达

目的探讨大鼠的不同纯度胰岛中干细胞标志物细胞角质蛋白19(CK-19)和胰十二指肠同源盒基因1(PDX-1)mRNA的表达。方法将30只雄性SD大鼠随机平均分成3组,均采用胶原酶Ⅴ型通过胰管对胰腺进行灌注,切取胰腺,剪碎、吹打、消化、离心获得胰岛细胞沉淀物。A组胰岛沉淀物不进行纯化;B组胰岛沉淀物中加入体积分数为25%的Ficoll-400液纯化;C组胰岛沉淀物依次加入25%及11%的Ficoll-400纯化。分别将上述3组纯化后的胰岛抽提RNA行逆转录聚合酶链反应(RT-PCR),分析胰岛干细胞标志物CK-19及PDX-1mRNA的表达。结果采用不同的纯化方法后,A、B、C三组获得胰岛的纯度分别为(43.6±6.29)%、(65.3±4.40)%和(77.6±6.36)%,三组间比较,差异有统计学意义(P<0.05)。A组胰岛中CK-19和PDX-1mRNA的表达明显高于B组和C组,而C组表达最弱。结论不同纯度的胰岛中均有CK-19及PDX-1mRNA的表达,表明在纯化胰岛中均有干细胞存在。     

成年猪胰岛的分离与纯化

目的　研究成年猪胰岛的分离与纯化方法。方法　胰岛的分离采用导管内连续灌注胶原酶消化法 ,纯化采用Nycodenz连续密度梯度离心。 结果　消化后每个胰腺平均可获得 (3970 0 0±185 96 3)个胰岛或 (986 0± 3 0 43)个胰岛 /克胰腺组织 ,纯化后为 (1480 0 0± 890 0 0 )个胰岛 /胰腺或(3 75 0± 493)个胰岛 /克胰腺组织 ,纯度 >95 %。体外培养示胰岛对糖刺激反应良好 ;免疫组织化学检查证实胰岛细胞存活。结论　经本法获得的胰岛具有较高的纯度及良好的功能 ,可为临床提供可靠的移植物来源。     

两种酶对猪胰岛消化分离效果的对比

目的　研究单纯酶 (胶原酶Ⅳ )与复合酶 (由胶原酶Ⅰ、Ⅳ、弹性蛋白酶、纤维素酶组成 )对成年猪胰岛的分离效果。方法　采用经胰管灌注消化酶的方法分离胰岛 ,用双硫腙染色做胰岛计数 ,台盼蓝染色判定胰岛活化水平 ,胰岛培养第 2d、第 4d、第 6d测定胰岛素含量 ,培养第 6d做胰岛活性评价 ,电镜观察胰岛的形态结构。结果　经复合酶消化法制备的胰岛数量为 (4 915±l0 42 )个胰岛 /克胰腺 ;而经单纯酶消化的胰岛数量为 (30 12± 989)个胰岛 /克胰腺。两组比较差异显著 (P <0 .0 5 )。结论　复合酶消化法是一种优于单纯酶消化法的成年猪胰岛分离方法。     

CTLA4Ig基因对大鼠胰岛移植后排斥反应的治疗作用

目的　研究CTLA4Ig基因在糖尿病大鼠体内表达及其产物对胰岛移植物存活的作用。方法　利用Lipofectin载体包裹CTLA4IgcDNA质粒后转染鼠胰岛和肌肉细胞 ,检测移植后CT LA4Ig表达和T淋巴细胞转化率。结果　胰岛移植术后 7dT淋巴细胞转化试验 ,实验组 (A组 )和对照组 (B组 )每分钟脉冲数 (cpm)分别为175 .7± 98.2 ,2 5 4.4± 116 .3 ,两组比较差异显著 (P <0 .0 5 )。A组胰岛移植第 7d ,2只大鼠血清CTLA4Ig呈阳性 (阳性率 2 0 % )。A、B两组胰岛移植后血糖维持正常时间分别为 (14.8± 12 .3)d和 (3 .6± 5 .1)d ,两组比较差异显著 (P <0 .0 5 )。A、B两组大鼠平均存活时间分别为 (2 4.0± 10 .8)d和 (10 .8± 4.8)d ,两组比较 ,差异有极显著性 (P <0 .0 1)。结论　脂质体包裹的CTLA4IgcDNA转染肌细胞和胰岛细胞 ,可以在受体大鼠胰岛细胞或肌肉组织中表达 ,其表达产物可使胰岛移植物和受体鼠存活时间明显延长 ,抑制细胞免疫活性 ,发挥其治疗排斥反应的作用     

