ATF3 and iNOS in Islet Distruction and Graft Rejection

目标和假设：本提案的长期目标是提高胰岛的效率， 移植虽然朝着这个目标已经取得了巨大的进展（如埃德蒙顿 方案），两个关键限制阻止胰岛移植成为广泛的临床现实：（1） 需要大量的免疫抑制，和（2）每个受体需要大量的胰岛。这 这项提案将通过检验压力诱导的细胞凋亡在细胞凋亡中起作用的假设来解决这些局限性。 在胰岛移植过程中8细胞破坏中起重要作用。两个应激诱导的促凋亡基因将 研究的重点：ATF3和iNOS。 目的1：为了检验ATF3在死亡受体中不起主要作用的假设， 介导的途径。Caspase 8是传递细胞凋亡信息的关键分子 受体：Fas和TNFR。将努力确定ATF3/iNOS介导的途径是否是 与死亡受体介导的途径不同。如果是这样的话，抑制半胱天冬酶8应该会进一步增强 ATFS/iNOS敲除胰岛抵抗应激诱导的细胞凋亡的能力。目标2：测试是否 缺乏ATF 3和/或iNOS的胰岛具有降低的移植排斥。三种实验性胰岛移植 模型将用于确定缺乏ATF3和/或iNOS是否减轻任何主要的 胰岛移植物存活的障碍：原发性无功能（通过同基因模型），同种免疫（通过同种异体模型）， 模型）和自身免疫（通过自身免疫模型）。目的3：检测RNA介导的胰岛基因沉默 干扰（RNAi）-使用ATF3和iNOS作为靶基因的可行性测试。DMA构建体表达 将产生U6启动子控制下的短发夹RNA以靶向降解ATF3 或/和iNOSmRNA。它们的效率将首先在产生胰岛素的MIN6 R细胞中测试，然后在原代细胞中测试。 小岛如果它们起作用，将测试通过RNAi "敲低" ATF 3和/或iNOS的野生型胰岛， 确定在没有敲除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：该提案将8细胞死亡的机制研究与技术开发相结合 基因沉默，目的是改善胰岛移植。如果成功，这项研究将 不仅提高了我们对胰岛破坏的理解，而且提高了我们改造胰岛的能力， 生存能力反过来，这将使每个受体能够以较低的数量移植胰岛。此外，本发明还提供了一种方法， 由于胰岛较不脆弱，它们可以耐受在这种条件下仍然存在的免疫攻击。 轻度免疫抑制，从而避免了严重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.