Pre-Conditioning Pancreatic Islets for Transplantation

描述（由申请人提供）： I型糖尿病是一种影响儿童和年轻人的毁灭性终身疾病，其原因是胰腺中胰岛的产生胰岛素的β细胞被破坏。尽管外源性胰岛素治疗是可行的，但糖代谢紊乱仍会引起许多危及生命的并发症。最近人类胰岛移植的成功导致80%的移植物存活率在一年内引发了热情，这种疗法将恢复许多糖尿病患者的长期健康。这种成功依赖于大胰岛块的移植，其通常涉及多次连续的胰岛移植。一般认为，需要大量的胰岛是必要的，因为只有一小部分移植的胰岛在移植后存活。细胞生物学的最新进展突出了环境氧作为影响细胞表型、基因表达和功能的分子信号的关键作用，这对于优化胰岛细胞功能和移植存活力可能具有至关重要的意义。维持“正常”范围的环境氧可最大限度地减少细胞氧化应激并保持细胞完整性。在哺乳动物器官中，存在宽范围的氧浓度，使得不同细胞类型的“常氧”是可变的。已知“低血糖”状况显著影响细胞信号传导和氧化应激反应。我们的研究团队最近发表的工作表明，“高氧”条件也显着影响细胞信号传导，基因表达，表型和活性氧的产生。鉴于胰岛对氧化应激的极端脆弱性，我们怀疑目前的临床方法涉及环境氧从“高氧”到“缺氧”条件的剧烈波动，对胰岛细胞功能和活力产生不利影响。缺氧预处理启动细胞保护级联反应，减少氧化还原应激的概念的证据已经建立了各种（非胰腺）细胞类型和组织。该建议将测试以下假设：移植前将胰岛的“常氧”设定点重新调整至其缺氧微环境的缺氧预处理将启动细胞保护性细胞信号传导途径，导致成功的胰岛移植，胰岛质量降低。在初步的数据中，我们表明，改善的生存能力和恢复胰岛缺氧预处理后发生。具体目的I：研究环境氧随时间对培养后胰岛细胞活力和恢复的影响。将使用体外试验和显微镜检查确定环境氧对培养后胰岛细胞活力、形态和恢复的影响。具体目的II：确定氧预处理对体内胰岛细胞功能的影响。将使用荧光显微镜和胰岛移植的边缘质量模型来确定氧预处理对体内胰岛活力和功能的影响。

DESCRIPTION (provided by applicant): Type I Diabetes is a devastating life-long disease affecting children and young adults which results from thedestruction of insulin-producing beta (beta) cells of the islets of Langerhans in the pancreas. The disturbance in glucose metabolism causes many life-threatening complications despite the availability of exogenous insulin therapy. The recent success with human islet transplantation resulting in an 80% graft survival rate at one year has sparked enthusiasm that this therapy will restore long-term euglycemia to many patients with diabetes. This success is dependent on transplantation of a large islet mass which usually involves multiple sequential islet transplants. It is generally believed that the need for a substantial mass of islets is necessary because only a fraction of the transplanted islets survive following transplantation. Recent advances in cell biology highlighting the critical role of ambient oxygen as a molecular signal which influences cell phenotype, gene expression and function may have critical importance for optimizing islet cell function and viability for transplant. Maintenance of a "normal" range of ambient oxygen minimizes cellular oxidative stress and preserves cellular integrity. In mammalian organs, there is a wide range of oxygen concentrations such that "normoxia" for different cell types is variable. "Hypoxic" conditions are known to significantly influence cellular signaling and oxidative stress responses. Recent published work from our research team demonstrates that "hyperoxic" conditions also significantly influence cellular signaling, gene expression, phenotype and production of reactive oxygen species. Given the extreme vulnerability of pancreatic islets to oxidative stress, we suspect that current clinical approaches which involve dramatic fluctuations in ambient oxygen from "hyperoxic" to"hypoxic" conditions adversely influence islet cell function and viability. The proof of concept that hypoxia preconditioning initiates a cytoprotection cascade which reduces redox stress has been established for a variety of (nonpancreatic) cell types and tissues. This proposal will test the hypothesis that hypoxia preconditioning to readjust the"normoxia" set point of pancreatic islets to their hypoxic microenvironment prior to transplant will initiate cytoprotective cellular signaling pathways, resulting in successful islet transplantation with decreased islet mass. In preliminary data we show that improved viability and recovery of islets occur after hypoxia pre-conditioning. Specific Aim I: To examine the effects of ambient oxygen over time on islet cell viability and recovery after culture. The influence of ambient oxygen on islet cell viability, morphology and recovery after culture will be determined using in vitro assays and microscopy. Specific Aim II: To determine the effect of oxygen pre-conditioning on islet cell function in vivo. The effect of oxygen pre-conditioning on islet viability and function in vivo will be determined using fluorescence microscopy and the marginal mass model of islet transplantation.