成人胰岛细胞的分离

目的　探讨成人胰岛细胞的分离及胰腺冷缺血时间与胰岛细胞活率的关系。方法　采取胰、肾联合切取 ,经腹主动脉插管 ,用自制的灌注器以高渗枸橼酸盐嘌呤溶液 (HC A液 ) 15 0 0～2 0 0 0ml进行原位灌洗。将肾与胰腺、十二肠、脾分离 ,采用胶原酶P消化胰腺 ,分离并纯化胰岛细胞 ,以双硫腙及丫腚橙染色 ,测定所得到的胰岛细胞的纯度及活率。结果　胰腺的冷缺血时间为 2 .5～ 8h ,温缺血时间为 0～ 3min。胰岛收获量为 (2 .38± 0 .6 7)× 10 3 IEQ/g ,纯化后胰岛细胞的活率为 19%～ 83% ,冷保存指数 (或输注指数 )为 9.8～ 6 6 .4。胰岛细胞的活率与胰腺的冷缺血时间呈负相关 ,冷保存指数和胰岛细胞活率呈负相关。结论　以高渗枸橼酸盐嘌呤溶液灌洗、胶原酶P消化胰腺所得到的胰岛细胞活率高 ,但胰腺的冷缺血时间不宜超过 5h。     

不同纯化方法对大鼠胰岛收获效果的研究

目的 探讨不同纯化方法对提高大鼠胰岛收获效果的作用。方法 随机选取SD雄性大鼠20只,按胰腺消化和胰岛纯化的方法的不同分为4组,消化过程中胆总管灌注胶原酶V,每组5只：①A组：Ficoll-400法纯化胰岛。②B组：光镜下移液吸移管手工挑拣纯化（手工法）胰岛。③C组：胰腺消化过程加入乌司他丁5000U／L．Ficoll-400法纯化胰岛。④D组：胰腺消化过程加入乌司他丁5000U／L,手工法纯化胰岛。结果 纯化后胰岛的收获量分别为A组（122±25．30）个,B组（179±21．02）个,C组（187±24．89）个和D组（276±24．85）个。乌司他丁灌注组（C组和D组）胰岛细胞的收获量与相应纯化方法的无乌司他丁灌注组（A组和B组）差异有统计学意义（P〈0．01）。手工法纯化组（B组和D组）胰岛的收获量与Ficoll-400纯化组（A组和C组）差异有统计学意义（P〈0．01）。结论 手工法可明显提高胰岛的收获量,乌司他丁灌注胰腺对大鼠胰岛分离具有保护作用。     

不同纯度大鼠供体胰岛中巢蛋白、神经元素3的表达

目的 观察巢蛋白（Nestin）、神经元素（Ngn）3在不同纯度大鼠胰岛中的表达。方法 将30只成年雄性SD大鼠随机分为3组，采用胰管内注入胶原酶消化法分离成年大鼠胰腺，Ⅰ组（n=10）：胰岛沉淀物不进行纯化；Ⅱ组（n=10）：胰岛沉淀物加入25％Ficoll-400纯化，Ⅲ组（n=10）：胰岛沉淀物依次加入25％、11％的Ficoll-400纯化。逆转录-聚合酶链反应（RT-PCR）检测Nes．tin和Ngn3mRNA在不同纯度胰岛细胞中的表达。结果 Ⅰ组获得胰岛纯度为（43．60±6．29）％，Ⅱ组获得胰岛纯度为（65．30±4．40）％，Ⅲ组获得胰岛纯度为（77．60±6．36）％，各组差异均有统计学意义（P〈0．05）。Ⅰ组中Nestin和Ngn3的mRNA表达明显高于Ⅱ组、Ⅲ组，Ⅲ组中表达最弱。结论 Nestin、Ngn3在不同纯度胰岛细胞中均有表达，但是在低纯度胰岛中表达最明显，提示低纯度胰岛中可能携带较多的干细胞。     

