小鼠胰岛的分离及胰岛移植

目的研究小鼠胰岛分离和移植的方法。方法在Gotoh等所介绍的小鼠胰岛分离方法的基础上作了一些改进,由原来经胆总管注入胶原酶消化液改为由胆囊注入,并在消化液和Ficoll分离液中加入了胰酶抑制剂和BSA。结果使分离纯化后的胰岛产量由原来方法的41.7±13.2个提高到了266.5±32.1个(P<0.01),活性在95%以上,除了少量导管外几乎不含腺泡组织。结论改进后的方法可以在肉眼条件下注入消化液,而不需要解剖显微镜,既方便了操作又提高了成功率;避免了整个消化和分离过程中胰酶对胰岛的消化作用,提高了得率,且具有很好的重复性。     

升清降浊胶囊对db/db小鼠肾组织PDGF-B表达的影响

目的:观察升清降浊胶囊改善自发性2型糖尿病db/db小鼠肾损伤的作用及对肾组织血小板衍化生长因子-B(PDGF-B)表达的影响.方法:将16只8周龄雄性db/db小鼠随机分为db/db模型组(n=8)和升清降浊组(n=8),选取db/m小鼠为空白对照组(n=8),升清降浊组给予升清降浊胶囊1800 mg·kg-1·d-...     

一种快速分离纯化小鼠胰岛的方法

目的 介绍一种快速分离纯化小鼠胰岛的方法及进行纯化后胰岛的活性、完整性和胰岛内结构的质量分析.方法 雄性ICR小鼠,采用2 mg/mL胶原酶V灌注和消化,并用Hanks液快速洗涤,用Histopaque（R）-1077和Histopaque（R）-1119密度梯度离心对胰岛进行纯化,用手工方法进行胰岛挑选,DTZ、FD-PI染色鉴定胰岛纯度及其活性,透射电镜观察胰岛内的结构.结果 胰岛开始消化至手工挑选前过程耗时＜ 25 min,每只小鼠得到胰岛数量为：128±36,当量为：145±42,纯度＞90％.透射电镜显示胰岛内部血管仍有损伤.结论 采用此方法可快速得到数量较多、结构较完整的小鼠胰岛,且活性高,为进一步进行胰岛的体外质量研究及体内移植奠定了基础.     

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.     

小鼠胰岛的分离纯化及体外功能检测的实验研究

目的：探讨获得高质量小鼠胰岛的分离纯化方法，评价其功能。方法：采用胆总管内胶原酶灌注膨胀消化胰腺的方法分离小鼠胰岛，不连续密度梯度离心法纯化胰岛，用双硫腙（Dithizone，DTZ）对胰岛进行特异性染色计算胰岛产量及纯度，以葡萄糖和茶碱刺激胰岛素释放检测胰岛功能。结果：胰岛的产量和活性主要与胰腺均匀膨胀和胶原酶的消化时间有关。平均每个小鼠胰腺能得到150～250个高质量胰岛，活性〉95％，纯度〉90％。葡萄糖及茶碱（carbachol，Cch）刺激后胰岛素释放量明显增加。结论：改良的胆总管内胶原酶灌注膨胀消化小鼠胰腺及不连续密度梯度Ficoll-400纯化胰岛的方法，可获得产量较高、纯度及功能较好的胰岛。     

