A Novel Approach to Identify the Mediators in Chronic Itch

识别慢性瘙痒介质的新方法

基本信息

批准号：
8933947
负责人：
ZHOUFENG CHEN
金额：
$ 22.88万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-15 至 2017-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8933947
关键词：
Acute Allergic Contact Dermatitis Animal Model Antihistamines Behavior Bombesin Bombesin Receptor Brain Candidate Disease Gene Cells Chloroquine Chronic Disease Drug Targeting Essential Genes Esthesia Future GRP gene Gene Expression Gene Expression Profiling Genes Genetic Health Histamine Human Image Immune In Situ Hybridization Injection of therapeutic agent Intervention Lasers Mediating Mediator of activation protein Medical Microscope Modeling Molecular Molecular Profiling Molecular Target Mus Mutant Strains Mice Nervous system structure Neurons Nociception Pain Pruritus Quality of life Receptor Signaling Reporting Resistance Role Sampling Signal Transduction Signaling Molecule Skin Small Interfering RNA Spinal Spinal Cord Spinal Ganglia Staining method Stains Stimulus System Testing Therapeutic Time base behavior test design differential expression drug discovery effective therapy genome-wide genome-wide analysis intradermal injection novel novel strategies novel therapeutics pain behavior prevent programs receptor receptor expression research study response screening translational study transmission process

项目摘要

DESCRIPTION (provided by applicant): Chronic itch is a presenting sign in numerous diseases associated with the skin, the immune, and the nervous systems. Chronic itch is largely resistant to conventional antihistamines, and few effective therapies are available. In order to develop novel therapeutics against chronic itch, there is a pressing need for identification of itc-specific signaling molecules involved. We have recently uncovered a small subset of itch-specific neurons which express gastrin-releasing peptide receptor (GRPR), an itch receptor, in the spinal cord. GRPR+ neurons are required for pruritoceptive but not for nociceptive transmission, indicating that they contain a myriad of itch-specific genes. To facilitate the identification of novel itch genes in GRPR+ neurons, we design a novel strategy that combines a genome-wide profiling of gene expression in GRPR+ neurons with functional screening, including behavioral tests and Ca2+ imaging. Gene expression profiles between the superficial laminae with normal GRPR+ neurons and without are compared. This strategy enables us to search for the genes that are expressed in GRPR+ neurons and are functionally required for mediating nonhistaminergic itch. Three sets of experiments will be performed to test the key hypothesis that there are distinct signaling signatures for histaminergic and nonhistaminergic itch in the spinal cord. First, genome-wide profiling of gene expression in GRPR+ neurons by microarray, qRT-PCR and in situ hybridization studies will be performed. The candidate genes expressed in GRPR+ neurons will be identified. Second, the involvement of the candidate genes in acute and chronic itch will be evaluated using a siRNA knockdown approach, and the key genes for mediating nonhistaminergic itch in acute or chronic itch will be identified. Third, the relationship between the candidate genes and GRPR will be determined by examining whether the knockdown of the gene would impair intrathecal GRP-evoked scratching and would further impact scratching behavior of GRPR mutant mice. In addition, whether the candidate genes are involved in GRPR-dependent Ca2+ signaling will be examined in a heterologous system using siRNA knockdown and Ca2+ imaging. The proposed studies are highly translational because the itch mediators are identified directly from chronic itch models, and likely to be itch-specific. Our proposed studies will significantly expand the pool of the targets that may be explored for future drug discovery program.

描述（由申请人提供）：慢性瘙痒是与皮肤，免疫和神经系统相关的许多疾病的表现。慢性瘙痒在很大程度上对常规抗组胺药具有抗药性，很少有有效的疗法可用。为了开发针对慢性瘙痒的新型治疗剂，需要鉴定涉及的ITC特异性信号分子的迫切需要。最近，我们发现了一小部分瘙痒特异性神经元，这些神经元表达脊髓中释放胃蛋白的肽受体（GRPR），一种瘙痒受体。 GRPR+神经元具有灵感需要，而不是伤害性传播，这表明它们包含无数瘙痒特异性基因。为了促进GRPR+神经元中新型瘙痒基因的鉴定，我们设计了一种新型策略，该策略结合了GRPR+神经元中基因表达的全基因组分析与功能筛选，包括行为测试和CA2+成像。比较具有正常GRPR+神经元的表面层之间的基因表达谱。该策略使我们能够搜索GRPR+神经元中表达的基因，并且在介导非组织胺能瘙痒所必需的功能上是必需的。将进行三组实验，以测试一个关键假设，即脊髓中的组蛋白能和非组织敏能迭代存在明显的信号特征。首先，将通过微阵列，QRT-PCR和原位杂交研究对GRPR+神经元中基因表达的全基因组分析。将确定在GRPR+神经元中表达的候选基因。其次，将使用siRNA敲除方法评估候选基因参与急性和慢性瘙痒，并确定介导非组织胺能瘙痒的关键基因。第三，候选基因与GRPR之间的关系将通过检查该基因的敲低是否会损害鞘内GRP诱发的划痕，并进一步影响GRPR突变小鼠的划痕行为。此外，将使用siRNA敲低和Ca2+成像在异源系统中检查候选基因是否参与GRPR依赖性CA2+信号传导。拟议的研究是高度转化的，因为瘙痒介质直接从慢性瘙痒模型中鉴定出来，并且可能是瘙痒特异性的。我们提出的研究将大大扩展可能探索未来药物发现计划的目标池。