ATF3 and iNOS in Islet Distruction and Graft Rejection

目标和假设：这项提议的长期目标是提高胰岛的效率 移植。虽然朝着这一目标(如埃德蒙顿)取得了巨大的进展 协议)，有两个关键限制阻碍了胰岛移植在临床上的广泛应用：(1) 需要大量的免疫抑制，以及(2)每个受体需要大量的胰岛。这 一项提案将通过测试应激诱导的细胞凋亡在 在胰岛移植中8细胞破坏中的重要作用。两个应激诱导的促凋亡基因将 成为研究的重点：ATF3和iNOS。 目的1：验证ATF3在死亡受体中不起主要作用的假设。 中介途径。Caspase8是传递细胞死亡信息的关键分子 受体：Fas和TNFR。将努力确定ATF3/iNOS介导的途径是否 有别于死亡受体介导的途径。如果是这样的话，对caspase 8的抑制应该会进一步加强。 ATFS/iNOS基因敲除的胰岛抵抗应激诱导的细胞凋亡的能力。目标2：测试 缺乏ATF3和/或iNOS的胰岛可减少移植物排斥反应。三种实验性胰岛移植 将使用模型来确定血管紧张素转换酶3和/或诱导型一氧化氮合酶的缺乏是否缓解(S)的任何主要 胰岛移植物存活的障碍：原发无功能(同基因模型)、同种异体免疫(异体模型) 模型)和自身免疫(通过自身免疫模型)。目的3：用RNA检测胰岛中的基因沉默 干扰(RNAi)-以ATF3和iNOS为靶基因的可行性测试。DMA构造表达 在U6启动子的控制下，将产生短发夹状RNA来靶向ATF3的降解 或/和iNOS基因的表达。他们的效率将首先在产生胰岛素的MIN6 R细胞中进行测试，然后在原代细胞中进行测试 小岛。如果它们奏效，野生型胰岛通过RNAi“敲除”ATF3和/或iNOS将被检测到 确定在没有这些促凋亡基因的情况下，它们是否比胰岛存活得更好。 意义：这项建议将8细胞死亡的机制研究与技术发展结合起来 基因沉默的研究，目的是改善胰岛移植。如果成功，拟议的研究将 不仅提高了我们对胰岛破坏的理解，而且提高了我们设计胰岛的能力 生存能力。反过来，这将使每个接收者能够以较低的数量移植胰岛。此外, 由于胰岛不那么脆弱，它们可能会容忍在这种情况下仍然存在的免疫攻击 轻微的免疫抑制，从而避免了通常重度免疫抑制的有害影响 用于移植。

Goal and Hypothesis: The long-term goal of this proposal is to improve the efficiency of islet transplantation. While tremendous progress has been made toward this goal (such as the Edmonton Protocol), two key limitations prevent islet transplantation from being a widespread clinical reality: (1) the need for heavy immunosuppression, and (2) the requirement of large numbers of islets per recipient. This proposal will address these limitations by testing the hypothesis that stress-inducedapoptosis plays an important role in 8 cell destruction during islet transplantation. Twostress-inducible, pro-apoptotic genes will be the focus of the studies: ATF3 and iNOS. Aim 1: To test the hypothesis that the ATF3 does not play a major role in the death receptor- mediated pathway. Caspase 8 is a key molecule to transmit the apoptotic information from the death receptors: Fas and TNFR. Efforts will be made to determine whether ATF3/iNOS-mediated pathway is distinct from death receptor mediated pathway. If they are, inhibition of caspase 8 should further enhance the ability of the ATFS/iNOS knockout islets to resist to stress-induced apoptosis. Aim 2: To test whether islets deficient in ATF3 and/or iNOS have reduced graft rejection. Three experimental islet transplant models will be used to determine whether the lack of ATF3 and/or iNOS alleviate(s) any of the main obstacles for islet graft survival: primary non-function (by syngeneic model), allo-immunity (by allogeneic model) andauto-immunity (by auto-immune model). Aim 3: To test gene silencing in the islets by RNA interference (RNAi) - feasibility test using ATF3 and iNOS as target genes. DMA constructs expressing short hairpin RNAs under the control of the U6 promoter will be generated to target the degradation of ATF3 or/and iNOS mRNA. Their efficiency will be tested in the insulin-producing MIN6 Rcells first then in primary islets. If they work, wild type islets with "knockdown" of ATF3 and/or iNOS by RNAi will be tested to determine whether they survive better than islets without the knockdown of these pro-apoptotic genes. Significance: This proposal combines mechanistic studies of 8 cell death with technological development of gene silencing, with the objective to improve islet transplantation. If successful, the proposed research will enhance not only our understanding of islet destruction but also our ability to engineer islets with improved survivability. This, in turn, will enable islets to be grafted at lower numbers per recipient. In addition, because the islets are less vulnerable, they may tolerate the immune attacks remained under the condition of mild immunosuppression, thus avoiding the deleterious effects of heavy immunosuppression commonly used in transplantation.