大豆胰蛋白酶抑制剂对大鼠胰岛分离纯化的影响

目的探讨大豆胰蛋白酶抑制剂（STI）在大鼠胰岛分离纯化中对胰岛产量及功能的影响。方法按胶原酶中是否加入STI将大鼠分为实验组和对照组，实验组在胶原酶消化液中按2．0mg／ml加入STI，对照组不添加STI。2组均运用胶原酶原位灌注大鼠胰腺的方法来分离胰岛，使用Ficoll-400用非连续梯度离心法纯化胰岛，将纯化前、后获得的胰岛进行计数，并对纯化后的胰岛进行形态、功能检查。同时进行大鼠同种异体胰岛移植观察其体内功能。结果实验组和对照组在消化后纯化前所得胰岛数量无明显差别[（624&#177;38．2）IEQVS（586&#177;37．7）IEQ，P〉0．053；在纯化后实验组与对照组所得胰岛数量[（408&#177;28．3）IEQVS（189&#177;27．1）IEQ，P〈0．05]和纯度[（93&#177;2．4）％VS（75&#177;2．1）％，P〈0．05；差异有统计学意义。实验组与对照组最终所得胰岛的体外功能差异无统计学意义（P〉0．05），体内功能实验结果差异亦无统计学意义（P〉0．05）。结论使用在胶原酶中加入STI的消化液原位灌注大鼠胰腺，可以明显提高大鼠胰岛的最终产量和纯度，但对胰岛的功能无明显影响。     

Fas配体阳性睾丸细胞和环孢素A对移植胰岛细胞存活起协同保护作用

目的　探讨Fas配体 (FasL)阳性睾丸细胞与胰岛细胞共移植后联用环孢素A(CsA)对移植胰岛细胞存活的协同保护作用。　方法　将同种大鼠胰岛细胞与睾丸Sertoli细胞同侧或异侧共移植于 4 1只糖尿病SD大鼠受体肾包膜下。实验大鼠分 7组 ,术后酌用CsA ,观察各组大鼠移植物存活情况。　结果　单纯胰岛细胞移植 (对照组 )后胰岛细胞的平均存活期为 (4 6± 1 1)d ,加用CsA存活期明显延长至 (2 1 8± 4 7)d(P <0 0 1)。与 1× 10 7个睾丸细胞同侧共移植的胰岛细胞平均存活期超过 (5 7 5± 4 0 )d ,但如移植前先封闭睾丸细胞表达的FasL后 ,移植的胰岛细胞平均存活期缩短为(5 8± 2 6 )d。胰岛细胞与 1× 10 5个睾丸细胞分别共移植于两侧肾包膜下 ,术后联用CsA ,胰岛细胞的平均存活期超过 (5 5 0± 6 5 )d ,与 1× 10 7个睾丸细胞同侧共移植的存活期相近 ,但比对照组或CsA组则显著延长 (P <0 0 1)。当胰岛细胞与 1× 10 6个睾丸细胞分别共移植且不用CsA时 ,胰岛细胞存活期平均仅为 (11 5± 3 1)d ,但仍较对照组延长 (P <0 0 5 )。　结论　表达FasL的睾丸细胞与CsA联用后可通过不同机制抑制胰岛细胞移植排斥反应而起到全身的协保护作用。     

