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Mechanism and Assessment of Hypoxia-Induced Islet Death

缺氧引起的胰岛死亡的机制和评估

基本信息

批准号：
6863682
负责人：
IAN R SWEET
金额：
$ 15.16万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-03-01 至 2008-02-29
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): Transplantation of human islets has the potential to cure Type I diabetes. However, death of human islets during isolation and after transplantation has been a major problem in the efficient transplantation of islets. A critical factor in islet survival may be hypoxia, which islets are subjected to at various degrees during all stages of islet transplantation including the storage of the pancreas, isolation, purification and culture of the islets, and after transplantation. Hypoxia results in death of islet cells, however it is not known what mechanism(s) mediate the process. We hypothesize that even short periods of hypoxia induce apoptosis in the islet cell, and that the islet death process may occur 24-48 hours after the hypoxic insult. The distinction between apoptosis and necrosis is clinically relevant since therapeutic strategies targeting the two processes would be different. In Specific Aim 1, we first quantify the time course of islet cell death and apoptosis in relation to different degrees and durations of hypoxia. It will be determined whether standard markers for apoptosis including activation of caspase 3 and translocation of cytochrome c from the mitochondria to the cytosol are observed prior to necrosis. In Specific Aim 2, a systems physiology approach to mitochondrial function will be applied to the assessment of islet viability. Sophisticated non-invasive detection technology combined with our recently developed flow culture system will be utilized to continuously assess oxygen consumption, redox state of cytochromes, and insulin secretion that will quantify transition states akin to vital signs in whole organisms. Mitochondrial function and the progression of apoptosis are linked, and our data shows that continuous assessment of metabolic state may distinguish the initiation and progression of apoptosis and necrosis. We will test the hypothesis that a metabolic threshold can be empirically derived that when exceeded will irreversibly result in islet cell death, loss of insulin secretory function and thereby transplant failure. This hypothesis embodies our notion that metabolical viability will be a better predictor of insulin secretory function of islets after transplantation than in vitro tests of insulin secretion which have been shown not to correlate with transplant success. Finally, we will use the assessment approach to evaluate whether improved oxygenation of the islets by flow culture prevents hypoxia-induced cell death observed in statically cultured islets. The benefits of the proposed research are evaluation of the contribution of hypoxia to islet death in islet isolation thereby laying the foundation for cytoprotective therapies against hypoxia induced islet death. In addition, the development of an islet flow culture system that both assesses and optimally maintains islets will be an important advancement in the field of transplantation based on its ability to predict and improve transplantation efficacy.

描述(申请人提供)：人类胰岛移植具有治愈I型糖尿病的潜力。然而，人胰岛在分离过程中和移植后死亡一直是胰岛有效移植的主要问题。胰岛存活的一个关键因素可能是低氧，在胰岛移植的所有阶段，包括胰腺的保存、胰岛的分离、纯化和培养，以及移植后，胰岛都会受到不同程度的损伤。低氧导致胰岛细胞死亡，但目前尚不清楚是什么机制(S)介导了这一过程。我们假设，即使是短时间的低氧也会诱导胰岛细胞的凋亡，并且胰岛的死亡过程可能发生在低氧损伤后24-48小时。细胞凋亡和坏死之间的区别在临床上是相关的，因为针对这两个过程的治疗策略是不同的。在具体目标1中，我们首先量化了胰岛细胞死亡和凋亡的时间进程与不同程度和持续时间的缺氧的关系。将确定在坏死之前是否观察到标准的凋亡标志物，包括caspase3的激活和细胞色素c从线粒体到胞浆的移位。在具体目标2中，线粒体功能的系统生理学方法将被应用于胰岛活性的评估。先进的非侵入性检测技术与我们最近开发的流动培养系统相结合，将被用于持续评估氧气消耗、细胞色素的氧化还原状态和胰岛素分泌，从而量化整个生物体的类似生命体征的过渡状态。线粒体功能和细胞凋亡的进程是相联系的，我们的数据表明，对代谢状态的持续评估可以区分细胞凋亡和坏死的启动和进展。我们将测试这样一个假设，即代谢阈值可以从经验上推导出来，当超过这个阈值时，将不可逆转地导致胰岛细胞死亡、胰岛素分泌功能丧失，从而导致移植失败。这一假设体现了我们的观点，即与胰岛素分泌的体外试验相比，代谢活性将更好地预测移植后胰岛的胰岛素分泌功能，后者已被证明与移植成功无关。最后，我们将使用评估方法来评估通过流动培养改善胰岛的氧合是否可以防止在静态培养的胰岛中观察到的缺氧诱导的细胞死亡。这项研究的好处是评估了低氧对胰岛分离后死亡的贡献，从而为针对低氧诱导的胰岛死亡的细胞保护治疗奠定了基础。此外，基于其预测和提高移植疗效的能力，开发一种既能评估胰岛又能以最佳方式维持胰岛的胰岛流式培养系统将是移植领域的重要进展。