Donation After Cardiac Death for Isolated Pancreatic Is*

心脏死亡后为孤立性胰腺捐献*

• 批准号: 7031599
• 负责人: LUIS Alberto FERNANDEZ
• 金额: $ 31.97万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7031599
• 关键词: NOD mouse; SCID mouse; apoptosis; calcium flux; catalase; cytokine; diabetes mellitus; flow cytometry; glucose metabolism; high performance liquid chromatography; human tissue; hypoxia; hypoxia inducible factor 1; membrane potentials; mitochondrial membrane; oxidation reduction reaction; oxidative stress; pancreatic islet transplantation; pancreatic islets; postmortem; pyridine nucleotide; superoxide dismutase; tissue /cell preparation; tissue donors

DESCRIPTION (provided by applicant): Islet transplantation is capable of restoring normal blood sugar control in patients with insulin dependent diabetes mellitus (DM). In spite of the great success shared by several islet transplant centers, one of limiting factors that has impaired the rapid success of this treatment is the shortage of organs. Donation after Cardiac Death (DCD) is a viable alternative that will aid in reducing this shortage. At present, islets for transplant into patients with type 1 DM are not typically isolated from DCD pancreata. Our goal is to provide a comprehensive assessment of human islets from DCD donors and compare them to islets from Donation after Brain Death (DBD) donors. The specific hypothesis is that islets from DCD donors are functionally equivalent to or even superior to islets from DBD. This hypothesis is based on observations that: a) long terms results of solid organ pancreas transplants with organs from DCD have provided excellent long term glycemia control and normalization of glycosylated hemoglobin; b) it is possible to isolate large number of islets, and to cure type 1 diabetes mellitus with a single donor islet transplant from a DCD pancreas; c) organs from DCD donors are not exposed to the hormonal and pro-inflammatory cytokine release which is typically present in DBD donors due to the physiological effect of brain death. These effects have been shown to contribute to apoptosis and even necrosis of the beta cells. The specific aims of this study are to compare isolated human islets from DCD donors to those from DBD donors by: 1) characterization of the physiological response to glucose as a rapid potency indicator of the quality of the islet preparation prior to transplantation, 2) determination of the effects of hypoxia on islet viability and function after isolation, 3) determination of the sensitivity of islets to cytokine induced apoptosis. Kinetic analysis of the pyridine nucleotide redox states, intracellular calcium, and mitochondrial membrane potential in response to glucose challenge will be measured. The effects of hypoxia after human islet isolation will be assessed through the quantification of hypoxia inducible transcription factor-1 (HIF-1) and the formation of reactive oxygen species (ROS), nitric oxide, and lipid peroxidation. Measurements of the mechanism of repair such as superoxide dismuatase and catalases upregulation will also be performed. The results obtained in this aim will aid in the development of therapies to protect against hypoxia induced islet death. The percentage of apoptotic islets from DBD and DCD will be assessed immediately post isolation and afterculture with IL-1beta, TNFalpha, and IFNgamma; mimicking the effects of ischemia/reperfusion injury after transplant. Determination of viability and apoptosis will be performed using a novel approach in which islets can be analyzed intact, preserving their complete architecture using Complex Object Parametric Analyzer and Sorter (COPAS) flow cytometry and results will be correlated in vivo. Increasing the donor pool available for islet isolation would significantly increase the availability of this therapy for type 1 diabetic patients. In addition, the development of rapid, accurate, and predictive tests of islet viability and function post isolation will contribute to significant improvements in post transplant success.

描述（由申请人提供）： 胰岛移植能够恢复胰岛素依赖性糖尿病（DM）患者的正常血糖控制。尽管几个胰岛移植中心取得了巨大的成功，但这种处理迅速成功的限制因素之一是器官的短缺。心脏死亡后捐赠（DCD）是可行的替代方法，可帮助减少这种短缺。目前，将移植到1型DM患者中的胰岛通常不是从DCD胰腺中分离出来的。我们的目标是对DCD捐赠者的人类胰岛进行全面评估，并将其与脑死亡（DBD）捐赠者捐赠的胰岛进行比较。具体假设是，来自DCD供体的胰岛在功能上等同于或什至优于DBD的胰岛。该假设是基于：a）具有DCD器官的固体器官胰腺移植的长期结果提供了极好的长期血糖控制和糖基化血红蛋白的归一化； b）可以分离大量胰岛，并从DCD胰腺中用单个供体胰岛移植治愈1型糖尿病； c）DCD供体的器官不暴露于激素和促炎性细胞因子释放，由于脑死亡的生理作用，该器官通常存在于DBD供体中。这些作用已被证明会导致β细胞的凋亡甚至坏死。 The specific aims of this study are to compare isolated human islets from DCD donors to those from DBD donors by: 1) characterization of the physiological response to glucose as a rapid potency indicator of the quality of the islet preparation prior to transplantation, 2) determination of the effects of hypoxia on islet viability and function after isolation, 3) determination of the sensitivity of islets to cytokine induced apoptosis.将测量吡啶核苷酸氧化还原态，细胞内钙和线粒体膜电位的动力学分析。人类胰岛分离后缺氧的影响将通过定量诱导性转录因子1（HIF-1）以及活性氧（ROS），一氧化氮和脂质过氧化的形成来评估。还将进行修复机理的测量，例如超氧化物溶酶和催化酶上调。在此目标中获得的结果将有助于开发疗法，以防止缺氧引起的胰岛死亡。 DBD和DCD的凋亡胰岛百分比将在分离后与IL-1Beta，TNFalpha和Ifngamma进行分离后立即评估；模仿移植后缺血/再灌注损伤的影响。将使用一种新的方法来确定生存力和凋亡的确定，可以将胰岛完整分析，并使用复杂对象参数分析仪和流式细胞仪保存其完整的体系结构，并将结果与​​体内相关。增加可用于胰岛分离的供体池将显着增加1型糖尿病患者的疗法的可用性。此外，胰岛生存力和功能隔离的快速，准确和预测性测试的发展将有助于显着改善移植后成功。

项目成果

A simplified approach to human islet quality assessment.

• DOI: 10.1097/tp.0b013e3181d54bce
• 发表时间: 2010-05-27
• 期刊: Transplantation
• 影响因子: 6.2
• 作者: Hanson MS;Park EE;Sears ML;Greenwood KK;Danobeitia JS;Hullett DA;Fernandez LA
• 通讯作者: Fernandez LA