ATF3 and iNOS in Islet Distruction and Graft Rejection

ATF3 和 iNOS 在胰岛破坏和移植物排斥中的作用

• 批准号: 8010462
• 负责人: TSONWIN HAI
• 金额: $ 8.92万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-14 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8010462
• 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; 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; Pathway interactions; Peptides; Play; Protocols documentation; RNA Interference; Research; Research Personnel; Resistance; Role; Stress; Symptoms; Technology; Testing; Transgenic Mice; Transplantation; Tumor Necrosis Factor Receptor; Universities; Uridine; Work; caspase-8; cytokine; diabetic; diabetic patient; improved; inhibitor/antagonist; islet; knock-down; non-diabetic; prevent; programs; promoter; receptor; response; small hairpin RNA; stem

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.

目标与假设：本提案的长期目标是提高胰岛的效率