实验大鼠胰岛分离移植技术方法的比较分析

目的 探索高效的大鼠胰岛分离移植技术方法.方法 应用Wistai-Furth大鼠,于体内或体外胶原酶经胰管灌注膨化胰腺,联合不同密度Ficoll液或Histopaque液纯化胰岛细胞,评估胰岛的数量、纯度、胰岛当量以及肾被膜下胰岛移植的有效性.结果 体外经胰管灌注膨化胰腺结合Histopaque液纯化提取胰岛的数量、纯度和胰岛当量值均显著高于体内灌注组各数值(P＜0.01),其提取时间无显著差别.1000个胰岛细胞移植进入左肾被膜下,有效的逆转了糖尿病大鼠高血糖,其远期糖耐受结果优于500和800个胰岛细胞移植组.结论 体外灌注膨化消化胰腺结合Histopaque液纯化胰岛的分离方法是一种满意的分离技术.1000个胰岛细胞是保证肾被膜下胰岛移植成功的最低有效数量.     

Significant progress in porcine islet mass isolation utilizing liberase HI for enzymatic low-temperature pancreas digestion.

BACKGROUND: Frequent success in human islet isolation is prevented by the large variability of scarce organ donors; this favors the future utilization of pigs as donors for clinical islet xenotransplantation. Porcine-specific difficulties of islet isolation are attributed to the intrinsic fragility of islets during pancreas digestion. METHODS: To preserve islet integrity during efficient pancreas dissociation, porcine pancreata (n=48) were distended after cold storage with cold University of Wisconsin solution containing Liberase HI and digested at 24-28 degrees C using digestion-filtration. Pancreata distended with University of Wisconsin solution containing well-proven crude collagenase and digested at 32-34 degrees C served as controls (n=46). Monolayer Ficolldiatrizoate gradient purification was performed in a Cobe 2991. RESULTS: Purified yield of islet equivalents per pancreas (mean+/-SEM) was almost doubled by Liberase HI compared with crude collagenase (526,480+/-46,560 vs. 270,270+/-19,420; P < 0.0001) and also significantly increased comparing islet equivalents per gram of pancreas (4,210+/-320 vs. 2,640+/-245; P=0.0004). Islet integrity was better preserved during Liberase HI digestion compared with crude collagenase digestion as indicated by isolation index (2.1+/-0.1 vs. 1.4+/-0.1; P<0.0001). Purity, viability, and in vitro function of islets did not differ between experimental groups. Preserved in vivo function of islets isolated by Liberase HI was demonstrated after subcapsular transplantation into 16 diabetic nude rats. CONCLUSIONS: If the problems related to xenograft rejection and xenosis could be solved, low-temperature digestion of porcine pancreata using Liberase HI could serve as an essential prerequisite for successful 1:1 xenotransplantation of pig islets into type 1 diabetic human recipients.     

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.     

Successful human islet isolation utilizing recombinant collagenase

The enzymatic dissociation of acinar tissue by collagenase is a substantial step in the isolation of pancreatic islets. Although essential collagenase components have been purified, the variability in the activity of different batches limits long-term reproducibility of isolation success. The utilization of purified recombinant proteases would solve this problem. In the present study, pancreases from multiorgan donors were dissociated by means of digestion-filtration using either Liberase HI (n = 51) or a recombinant collagenase blend (n = 25). No significant differences were found regarding islet yield before and after purification, the percent of exocrine-attached islets, and final purity. However, the ratio between islet equivalents and islet numbers indicated a lesser fragmentation in islets isolated with recombinant collagenase (P < 0.01). In contrast, viability was slightly higher in islets isolated with Liberase (92.3 +/- 0.8 vs. 85.6 +/- 2.9%; P < 0.05). Insulin release during static glucose incubation was not different between experimental groups. Islet transplantation into diabetic nude mice resulted in sustained normoglycemia in either group until the graft was removed. These results demonstrated that viable human islets can be isolated using recombinant collagenase. Final optimization of this enzyme blend would offer continuous reproducibility of isolation success.     

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.     

