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

1. 改良的繁殖猪胰岛的大规模分离-从非心脏供体中收获的可能性-建立一种成年猪胰岛的大规模分离程序，可作为异种移植的供体来源，并作为人类胰岛分离的模型。我们改进了常规隔离程序的几个特点。在屠宰场，我们首先选择1.5岁以上（体重超过200公斤）、热缺血时间（WIT）为1.5*2分钟的种猪作为非心脏跳动供体。然后，我们制作了一种特殊的酶混合物，其主要含量为胶原酶S-1 （260 U/mg, NittaZelatin，日本）和胶原酶P （1.86 U/ml, Lyo Boehringer-Mannheim）。美国),DNase (Sigma。美国)。Disparse （NittaZelatin，日本），蛋白酶抑制剂（Sigma，美国）在胰腺收获时，将这种混合物轻轻地注射到胰管中。为了防止胰腺的过度消化，首先将混合物冷却到低于10‰。第四，用COBE 2991细胞处理机用葡聚糖70梯度法纯化胰岛细胞。因为右旋糖酐70非常便宜，并且与菲柯尔梯度具有相同的纯化效果。结果经常报道连续种猪胰岛分离。双硫腙（DTZ）染色后最长直径大于50 μ m的胰岛总分离产量为85,900 * 19,954个。291667 * 240452 IEQ （2900 * 2324 IEQ/g）。分离的胰岛纯度很高：90.2 * 3.8%。体外培养过程中的葡萄糖刺激诱导离体种猪胰岛显著释放胰岛素。两只糖尿病大鼠接受用网状增强聚乙乙醇水凝胶袋（MRPB）包裹的胰岛移植，血清葡萄糖水平显著降低（低于200 mg/dl）持续了13天和19天。分别腹腔异种移植离体种猪胰岛后无免疫抑制。总之，我们成功地从一个不跳动的供体中高效、廉价地分离出了大量的成年猪胰岛。酸性氧化电位水预防胰岛移植细菌感染的效果“酸性氧化电位水”（又称“电解强酸溶液”、“功能水”）是日本开发的，是将含有少量氯化钠的水电解后在阳极上形成的一种强酸。它具有以下物理性质：pH值从2.3到2.7；氧化还原电位，从1000到1100 mV；溶解氯。从30到40 PPM；还有溶解氧。从10到30 ppm。由于这些特性，酸性氧化电位水对所有细菌都有很强的杀菌作用，包括耐甲氧西林葡萄球菌（MRSA），病毒包括HIV、HBV、HCV、CMV和真菌，这是由于它含有活性氧和活性氯的作用。本研究通过蘸片法测定猪胰岛表面细菌数量和胰岛培养结果，评价酸性氧化电位灌溉对猪胰岛异种移植过程中细菌污染的影响；并通过静态培养试验和形态学检查，评价其对离体胰岛预防细菌污染的直接效果。研究发现，酸性氧化电位水直接冲洗胰腺对防止细菌污染非常有效，但直接冲洗离体胰岛会略微降低其活力和功能。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