新型胰岛分离液对小鼠胰岛分离效果的研究

目的  探讨新型胰岛分离液（IPS液）在小鼠胰岛分离中的分离效率及胰岛保护作用。方法  将消化后的小鼠胰腺按体积平均分为两组（IPS组和UW组）,分别应用IPS-Optiprep液及UW-Optiprep液进行连续梯度密度离心分离胰岛,比较两组分离液的分离纯化效率和分离后的胰岛活性。将诱导成功的糖尿病小鼠随机分为3组：实验组（n=10）,接受采用IPS-Optiprep液分离纯化的胰岛移植;对照组（n=10）,接受采用UW-Optiprep液分离纯化的胰岛移植;假性移植组（n=5）,仅给予手术但并不进行胰岛移植。分析3组小鼠术后血糖水平以及测实验组和对照组小鼠术后21 d的腹腔葡萄糖耐量试验的血糖水平。比较两种分离液的配制成本。结果  与UW组相比,IPS组的IPS-Optiprep液可提供更高的胰岛当量（IEQ）、胰岛纯度、回收率及胰岛完整度。胰岛形态观察可见,IPS组胰岛被膜完整,直径明显大于UW组。UW组纯化后的胰岛活性率高于IPS组[（88±5）%比（84±3）%,P < 0.01]。与UW-Optiprep液相比,IPS-Optiprep液可获得相当的在体胰岛功能。IPS-Optiprep液可显著降低胰岛分离纯化成本。结论  新型胰岛分离液IPS-Optiprep液在胰岛分离提纯中显示出较好的分离效率,增加了胰岛的纯度、完整度与回收率,并显著降低了纯化成本,但对胰岛细胞活性的保护作用稍逊,可能与胰岛的高完整度及IPS-Optiprep液中的内毒素有关。     

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。     

大鼠胰岛细胞的分离培养与鉴定

目的 探讨胰岛细胞的分离、培养与鉴定。方法 从实验室中挑选出30只成年雄性大鼠,将胶原酶注入大鼠的胰腺导管,聚蔗糖梯度离心法分离胰岛细胞,并培养与鉴定胰岛细胞。培养1周后用双硫腙（DTZ）行胰岛细胞鉴定。用丫啶橙（AO）、碘丙啶（PI）检测胰岛细胞活性。结果 经过分离与纯化,大鼠胰岛细胞的获得率较高且较稳定;分离后的胰岛细胞在适宜环境中生长,生长状态较佳,在培养后的12~24 h可贴壁,在培养后4~5 d细胞生长状态达到顶峰,细胞完好,且折光性能优异。利用免疫组化法鉴定胰岛细胞,分离的细胞SMA及PDGFR表达呈阳性,少数细胞vWF表达呈阳性。结论 我科此次分离纯化大鼠胰岛细胞的方法为逆行灌注胶原酶+原位消化+Ficoll液梯度分离法,实验结果显著,能在一定程度上获取纯度较高的大鼠胰岛细胞。分离后的胰岛细胞在培养4~5 d后达到最佳状态,适用于作为实验室胰岛细胞功能深入研究的实验材料。     

几种小鼠胰岛的分离纯化方法比较

目的探讨几种小鼠胰岛的分离纯化方法及进行纯化后胰岛的计数和完整性的分析。方法选用ICR小鼠,采用不同胶原酶消化方法和不同的胶原酶种类,并用Ficoll-PaqueTMPLUS密度梯度离心法对胰岛进行纯化,并对获得的胰岛进行DTZ染色计数和计算当量,扫描电镜考察胰岛的完整性。结果采用胶原酶V和P胰管灌注、内外消化结合,消化需时较短,胶原酶V和P在胰岛纯化前后每只小鼠收获的胰岛细胞数量和当量无差别(P>0.05);扫描电镜结果显示消化较好的胰岛表面被膜完整,消化过度的胰岛导致被膜不完整,易致外层细胞损伤。结论小鼠逆行胰管灌注胶原酶、内外消化相结合的胰岛消化方法所需时间较短,但同时要注意防止消化过度。     

Isolation of porcine pancreatic islets for xenotransplantation studies: Effects of low collagenase digestion temperatures

Abstract: Islets of Langerhans were isolated from porcine pancreata by a modification of our previously described method. The modification involved the use of a low temperature of collagenase digestion (30°C) during the process of islet isolation. The resulting islets were then evaluated in vitro and in vivo and compared to islets isolated at the regular 37°C temperature.
The islets produced at the low temperature were more compact compared to the control islets. In the dextran density gradient these islets were deposited at the interface of the 1.060 and 1.068 g/ml density bands as compared to 1.050 and 1.060 g/ml for the control islets. In addition, the experimental islets contained a higher proportion of compact, unfragmented islets (68%) compared to the regular islets (55%), and their uptake of the dithizone stain was considerably slower than with the control islets. All ten batches of freshly isolated microencapsulated islets produced at both temperatures responded to the glucose stimulation. After 4 weeks of in vitro culture the islets of both groups microencapsulated in alginate-polylysine-alginate (APA) microcapsules still retained glucose responsiveness, with the experimental islets demonstrating significantly higher responsiveness to the high glucose (16.7 mM) and 0.1 mM IB MX stimulation. The morphology of unencapsulated islets in the experimental group following 4 weeks of in vitro culture indicates much firmer islet structure compared to the control islets. In addition, the unencapsulated experimental islets following the 4 week culture were still found to have secreted insulin when exposed to glucose. In transplantation studies both the experimental and the control islets normalized diabetic hyperglycemia in diabetic mice in a comparable fashion. In general, the low temperature digestion results in superior islets in terms of their morphology, viability, and physiological function.     

