ATF3 and iNOS in Islet Destruction and Graft Rejection

ATF3 和 iNOS 在胰岛破坏和移植物排斥中的作用

• 批准号: 7184442
• 负责人: TSONWIN HAI
• 金额: $ 23.77万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-02-15 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7184442
• 关键词: Address; Aftercare; Allogenic; Antibodies; Apoptosis; Apoptotic; B-Lymphocytes; Biological Assay; CD95 Antigens; Cell Death; Cell Survival; Cells; Cessation of life; Clinical; Complementary DNA; Condition; DNA; Data; Development; Diabetes Mellitus; Engineering; Gene Expression; Gene Silencing; Gene Targeting; Genes; Goals; Graft Rejection; Graft Survival; Immune; Immunity; Immunosuppression; In Vitro; Insulin; Islets of Langerhans Transplantation; Knock-out; Knockout Mice; Letters; Mediating; Mediator of activation protein; Messenger RNA; Mitochondria; Modeling; Nitric Oxide; Nucleotides; Numbers; Pathway interactions; Patients; Peptides; Play; Protocols documentation; RNA Interference; Research; Research Personnel; Resistance; Role; Stress; Symptoms; Technology; Testing; Therapeutic immunosuppression; Transgenic Mice; Transplantation; Tumor Necrosis Factor Receptor; Universities; Uridine; Work; caspase-8; cytokine; diabetic; improved; inhibitor/antagonist; islet; isoleucyl-glutamyl-threonyl-aspartic acid fluoromethyl ketone; knock-down; non-diabetic; prevent; programs; promoter; receptor; response; small hairpin RNA; stem

DESCRIPTION (provided by applicant): 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-induced apoptosis plays an important role in 8 cell destruction during islet transplantation. Two stress-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) and auto-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 R cells 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.

描述（由申请人提供）： 目标和假设：该提案的长期目标是提高胰岛移植的效率。虽然在实现这一目标方面已经取得了巨大进展（例如《埃德蒙顿方案》），但有两个关键限制阻碍了胰岛移植成为广泛的临床现实：（1）需要重度免疫抑制，（2）每个接受者需要大量胰岛。该提案将通过测试应激诱导的细胞凋亡在胰岛移植过程中 8 细胞破坏中发挥重要作用的假设来解决这些局限性。两个应激诱导的促凋亡基因将成为研究的重点：ATF3 和 iNOS。目标 1：检验 ATF3 在死亡受体介导的途径中不起主要作用的假设。 Caspase 8 是从死亡受体 Fas 和 TNFR 传递细胞凋亡信息的关键分子。将努力确定 ATF3/iNOS 介导的途径是否不同于死亡受体介导的途径。如果是的话，抑制 caspase 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：测试缺乏 ATF3 和/或 iNOS 的胰岛是否可以减少移植物排斥。将使用三种实验性胰岛移植模型来确定ATF3和/或iNOS的缺乏是否减轻胰岛移植物存活的任何主要障碍：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）和自身免疫（通过自身免疫模型）。目标 3：通过 RNA 干扰 (RNAi) 测试胰岛中的基因沉默 - 使用 ATF3 和 iNOS 作为靶基因进行可行性测试。将生成在 U6 启动子控制下表达短发夹 RNA 的 DMA 构建体，以靶向 ATF3 或/和 iNOS mRNA 的降解。它们的效率将首先在产生胰岛素的 MIN6 R 细胞中进行测试，然后在初级胰岛中进行测试。如果它们起作用，将测试通过RNAi“敲低”ATF3和/或iNOS的野生型胰岛，以确定它们是否比没有敲低这些促凋亡基因的胰岛存活得更好。意义：该提案将8细胞死亡的机制研究与基因沉默技术的发展相结合，旨在改善胰岛移植。如果成功，拟议的研究不仅将增强我们对胰岛破坏的理解，还将增强我们设计具有更高生存能力的胰岛的能力。反过来，这将使每个受体能够以更少的数量移植胰岛。此外，由于胰岛不易受到伤害，因此可以在轻度免疫抑制的情况下耐受残留的免疫攻击，从而避免移植中常用的重度免疫抑制的有害影响。