The use of iodixanol for the purification of rat pancreatic islets

Transplantation of pancreatic islets is a promising therapeutic treatment for type 1 diabetes mellitus. For clinical and experimental transplantation, a large number of pure pancreatic islets are required for transplantation. Thus, the improvement of islet isolation and purification techniques are crucial. In this context, iodixanol-based solution, successfully used for the purification of porcine islets, seems to be a possible alternative to Ficoll for purification of islets. The aim of this study was to test the efficacy of iodixanol compared with Ficoll density gradients for the purification of rat pancreatic islets. Twelve Wistar rats were used for isolation and purification of pancreatic islets. Pancreata were digested with Liberase R1 and islets purified by two gradients: Ficoll or iodixanol gradient. The number and the purity of the pancreatic islets were assessed. To analyze the response of isolated pancreatic islet to glucose challenge, in vitro experiments were performed by measuring the insulin concentration in the Supernatant. The results demonstrated that the iodixanol gradient provided a higher purity of pancreatic islets compared to the Ficoll gradient. In addition, the rat islet yield by iodixanol gradient was significantly higher compared to a Ficoll gradient (751 +/- 16 versus 464 +/- 19 pancreatic islets, respectively; P < .001). The viability of pancreatic islets isolated by an iodixanol gradient was confirmed by high glucose challenge, with more than twofold higher increase in insulin secretion. The present study demonstrated that iodixanol density gradient overcomes Ficoll density gradient, providing a greater number of pure and functional rat pancreatic islets.     

Bovine serum albumin density gradient isolation of rat pancreatic islets

The use of a bovine serum albumin (BSA) density gradient for isolation of rat pancreatic Islets of Langerhans after collagenase digestion has been compared with the standard Ficoll separation technique. The criteria studied were islet yield (insulin extraction of the islet interfaces and pellet), purity of preparation (amylase content of the islet preparation), insulin release characteristics, and the result of isologous transplantation in diabetic rats. The islet interface of the BSA gradient contained 62.2% of the total insulin, whereas the corresponding interface of the Ficoll gradient contained only 36.6% (P less than 0.001). The amylase content of the Ficoll-separated islet preparation was 6133 U/L, as opposed to 1230 U/L with BSA (P less than 0.001). BSA-isolated islets gave similar insulin release characteristics to non-density-gradient-isolated islets, whereas Ficoll-separated islets showed suboptimal insulin release. Single-donor-single-recipient transplantation was successfully performed with BSA-isolated islets whereas multiple donors were required with Ficoll-separated islets. Thus significantly improved results were found with the bovine serum albumin density gradient separation in all criteria and consequently the use of this gradient represents an advance in islet isolation techniques.     

Effective islet isolation method with extremely high islet yields from adult pigs

Achieving good islet isolation is one of the most important factors for successful islet transplantation. Porcine pancreas is suitable for islet isolation research due to its anatomical and physiological similarities to human pancreas. In this study, we evaluated a new porcine islet isolation method designed to maximize islet yield and compared it with our previous open pan method and the standard method using a Ricordi chamber (Ricordi method). We performed 15 porcine islet isolations, five each with the new method, the open pan method, and the Ricordi method. The new method features several important improvements. Pancreata remain uncut and are kept intact during collagenase intraductal injection, a large filtration chamber to handle whole pancreata, low concentration of collagenase (Liberase HI) for digestion, and large plastic containers for large-scale islet purification. All isolated islets were assessed for yield, purity, viability and in vitro function. Islets isolated with this new method were transplanted under the kidney capsules of SCID mice with chemically induced diabetes for in vivo functional assessment (n = 8). With the new method, we obtained on average more than 1,000,000 islet equivalents (IE) (1,236,266 +/- 213,486 IE) (mean +/- SE) before purification and 800,000 IE (879,815 +/- 222,729 IE) after purification from one adult pig. Islet yield per pancreas was significantly higher compared with our previous open pan method (30,666 +/- 11,532 IE, p < 0.01) and the Ricordi method (317,073 +/- 86,093 IE, p < 0.05). All mice, transplanted with 1000 islets from the new method, returned to normoglycemia within 4 days after transplantation. Our new method makes it possible to obtain extremely high porcine islet yield with good function. It should produce useful information for human islet isolation and transplantation, and might be applied to single donor clinical xenogeneic transplantation.     

