Islet Transplantation of IL-10 gene transfection into Thymus and Testis

1.改进的种猪胰岛的大规模分离-从无心跳供体收获的可能性-建立成年猪胰岛的大规模分离程序，可用作异种移植的供体来源和人类胰岛分离的模型。我们改进了传统分离方法的几个特点。在屠宰场，我们首先选择1.5岁以上（体重超过200 kg）的种猪，其热缺血时间（WIT）为1.5*2分钟作为无心跳供体。以胶原酶S-1（260 U/mg，NittaZelatin，Japan）、胶原酶P（1.86 U/ml，LyoBoehringer-Mannheim）为主要成分，配制成一种特殊的酶制剂。美国），DNA酶（Sigma. U.S.A.）。Disparse（NittaZelatin，日本），蛋白酶抑制剂（Sigma，美国）。在胰腺收获时，将该混合物非常轻柔地注射到胰管中。为了防止胰腺过度消化，首先将混合物冷却至低于10摄氏度。 关于我们 用COBE 2991细胞处理器通过葡聚糖70梯度法纯化胰岛。因为右旋糖酐7 O非常便宜并且具有与Ficoll梯度相同的纯化效果。结果往往连续种猪胰岛分离的报告。用双硫腙（DTZ）染色后，最长直径大于50 μ m的胰岛分离的总产量为85，900 × 19，954个胰岛。291，667 * 240，452 IEQ（2，900 * 2，324 IEQ/g）。分离的胰岛的纯度非常高：90.2 * 3.8%。在体外孵育过程中，葡萄糖刺激诱导分离的种猪胰岛显著释放胰岛素。在两个糖尿病大鼠接受封装的胰岛移植物使用am esh增强的聚乙烯醇水凝胶袋（MRPB），血清葡萄糖水平的显着降低（低于200毫克/分升）持续13和19天。分别在无免疫抑制的分离的种猪胰岛的腹膜内异种移植后。总之，我们成功地从无心跳供体中高效、廉价地分离了大量的成年猪胰岛。酸性氧化电位水在胰岛移植中预防细菌感染的有效性日本开发的“酸性氧化电位水”（也称为“电解强酸溶液”、“功能水”）是在电解含有少量氯化钠的水时在阳极上形成的强酸。它具有以下物理性质：pH值为2.3至2.7 ;氧化还原电位为1，000至1，100 mV ;溶解氯。30至40 ppm ;和溶解氧。从10到30 ppm。由于这些特性，酸性氧化电位水对所有细菌具有强烈的杀菌作用，包括耐甲氧西林金黄色葡萄球菌（MRSA），病毒包括HIV，HBV，HCV，CMV和真菌，这是由于其所含的活性氧和活性氯的作用。本研究通过采用玻片法计数胰腺表面细菌数，评价酸性氧化电位水冲洗对猪胰岛移植中细菌污染的影响，以及对胰岛培养结果的影响;并通过静态孵育试验和形态学检查来评价其用于防止细菌污染时对分离胰岛的直接影响。用酸性氧化电位水直接冲洗胰腺被发现通过防止细菌污染非常有效，但是直接inigation分离的胰岛稍微降低它们的活力和功能。少

1. Improved large-scale isolation of breeder porcine islets - Possibility of harvesting from non-heart-beating donor -To establish a large-scale isolation procedure for adult porcine islets usable as a donor source for xenotransplantation and as a model of human islet isolation. we improved several characteristics of the coventional isolation procedure. At a slaughterhouse we first selected breeder pig over 1.5 years old (and over 200 kg in weight) whose warm ischemic time (WIT) were 1.5*2 minutes as non-heart-beating donors. Then, we made a special enzymic mixture whose maincontents were collagenase S-1 (260 U/mg, NittaZelatin, Japan), collagenase P (1.86 U/ml Lyo Boehringer-Mannheim. U.S.A.), DNase (Sigma. U.S.A.). Disparse (NittaZelatin, Japan), Protease inhibitor (Sigma, USA). Neat, this mixture was injected very gently into the pancreatic duct at the time of pancretic harvesting. To prevent overdigestion of the pancreas, the mixture was first cooled to less than 10ﾟC.Fourth by, we … More purified the islets with a COBE 2991 cell processor by the Dextran 70 gradient method. because Dextran 7O is very cheap and has the same purification effect as the Ficoll gradient. The results often consecutive breeder porcine islet isolations are reported. The total yield of isolations of islets over 50 mu m in the longest diameter after staining wish Dithizone (DTZ) was 85,900 * 19,954 islets. 291,667 * 240,452 IEQ (2,900*2,324 IEQ/g). The purity of the isolated islets was very high : 90.2 * 3.8%. Glncose stimulation during in vitro incubation induced signiliesnt insulin release from isolated breeder porcine islets. In two of the diabetic rats receiving encapsulated islets grafts using am esh-reinforced polyvinyl alcohol hydrogel bag (MRPB), a prominent reduction in serum glucose levels (less than 200 mg/dl) persisted for 13 and 19 days. respectively, after intraperitoneal xenotransplantation of isolated breeder porcine islets without immunosuppression. In conclusion, we succeeded in efficiently and cheaply isolating a large amount of adult porcine islets from a non-heart-beating donor.2. Effectiveness of acidic oxidative potential water in preventing bacterial infection in islet transplantation"Acidic oxidative potential water" (also termed "electrolyzed strong acid solution", "function water") which was developed in Japan, is a strong acid formed on the anode in the electrolysis of water containing a small amount of sodium chloride. It has these physical properties : pH, from 2.3 to 2.7 ; oxidative-reduction potential, from 1,000 to 1,100 mV ; dissolved chlorine. from 30 to 40 ppm ; and dissolved oxygen. from 10 to 30 ppm. Because of these properties, acidic oxidative potential water has strong bactericidal effects on all bacteria including methicillin-resistant staphylococcus oureus (MRSA), viruscs including HIV, HBV, HCV, CMV, and fungi as a result of the action the active oxygen and active chlorine that it contains. We conducted this study to evaluate the effect of acidic oxidative potential water irrigation on bacterial contamination on the harvesting of porcine pancreata from slaughterhouses for islet xenofransplantation by counting the number of pancreatic surface bacteria using the Dip-slide method, and on the results of islet culture ; and to evaluate the direct effect on isolated islets when it is used to prevent bacterial contamination by the static incubation test and by morphorogical examination. Direct irrigation of the pancreas by acidic oxidative potential water was found to very effective by prevent bacterial contamination, but direct inigation of isolated islets slightly decreased their viability and function. Less