Donation After Cardiac Death for Pancreatic Islet Cells

描述(由申请人提供)： 胰岛移植能够恢复胰岛素依赖型糖尿病(DM)患者的正常血糖控制。尽管几个胰岛移植中心都取得了巨大的成功，但限制这种治疗快速成功的因素之一是器官短缺。心脏死亡后捐献(DCD)是一种可行的替代方案，将有助于减少这种短缺。目前，用于移植到1型糖尿病患者体内的胰岛通常不是从DCD胰腺分离出来的。我们的目标是提供对DCD捐赠者的人类胰岛的全面评估，并将它们与脑死亡(DBD)捐赠者的胰岛进行比较。具体的假设是来自DCD供体的胰岛在功能上等同于甚至优于来自DBD的胰岛。这一假说基于以下观察：a)使用DCD胰腺器官进行实体器官移植的长期结果提供了良好的长期血糖控制和糖化血红蛋白正常化；b)有可能分离大量胰岛，并通过单一供体胰岛移植治愈1型糖尿病；c)由于脑死亡的生理作用，DCD供者的器官不会暴露在DBD供者体内通常存在的激素和促炎细胞因子释放的影响下。这些效应已被证明有助于β细胞的凋亡甚至坏死。本研究的具体目的是比较DCD供体和DBD供体分离的人胰岛：1)移植前对葡萄糖的生理反应作为胰岛准备质量的快速效力指标；2)分离后低氧对胰岛活力和功能的影响；3)胰岛对细胞因子诱导的凋亡的敏感性。将测量响应葡萄糖挑战的吡啶核苷酸氧化还原状态、细胞内钙和线粒体膜电位的动力学分析。通过测定缺氧诱导转录因子-1(HIF-1)的含量以及活性氧(ROS)、一氧化氮(NO)和脂质过氧化反应的形成来评估人胰岛分离后缺氧的影响。还将进行修复机制的测量，如超氧化物歧化酶和过氧化氢酶上调。这一目的的结果将有助于开发预防低氧诱导的胰岛死亡的治疗方法。DBD和DCD胰岛的凋亡百分比将在分离后立即评估，并在IL-1β、TNFpha和IFNGamma的作用下培养；模拟移植后缺血/再灌注损伤的影响。将使用一种新的方法来确定存活和凋亡，在这种方法中，可以完整地分析胰岛，使用复杂对象参数分析仪和分类器(Copas)流式细胞术保留其完整的结构，结果将在体内相互关联。增加可用于胰岛分离的供体池将显著增加该疗法对1型糖尿病患者的可获得性。此外，分离后胰岛活性和功能的快速、准确和预测性测试的发展将有助于显著提高移植后的成功率。

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.