Renoprotective effect of COMP-angiopoietin-1 in db/db mice with type 2 diabetes

Background. Inflammatory processes have been recently seen asunderlying the pathogenesis of diabetic nephropathy. Angiopoietin-1(Ang1) plays essential roles in regulating vascular growth,development, maturation, permeability and inflammation. We havedeveloped a soluble, stable and potent Ang1 variant, cartilageoligomeric matrix protein (COMP)-Ang1. Methods. In this study, db/db mice were treated with recombinantadenovirus expressing either COMP-Ang1 or LacZ. Histology, inflammatory,metabolic, and fibrotic parameters and signalling pathway wereevaluated. Results. COMP-Ang1 reduced albuminuria and decreased mesangialexpansion, thickening of the glomerular basement membrane andpodocyte foot process broadening and effacement. COMP-Ang1 suppressedboth renal expression of intercellular adhesion molecule-1 andmonocyte chemoattractant protein-1 and monocyte/macrophage infiltrationin diabetic db/db mice. COMP-Ang1 also reduced renal tissuelevels of transforming growth factor-ß1 (TGF-ß1),-smooth muscle actin, fibronectin, as well as Smad 2/3 expression,but increased Smad 7 expression. In human umbilical vein endothelialcells (HUVECs) grown in high glucose concentrations of glucose,recombinant COMP-Ang1 protein decreased nuclear factor-B (NF-B)expression. COMP-Ang1-mediated inhibition of increased NF-B-DNAbinding in nuclear extracts from HUVECs grown in high glucosewas significantly blocked by soluble Tie2 receptor-Fc. In addition,COMP-Ang1 significantly decreased fasting blood glucose level,epididymal fat weight to body weight ratio, and epididymal adipocytesize in diabetic db/db mice. After intraperitoneal glucose challenge,COMP-Ang1 significantly lowered plasma glucose levels. However,there was no difference in serum insulin levels. Conclusion. We conclude that COMP-Ang1 delayed the fibroticchanges in the kidney of diabetic db/db mice through its anti-inflammatoryor metabolic effects.     

Culture of the islets of Langerhans for transplantation

The isolated islets of Langerhans are the most available donors for transplantation. As the preservation of the isolated islets is difficult, we attempted to keep these tissues viable by use of an organ culture. Islets of Langerhans from adult Wistar rats were isolated by a collagenase technique and cultured in air-CO₂ (95-5%) incubator at 37°C. Insulin contents of the culture media which was changed every 3 days ranged from 1097 to 1434 μU/ml during the 80 days' culture period. Transplantation of these islets into the portal vein of streptozotocin-induced diabetic rats resulted in a good recovery from the diabetic state. These studies indicate that cultured islets do preserve their original biological abilities.     

Regulation of xenogeneic porcine pancreatic islets

The use of xenogeneic porcine pancreatic islets has been shown to be a potentially promising alternative to using human allogeneic islets to treat insulin-dependent type 1 diabetes (T1D). This article provides an overview of the existing FDA regulatory framework that would be applied to the regulation of clinical trials utilizing xenogeneic porcine pancreatic islets to treat T1D.     

