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背景:近来,从大动物获得大量存活的胰岛细胞技术已经日趋成熟,并且已经应用到了临床。然而大量研究表明,同小动物相比,猪胰岛细胞的收获量,还是相对过低,究其原因,是否与猪胰腺外分泌组织生化成分有关?此研究旨在为糖尿病患者提供可靠的胰岛细胞来源。目的:探讨单纯酶和复合酶消化液对成猪胰岛细胞分离的作用及其机制,为糖尿病患者的二级康复措施介入提供理论依据。设计:以动物为研究对象,随机对照实验研究。单位:一所大学医院的普外科和老年病科。材料:实验于2003-06/2004-05在哈尔滨医科大学附属第二医院完成。哈尔滨市哈达屠宰厂提供成年杂种活猪20头,猪龄12～24个月,体质量100kg左右。干预:屠宰放血后在相对无菌情况下,仔细取出胰腺脾部放在冷RP-MI1640液内,迅速送到实验室。快速去除胰周组织,脂肪、血管等。用1∶5000洗必泰浸泡3min,然后用冷RPMI1640液冲洗,4℃条件下在平皿中用眼科剪剪成1mm×1mm×1mm左右的碎片,均分成两份,分别放入含有单纯酶溶液和和复合酶溶液的锥形瓶中,分级消化获得胰岛细胞。主要观察指标:经双硫腙染色计数胰岛细胞,并做椎虫蓝染色测定胰岛细胞活性,胰岛素释放试验观察胰岛细胞的功能;电子显微镜对胰岛细胞团检查,检验其结构的完整性。结果:复合酶消化制备物在平均产量上?
Background: Recently, techniques for obtaining large numbers of islet cells that survive from large animals have matured and have been applied clinically. However, a large number of studies have shown that, compared with small animals, the amount of porcine islet cells harvest is still relatively low, the reason is related to porcine pancreatic exocrine biochemical composition? This study aims to provide reliable sources of islet cells in diabetic patients . OBJECTIVE: To investigate the effect and mechanism of simple enzyme and compound enzyme digestive juice on the isolation of islets from piglets and to provide a theoretical basis for the intervention of secondary rehabilitation measures in diabetic patients. Design: Animals as research subjects, randomized controlled experimental study. Unit: General Surgery and Geriatrics in a University Hospital. Materials: The experiment was performed at the Second Affiliated Hospital of Harbin Medical University from June 2003 to May 2004. Harbin Hatha slaughter plant to provide 20 hybrid pigs adult pigs, pigs aged 12 to 24 months, the body mass of about 100kg. Intervention: After slaughtering and bleeding, in a relatively aseptic situation, carefully remove the spleen of the pancreas and place it in cold RP-MI1640 solution and send it to the laboratory promptly. Rapid removal of pancreatic tissue, fat, blood vessels and so on. Wash with 1: 5000 chlorhexidine 3min, then rinsed with cold RPMI1640 solution, and cut into 1mm × 1mm × 1mm fragments with ophthalmic scissors in a petri dish at 4 ℃, divided into two, were placed in a solution containing simple enzyme solution And complex enzyme solution conical flask, grading digestion to obtain islet cells. MAIN OUTCOME MEASURES: Islet cells were counted by dithizone staining, and islet cell staining was performed to determine the activity of islet cells. Insulin release assay was used to observe the function of islet cells. Insulin cell mass was examined by electron microscopy to check its structural integrity. Results: The compound enzyme digestion preparation in the average yield?