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

IL-10基因转染的胰岛移植进入胸腺和睾丸

• 批准号: 07671344
• 负责人: MIYAMOTO Masaaki
• 金额: $ 1.54万
• 依托单位: Kinki University, School of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07671344/
• 关键词: Islet transplantation; porcine islets; IL-10; gene transfection; A cidic oxidative potential water; non-heart-beating donor; 膵ラ島; 膵ラ島分離培養法; 凍結保存; Islet bant; 胸腺; 睾丸; Immunomodulation; 分離培養法; 膵ラ島凍結保存法; islet bank; vector

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

1. 改进种猪胰岛的大规模分离 - 从无心跳供体获取的可能性 - 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200公斤以上）、温缺血时间（WIT）为1.5*2分钟的种猪作为无心跳供体。然后，我们制作了一种特殊的酶混合物，其主要成分是胶原酶S-1（260 U/mg，NittaZelatin，日本），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）。纯粹地，在胰腺收获时，将这种混合物非常轻轻地注射到胰管中。为了防止胰腺过度消化，首先将混合物冷却至 10°C 以下。第四，我们使用 COBE 2991 细胞处理器通过右旋​​糖酐 70 梯度法纯化胰岛。因为Dextran 7O非常便宜并且具有与Ficoll梯度相同的纯化效果。经常报道连续种猪胰岛分离的结果。双硫腙(DTZ)染色后最长直径超过50μm的胰岛分离总产量为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%。体外培养期间的葡萄糖刺激诱导分离的种猪胰岛显着释放胰岛素。在两只接受使用网状增强聚乙烯醇水凝胶袋 (MRPB) 封装胰岛移植物的糖尿病大鼠中，血糖水平显着降低（低于 200 mg/dl），持续 13 天和 19 天。分别在腹腔内异种移植分离的种猪胰岛（不使用免疫抑制）后。总之，我们成功地、高效、廉价地从无心跳供体中分离出大量成年猪胰岛。2．酸性氧化电位水在预防胰岛移植中细菌感染的功效日本开发的“酸性氧化电位水”（也称为“电解强酸溶液”、“功能水”）是电解含有少量氯化钠的水在阳极上形成的强酸。它具有以下物理特性：pH值，从2.3到2.7；氧化还原电位，从 1,000 到 1,100 mV；溶解氯。 30 至 40 ppm；和溶解氧。 10 至 30 ppm。由于这些特性，酸性氧化电位水通过其所含的活性氧和活性氯的作用，对包括耐甲氧西林金黄色葡萄球菌（MRSA）在内的所有细菌、包括HIV、HBV、HCV、CMV和真菌在内的病毒和真菌具有强烈的杀菌作用。我们进行这项研究是为了评估酸性氧化电位水冲洗对从屠宰场收获用于胰岛异种移植的猪胰腺的细菌污染的影响，通过使用浸玻片法计数胰腺表面细菌的数量以及胰岛培养的结果；并通过静态培养试验和形态学检查来评估用于防止细菌污染时对孤立胰岛的直接影响。研究发现，用酸性氧化电位水直接冲洗胰腺可以非常有效地防止细菌污染，但直接冲洗孤立的胰岛会略微降低其活力和功能。较少的

项目成果

宮本正章 他: "ヒト,ブタ(adult,fetus)膵ラ島に対する自動化凍結保存法の進歩とそのimmunomodulationとしての意義" 低温医学. 22. 54-60 (1996)

Masaaki Miyamoto 等人：“人类和猪（成人、胎儿）胰岛自动冷冻保存的进展及其作为免疫调节的意义”Cryogenic Medicine，22. 54-60 (1996)。

T Kenmochi, M Miyamoto and Y Mullen: "Protection of mouse islet isografts from nonspecific inflammatory damage by recipient treatment with nicotinamide and 15-deoxyspergualin" Cell Transplantation. 5. 41-47 (1996)

T Kenmochi、M Miyamoto 和 Y Mullen：“通过烟酰胺和 15-脱氧精胍菌素治疗受体，保护小鼠胰岛同种移植物免受非特异性炎症损伤”细胞移植。

M.Miyamoto, K.Inoue, M.Hoki, Y.J.Gu, W.X.Cui, and H.Ohyanagi: "Effect of acidic oxidative potential water for microbial contaminations at harvesting of porcine pancreas in islet xenotransplantation." Transplant Proc. (in press).

M.Miyamoto、K.Inoue、M.Hoki、Y.J.Gu、W.X.Cui 和 H.Ohyanagi：“酸性氧化电位水对胰岛异种移植中收获猪胰腺时微生物污染的影响”。

P Y Benhamou, E Stein, C Hober, M Miyamoto: "Ultraviolet ligh irradiation reduces human islet immunogenicity without altering islet function." Horm.metab.res. 27. 113-120 (1995)

P Y Benhamou、E Stein、C Hober、M Miyamoto：“紫外线照射可降低人类胰岛免疫原性，但不会改变胰岛功能。”

Wanxing Cui, Yuanjun Gu, Masaaki Miyamoto, et al.: "A novel isolation method of adult porcine pancreatic islet with 2-stage digestion procedure." Cell Transplantation. (in press).

Wanxing Cui、Yuanjun Gu、Masaaki Miyamoto 等人：“一种采用两阶段消化程序分离成年猪胰岛的新型方法。”