Effects of collagenase concentration on the purity and viability of isolated porcine pancreatic islets for use in xenotransplantation studies

Abstract: The use of lower concentrations of collagenase (0.8 mg/ml as compared to 1.25 and 1.5 mg/ml) resulted in isolation of porcine pancreatic islets retaining their original compact, wholesome appearance. In in vitro experiments, 100% of islet batches isolated with the low collagenase concentration responded to glucose stimulation, while only 63.1% of islet batches isolated with the high concentration of the enzyme did. The response to high glucose challenge of islets isolated with the low enzyme concentration was considerably higher compared to high collagenase concentration islets.
In in vivo experiments, intraperitoneal transplants of microencapsulated islets isolated using 0.8 mg/ml of collagenase reversed diabetes in diabetic BALB-c mice in a significantly larger proportion of recipients (87.5%) as compared to 33.3% of recipients grafted with 1.5 mg/ml collagenase islets.
The results of this study demonstrate that the use of a low collagenase concentration is conducive to isolation of islets of a superior morphology, purity, viability, and functioning ability.     

Effect of cooling rate and its interaction with pre-freeze and post-thaw tissue culture on the in vitro and in vivo function of cryopreserved pancreatic islets

Rapid cooling destroys passenger lymphoid cells during cryopreservation. We now describe improved in vivo survival of rat islets after rapid cooling by adding pre-freeze tissue culture. Islets were equilibrated with 2m dimethylsulphoxide, cooled at 0.3°, 20°, or 70°C/min, and stored at-196°C. The culture was kept at 37°C for 2 or 72 h before and/or after preservation. When cooled at 0.3°C/min, keeping the culture for 72 h gave the highest proportion of dye-excluding cells, but more than 50% were viable under all culture conditions. Islets cooled at 20° or 70°C/min (rapidly) required 72 h of culture for a survival rate of more than 50%. When islets were cultured for 72 h before cryopreservation, their in vitro insulin secretory ability was similar to that of slowly cooled islets and they were able to sustain normoglycaemia in diabetic animals, although more islets were needed. Extended tissue culture before freezing improves the survival of rapidly cooled islets and is therefore important for combined immunomodulation and cryopreservation.     

Cryopreservation of hamster pancreatic islets using a rapid cooling rate

To clarify the possibility of developing a rapid colling rate for islet cryopreservation, we used a cooling rate of 25°C/min for hamster pancreatic islet cryopreservation using 15 per cent dimethylsulfoxide as a cryoprotectant. After preservation, these islets were examined for their morphology and function by assaying the insulin release after glucose stimulation and the contents of the insulin and DNA in 10 islets. In addition, islet cell replicatory activity was investigated by an autoradiographic technique. The effects of transplantation of the islets upon isogeneic and xenogeneic transplantation were also examined. Freezing using a rapid cooling rate of 25°C/min was found to be as effective as a slow cooling rate of 1°C/min for hamster islet cryopreservation. Morphologically, the cryopreserved islets appeared to be similar to the non-frozen cultured islets. The glucose-stimulated insulin release and cell replicatory activitiesin vitro also remained unchanged, whereas the number of cells per islet decreased slightly after cryopreservation. The grafting of cryopreserved islets normalized streptozotocin induced hyperglycemia following isogeneic transplantation. On the other hand, no prolongation of graft survivals in the case of the xenogeneic transplantation of hamster islets to rats was observed. The isogeneically transplanted islets exhibited the same cell replicatory activitiesin vivo, which was even higher compared that of normal hamster pancreatic isletsin situ. In conclusion, the present findings indicate that hamster pancreatic islets can be successfully cryopreserved using a rapid cooling rate, however, it does not appear that this treatment reduces islet vulnerability to xenogeneic graft rejection.     

Experimental studies on cryopreservation combined with the cultural process of isolated rat pancreatic islets

Recently many investigators have attempted transplantation of isolated islets of Langerhans in an expectation of therapeutic effects in patients with diabetes. In the course of time, preservation of the islets was found to be a prerequisite for successful transplantation. In the present studies, we used our newly designed method of preserving islets, i.e., cryopreservation combined with the cultural process before and after freezing, and we examined the insulin-releasing activity of the islets and attempted transplantation of the islets so-obtained. As to the insulin-releasing activity, the islets cryopreserved after short-term culture increased insulin output as compared with those cryopreserved without culture. Damage to Langerhans’ islets resulting from isolation and cryopreservation was overcome during the culture. Transplantation of Langerhans’ islets cultured for 3–4 days before and after the cryopreserving process produced favorable results.     

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.