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GENE EXPRESSION IN AXOTOMIZED RETINAL GANGLION CELLS

轴切视网膜神经节细胞中的基因表达

基本信息

批准号：
2710767
负责人：
Leonard A Levin
金额：
$ 8.92万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
1994
资助国家：
美国
起止时间：
1994-08-01 至 1999-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2710767
关键词：
RNase protection assay apoptosis denervation developmental genetics electron microscopy fusion gene gene expression genetic library immunocytochemistry laboratory rat neurogenetics neuronal transport northern blottings retinal ganglion

项目摘要

Death of retinal ganglion cells is the final common pathway underlying virtually all diseases of the optic nerve, including anterior ischemic optic neuropathy, optic neuritis, and compressive optic neuropathy. Despite a wealth of studies examining the direct effect of hypoxia, excitotoxins, and other injuries on retinal ganglion cells, the effect on these cells of axonal injury, which underlies most optic neuropathies, is far less clearly understood. Attempts to promote regeneration of ganglion cell axons after axotomy presupposes an intact ganglion cell soma; only by preventing ganglion cell death can it be expected that regenerative strategies will succeed. Therefore the overall goal of this proposal is to use recently developed techniques to better understand the subcellular events underlying death of retinal ganglion cells due to axonal injury. The working hypothesis is that axotomy of retinal ganglion cells causes expression of genes that turn on a program for cell death, and that these genes and their products can be characterized. The long-term goal is to modulate these genes or their products and therefore allow rescue of retinal ganglion cells when the optic nerve is injured. The first specific aim is to identify genes differentially expressed in axotomized retinal ganglion cells. A subtracted copy DNA (cDNA) library will be made from messenger RNA (mRNA) extracted from axotomized and control rat retinas. Candidate cDNAs will be screened with Northern blotting or ribonuclease protection assay using RNA probes. The second specific aim is to study the tissue, species, and developmental expression of the identified genes. Gene sequences will be used to look for homologies to other genes associated with cell death. The third specific aim is to express the protein products of the genes and determine their localization and possible function. The health-relatedness of this project is to diseases affecting the anterior and retrobulbar optic nerve. While these disorders directly damage axons contained within the optic nerve, the irreversible loss of vision reflects the death of ganglion cells contained within the retina. Understanding the subcellular nature of the ganglion cell response to axonal injury will allow development of therapies for these diseases. The opportunity to receive formal training in molecular biology and carry out this research program will also further the applicant's goal of becoming an independent investigator.

视网膜神经节细胞的死亡是视网膜病变的最终共同途径。几乎所有的视神经疾病，包括前部缺血性视神经病变、视神经炎和压迫性视神经病变。尽管有大量的研究探讨了缺氧的直接影响，兴奋性毒素等对视网膜神经节细胞的损伤，这些轴突损伤的细胞是大多数视神经病变的基础，更不清楚的是。促进神经节再生的尝试轴突切断后的细胞轴突以完整的神经节细胞索马为前提;只有通过防止神经节细胞死亡，战略将取得成功。因此，本提案的总体目标是使用最近开发的技术，以更好地了解亚细胞轴突损伤导致视网膜神经节细胞死亡的潜在事件。目前的假设是，视网膜神经节细胞的轴突切断导致这些基因的表达开启了细胞死亡的程序，基因及其产物可以被表征。长期目标是调节这些基因或它们的产物，因此可以拯救视神经受损时，视网膜神经节细胞。第一个具体的目标是确定差异表达的基因，轴突切断的视网膜神经节细胞。消减拷贝DNA（cDNA）文库将由从轴突切断和对照大鼠视网膜。候选cDNA将用北方引物进行筛选。印迹或使用RNA探针的核糖核酸酶保护测定。第二具体目标是研究组织，物种和发育表达已识别的基因。基因序列将被用来寻找与细胞死亡相关的其他基因的同源性。第三特定目的是表达基因的蛋白质产物，并确定其定位和可能的功能。该项目与健康有关的是影响儿童健康的疾病，前和球后视神经。虽然这些疾病直接损伤视神经内所含的轴突，视觉反映了视网膜内神经节细胞的死亡。了解神经节细胞对轴突损伤将允许开发用于这些疾病的疗法。的有机会接受分子生物学的正式培训，这项研究计划也将进一步申请人的目标，成为独立调查员