Evaluation of viable β-cell mass is useful for selecting collagenase for human islet isolation: comparison of collagenase NB1 and liberase HI

The selection of enzyme blend is critical for the success of human islet isolations. Liberase HI collagenase (Roche) was introduced in the 1990s and had been widely used for clinical islet transplantation. More recently, a blend collagenase NB1 has been rendered available. The aim of this study was to evaluate the isolation outcomes and islet quality comparing human islet cells processed using NB1 and Liberase HI. A total of 90 isolations processed using NB1 (n = 40) or Liberase HI (n = 50) was retrospectively analyzed. Islet yield, function in vitro and in vivo, cellular (including β-cell-specific) viability and content, as well as isolation-related factors were compared. No significant differences in donor-related factors were found between the groups. There were also no significant differences in islet yields (NB1 vs. Liberase: 263,389 ± 21,550 vs. 324,256 ± 27,192 IEQ; p = n.s., respectively). The pancreata processed with NB1 showed a significantly longer digestion time (18.6 ± 0.7 vs. 14.5 ± 0.5 min, p < 0.01), lower β-cell viability (54.3 ± 3.4% vs. 72.0 ± 2.1%, p < 0.01), β-cell mass (93,671 ± 11,150 vs. 148,961 ± 12,812 IEQ, p < 0.01), and viable β-cell mass (47,317 ± 6,486 vs. 106,631 ± 10,228 VβIEQ, p < 0.01) than Liberase HI. In addition, islets obtained with Liberase showed significantly better graft function in in vivo assessment of islet potency. The utilization of collagenase NB1 in human islet isolation was associated with significantly lower β-cell viability, mass, and islet potency in vivo in our series when compared to Liberase HI, even though there was no significant difference in islet yields between the groups. Evaluation of viable β-cell mass contained in human islet preparations will be useful for selecting enzyme blends.     

大鼠胰岛的分离纯化方法改进与功能鉴定

目的 通过改进胰腺消化和分离的技术条件,提高成年大鼠胰岛分离纯化产率和质量. 方法 用胶原酶Ⅺ液灌注消化成年SD大鼠胰腺,对胰岛分离纯化方法加以改进:以 4 种比重的 Euro- Ficoll (F1∶D=1.132,F2∶D=1.108,F4∶D=1.069) 和 Hank's 液(F5∶D=1.023) 不连续密度梯度离心,以离心半径 15 cm,2 000 r/min 于4℃缓慢升降离心 20 min,收集位于F1 和 F2界面的胰岛.双硫腙特异染色法鉴定胰岛纯度;二醋酸酯荧光素/碘化丙啶染色法计算胰岛成活率;放射免疫分析法检测葡萄糖刺激的胰岛素分泌量,计算刺激指数.将胰岛当量(islets equivalent quantity,IEQ) 为 1000 的胰岛移植于同品系糖尿病大鼠肾包膜下,9d 内隔日观察动物血糖的变化,评价胰岛功能.比较分离条件优化前后收获胰岛的产率和质量. 结果 改进纯化方法后每只大鼠胰岛收获量为(920±122) IEQ,胰岛纯度> 90%,胰岛细胞成活率为 91%±2%.胰岛细胞功能良好,在低糖和高糖刺激后培养液中胰岛素浓度分别为(18.25±0.32) mU/L 和(36.70±3.57)mU/L,刺激指数为 2.01±0.15.1000 IEQ 胰岛移植于糖尿病大鼠肾包膜下,观察期内可维持动物血糖水平正常. 结论 改进后的胶原酶灌注消化和不连续梯度离心方法提高了胰岛的产率,保证了胰岛的高纯度及高成活率.