Mechanism and Assessment of Hypoxia-Induced Islet Death

描述（由申请人提供）：人类胰岛移植有可能治愈I型糖尿病。然而，人胰岛在分离期间和移植后的死亡一直是胰岛有效移植的主要问题。胰岛存活的关键因素可能是缺氧，在胰岛移植的所有阶段，包括胰腺的储存、胰岛的分离、纯化和培养，以及移植后，胰岛都经历不同程度的缺氧。缺氧导致胰岛细胞死亡，但尚不清楚是什么机制介导了这一过程。我们推测，即使是短时间的缺氧也会诱导胰岛细胞凋亡，并且胰岛死亡过程可能发生在缺氧损伤后24-48小时。细胞凋亡和坏死之间的区别是临床相关的，因为靶向这两个过程的治疗策略将是不同的。 在具体目标1中，我们首先量化了与不同程度和持续时间的缺氧相关的胰岛细胞死亡和凋亡的时间过程。将确定在坏死之前是否观察到细胞凋亡的标准标志物，包括半胱天冬酶3的活化和细胞色素c从线粒体到胞质溶胶的易位。在具体目标2中，线粒体功能的系统生理学方法将用于评估胰岛活力。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，将用于连续评估耗氧量，细胞色素的氧化还原状态和胰岛素分泌，这将量化整个生物体中类似于生命体征的过渡状态。线粒体功能和细胞凋亡的进展是相关的，我们的数据表明，代谢状态的持续评估可以区分细胞凋亡和坏死的开始和进展。我们将检验一个假设，即代谢阈值可以根据经验得出，当超过该阈值时，将不可逆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。这一假说体现了我们的观点，即代谢活力将是一个更好的预测移植后胰岛的胰岛素分泌功能比体外测试的胰岛素分泌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防止在静态培养的胰岛中观察到的缺氧诱导的细胞死亡。本研究的益处在于评估缺氧对胰岛分离中胰岛死亡的贡献，从而为针对缺氧诱导的胰岛死亡的细胞保护疗法奠定基础。此外，评估和最佳维持胰岛的胰岛流动培养系统的开发将是基于其预测和改善移植功效的能力的移植领域中的重要进步。

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.