The Pain Neural Transcriptome

疼痛神经转录组

• 批准号: 8552558
• 负责人: Michael J. Iadarola
• 金额: $ 328.03万
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8552558
• 关键词: Acute Pain; Address; Advanced Malignant Neoplasm; Analgesics; Animal Model; Arthritis; Autopsy; Axotomy; Biochemistry; Bioinformatics; C Fiber; Calcium; Calcium Channel Blockers; Canis familiaris; Cell Separation; Cells; Clonidine; Collaborations; Complement; Computational Biology; Computer Workstations; Contracts; Control Animal; Data; Data Set; Devices; Doctor of Philosophy; Dorsal; Dyes; Equipment; Excision; Face; Fluorescence; Fluorescent Dyes; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Genes; Genetic; Goals; Head; Human; Human Resources; Individual; Inflammation; Institutes; Intractable Pain; Isolectin; Knowledge; Label; Laboratories; Libraries; Malignant Bone Neoplasm; Mediating; Messenger RNA; Methods; Modeling; Molecular; Molecular Neurobiology; Molecular Profiling; Molecular Target; Monkeys; Morphine; Mus; National Institute of Mental Health; Nerve; Neurons; Nociception; Nociceptors; Occupations; Opioid Receptor; Pain; Pain management; Pathway interactions; Pennsylvania; Peripheral; Peripheral Nerves; Peripheral nerve injury; Persistent pain; Polyribosomes; Population; Procedures; Process; Protocols documentation; Publications; RNA; Rattus; Recruitment Activity; Reporting; Research; Research Infrastructure; Research Methodology; Research Personnel; Research Project Grants; Resiniferatoxin; Reverse Transcriptase Polymerase Chain Reaction; Ribosomal RNA; Running; Sampling; Scientist; Sensory; Sensory Ganglia; Sequence Alignment; Sequence Analysis; Signal Transduction; Site; Sodium Channel; Sorting - Cell Movement; Specific qualifier value; Speed; Spinal Cord; Spinal Ganglia; Staging; Stimulus; Structure; Structure of trigeminal ganglion; TRPV1 gene; Testing; Time; Tissues; Training; Transcript; Trigeminal System; Universities; Work; Writing; base; cDNA Library Construction; chronic pain; data sharing; dorsal horn; experience; injured; interest; meetings; nerve injury; novel; osteosarcoma; pre-doctoral; receptor; relating to nervous system; time use; translational study; web site

Summary: This project is in the beginning stages. At this point we have established several research methods and protocols, built the supporting infrastructure in terms of equipment, space in the lab, collaborative arrangements, hired and begun to train a team of scientists and support personnel to conduct the research project. Over the course of this year we have performed over 150 sequencing runs on the Illumina HiSeq 2000 and examined peripheral inflammation, peripheral nerve damage, models of arthritis, sequencing of genetically labeled neuronal populations and completed the trigeminal transcriptome in several species. We are in the process of writing multiple reports on these data. Some of the most interesting observations relate to the expression of the exact genes that mediate the actions of known analgesic drugs such as morphine, clonidine and sodium and calcium channel blockers. We can now assign which nociceptive neuronal population is controlled by which analgesic drug and, because we are examining both model species and human post-mortem tissue, we can generalize to the human and focus analgesic research on the most relevant known and novel molecular targets. The personnel now include a Ph.D. research fellow trained in biochemistry and bioinformatics, a predoctoral fellow trained in bioinformatics and computational biology, and a post-BAC IRTA with experience in RNA extraction who also will be trained in cDNA library construction and RT-PCR analysis. The laboratory has purchased two dedicated computer workstations for high-speed sequence alignment and analysis of the transcriptome libraries and constructed a third. We upgraded our Bioanalyzer for RNA quality assessment and utilize 96 and 384 well real-time PCR devices for independent verification of mRNA alterations seen with the RNA-seq methods. For sequencing we work with investigators at the National Institutes Sequencing Center (NISC) and mainly use the Illumina High-Seq 2000 platform for deep sequencing, generally in multiplex mode to analyze 6 independent samples/lane. We determined that polyA+ selection was superior to ribosomal RNA depletion for construction of the sequencing libraries. We contracted with the University of Pennsylvania to obtain canine tissue for the RNA studies. These tissues will be obtained from controls and animals with osteosarcoma euthanized because of inadequate pain control or treated with resiniferatoxin and tissues obtained at autopsy. The data obtained will allow us to test for genes activated by nociceptive input from naturally occurring bone cancer and modulated by treatment. Accrual in the canine study is nearly complete. The data will also be used for comparison to parallel studies in mouse, rat and human (although the exact models will be different). The transcriptome project is systematically investigating the first three steps in the pain pathway beginning with injured peripheral tissue or nerve, the dorsal root ganglion and the dorsal (sensory) spinal cord. The equivalent structures for the face and head are the trigeminal ganglia and the medulla (medullary dorsal horn). We have obtained and analyzed these two tissues in collaboration with Dr. Joel Kleinman of NIMH. These same tissues will be analyzed from rat, dog and monkey. Using the Bioanalyzer, we have analyzed total RNA extracted from the human medullary dorsal horn and trigeminal ganglia. After several technical improvements, analysis yielded excellent RNA integrity numbers. We have nearly completed the initial study on inflammation-induced genes in rats. This includes a systematic examination of the time course of gene regulation in inflamed peripheral tissue, DRG and dorsal horn. Comparisons will be made to several nerve injury models since these are distinct from the inflammation and the analysis of the time course of DRG gene expression following peripheral nerve injury (axotomy) has also been partially completed. The results are being assembled for publications. We have also created a web site for data sharing and this will form the basis for a publically accessible site for other groups to access the data.

