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取得了巨大的成功，但阻碍这种治疗快速成功的限制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DM患者中的胰岛通常不是从DCD胰腺中分离的。我们的目标是对DCD供体的人类胰岛进行全面评估，并将其与脑死亡后捐赠（DBD）供体的胰岛进行比较。具体假设是来自DCD供体的胰岛在功能上等同于或甚至上级来自DBD的胰岛。该假设基于以下观察结果：a）用来自DCD的器官进行实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化血红蛋白的正常化; B）可以分离大量胰岛，并且可以用来自DCD胰腺的单个供体胰岛移植治愈1型糖尿病; c）来自DCD供体的器官不暴露于激素和促炎细胞因子释放，这通常存在于DBD供体中，这是由于脑死亡的生理效应。这些作用已被证明有助于β细胞的凋亡甚至坏死。本研究的具体目的是通过以下方式比较从DCD供体分离的人胰岛与从DBD供体分离的人胰岛：1）表征对葡萄糖的生理反应，作为移植前胰岛制备质量的快速效力指标，2）测定分离后缺氧对胰岛活力和功能的影响，3）测定胰岛对细胞因子诱导的细胞凋亡的敏感性。将测量吡啶核苷酸氧化还原状态、细胞内钙和线粒体膜电位对葡萄糖激发的响应的动力学分析。将通过定量缺氧诱导转录因子-1（HIF-1）和活性氧（ROS）、一氧化氮和脂质过氧化的形成来评估人胰岛分离后缺氧的影响。还将测量修复机制，如超氧化物歧化酶和过氧化氢酶上调。在这一目标中获得的结果将有助于开发防止缺氧诱导的胰岛死亡的疗法。在分离后立即评估来自DBD和DCD的凋亡胰岛的百分比，并用IL-1 β、TNF α和IFN γ培养后评估;模拟移植后缺血/再灌注损伤的影响。将使用一种新方法进行活力和凋亡的测定，其中可以使用复杂对象参数分析仪和分选仪（COPAS）流式细胞术完整分析胰岛，保留其完整结构，并将结果与体内相关。增加可用于胰岛分离的供体库将显着增加该疗法对1型糖尿病患者的可用性。此外，开发快速、准确和预测性的分离后胰岛活力和功能测试将有助于显著改善移植后的成功率。

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.