摘要：该项目处于起步阶段。 在这一点上，我们已经建立了几种研究方法和协议，在设备，实验室空间，合作安排方面建立了支持基础设施，聘请并开始培训一支科学家和支持人员团队来进行研究项目。 在今年的过程中，我们已经在Illumina HiSeq 2000上进行了超过150次测序，并检查了外周炎症，外周神经损伤，关节炎模型，遗传标记神经元群体的测序，并完成了几个物种的三叉神经转录组。我们正在编写关于这些数据的多份报告。 一些最有趣的观察结果与介导已知镇痛药物（如吗啡、可乐定和钠钙通道阻滞剂）作用的确切基因的表达有关。 我们现在可以分配哪些伤害性神经元群体由哪种镇痛药物控制，因为我们正在检查模型物种和人类死后组织，我们可以推广到人类，并将镇痛研究集中在最相关的已知和新的分子靶点上。 现在的员工包括一名博士。一名接受过生物化学和生物信息学培训的研究员，一名接受过生物信息学和计算生物学培训的博士前研究员，以及一名具有RNA提取经验的后BAC IRTA，他也将接受cDNA文库构建和RT-PCR分析培训。实验室购买了两台专用计算机工作站，用于高速序列比对和转录组文库分析，并建造了第三台。我们升级了用于RNA质量评估的生物分析仪，并利用96孔和384孔实时PCR装置对RNA-seq方法观察到的mRNA变化进行独立验证。对于测序，我们与美国国立研究院测序中心（NISC）的研究人员合作，主要使用Illumina High-Seq 2000平台进行深度测序，通常以多重模式分析6个独立样品/泳道。 我们确定，polyA+选择上级核糖体RNA耗竭的测序文库的构建。 我们与宾夕法尼亚大学签订合同，获得犬组织用于RNA研究。 这些组织将从对照组和因疼痛控制不充分而实施安乐死或用树脂毒素处理的骨肉瘤动物以及尸检时获得的组织中获得。所获得的数据将使我们能够测试由来自自然发生的骨癌的伤害性输入激活并通过治疗调节的基因。犬研究的累积接近完成。 这些数据还将用于与小鼠、大鼠和人的平行研究进行比较（尽管确切的模型不同）。 转录组项目正在系统地研究疼痛通路的前三个步骤，从受损的外周组织或神经、背根神经节和背侧（感觉）脊髓开始。 面部和头部的等效结构是三叉神经节和延髓（延髓背角）。 我们与NIMH的Joel Kleinman博士合作获得并分析了这两种组织。将对大鼠、犬和猴的相同组织进行分析。 利用生物分析仪，我们分析了总RNA提取的人延髓背角和三叉神经节。经过几次技术改进，分析产生了极好的RNA完整性数字。我们已经基本完成了大鼠炎症诱导基因的初步研究。 这包括对发炎的外周组织、背根神经节和背角中基因调控的时程的系统检查。 将进行比较，以几个神经损伤模型，因为这些是不同的炎症和DRG基因表达的时间过程的分析后，周围神经损伤（轴突切断术）也已部分完成。 目前正在汇总结果，以便出版。 我们还建立了一个数据共享网站，这将成为其他团体访问数据的可访问网站的基础。