EFFECTS OF OVEREXPRESSION OF NERVE GROWTH FACTOR

神经生长因子过度表达的影响

• 批准号: 2269774
• 负责人: BRIAN M DAVIS
• 金额: $ 14.78万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-05-01 至 1997-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2269774
• 关键词: afferent nerve; apoptosis; autoradiography; axon; calcitonin gene related peptide; cell population study; cholecystokinin; complementary DNA; complementary RNA; gene expression; genetically modified animals; immunocytochemistry; in situ hybridization; innervation; keratin; laboratory mouse; messenger RNA; neural degeneration; neurofilament; neurogenesis; neurotrophic factors; northern blottings; nucleic acid probes; peptide hormone biosynthesis; polymerase chain reaction; recombinant proteins; skin; somatostatin; spinal ganglion; tachykinin; trigeminal nerve; tyrosine 3 monooxygenase; western blottings

NGF is a neurotrophic factor known to play a central role in neuron survival, axon growth and gene expression during development and in the adult. NGF can also modulate the physiology of primary sensory neurons involved in pain and temperature sensation. We have developed a new approach to study the role of NGF in neuronal development that utilizes a transgenic mouse model system. Three lines of transgenic mice have been isolated that express increased levels of NGF in the skin, a peripheral target tissue that normally produces NGF. These mice express a transgene containing tissue-specific regulatory sequences from a human epidermal keratin gene (K14) linked to the mouse NGF cDNA. The K14-NGF transgene targets NGF expression to the basal cells of the epidermis and other stratified, squamous epithelium. Expression in the skin begins at approximately E14, the time that endogenous NGF levels decline and neuronal cell death is initiated. We have found that the forced upregulation of NGF in the skin during this critical period has profound effects on the development of the peripheral nervous system. The goal of the proposed research is to elucidate the effect of NGF over- expression on trigeminal and dorsal root ganglia, focusing on changes in cell number, neuronal phenotype, and axon growth. The specific aims of these studies are to: 1) Determine if the amount of NGF affects the number and size of primary sensory neurons in trigeminal and dorsal root ganglia. NGF mRNA will be measured using reverse PCR and in situ hybridization analysis. Relative levels of the NGF polypeptide will be measured using western blot analysis. 2) Determine if increased NGF expression induces changes in primary sensory projections to peripheral and central targets. Immunocytochemistry and western blotting for neurofilaments and NGF will be used to examine the density of innervation of the transgenic skin. In addition, tract tracing will be used to determine if central projections of sensory afferents are affected. 3) Determine if over-expression of NGF differentially affects subpopulations of sensory neurons. In situ hybridization will be used to neurochemically identify primary sensory neurons in the trigeminal and dorsal root ganglia in normal and transgenic mice. Once identified the number and somal size of these subpopulations will be determined. Initial studies will use cRNA and oligonucleotide probes for mRNAs known to be regulated by NGF, including preprotachykinin, calcitonin gene- related peptide, cholecystokinin, somatostatin and tyrosine hydroxylase mRNAs. This model provides a unique means to test the major tenet of the neurotrophic hypothesis in an in vivo system in which changes in the level of NGF are restricted to a single target tissue of peripheral neurons.

NGF是一种已知在神经元中发挥中枢作用的神经营养因子 发育过程中的存活、轴突生长和基因表达 成年人。神经生长因子也可以调节初级感觉神经元的生理。 有痛感和体温感的。我们开发了一种新的 研究神经生长因子在神经元发育中作用的方法 转基因小鼠模型系统。三个转基因小鼠品系已经 被分离出来，在皮肤中表达增加的NGF水平， 正常情况下产生神经生长因子的外周靶组织。这些小鼠表达了 含有人类组织特异性调控序列的转基因 表皮角蛋白基因(K14)与小鼠神经生长因子(NGF)基因连锁。K14-NGF 转基因靶向表皮的基底细胞表达NGF和 其他复层鳞状上皮。皮肤中的表情开始于 大约E14，内源性NGF水平下降和 神经细胞死亡是启动的。我们发现，被强迫的 在这一关键时期，皮肤中NGF的上调具有深远的意义 对周围神经系统发育的影响。目标是 这项拟议研究的目的是阐明NGF在体内的作用。 在三叉神经节和背根神经节的表达，主要集中在 细胞数量、神经元表型和轴突生长。的具体目标 这些研究是为了：1)确定NGF的数量是否影响 三叉神经根和背根区初级感觉神经元的数量和大小 神经节。用逆转录-聚合酶链式反应和原位杂交检测神经生长因子基因的表达 杂交分析。NGF多肽的相对水平将是 采用蛋白质印迹分析进行检测。2)确定NGF是否增加 表达诱导初级感觉投射到外周的变化 和中心目标。免疫细胞化学和免疫印迹检测 神经丝和神经生长因子将被用来检测神经的密度。 转基因皮肤的。此外，还将使用轨迹跟踪来 确定感觉传入的中枢投射是否受到影响。3) 确定NGF的过度表达是否会对亚群产生不同影响 感觉神经元。将使用原位杂交技术 神经化学鉴定三叉神经和三叉神经的初级感觉神经元 正常和转基因小鼠的背根神经节。一旦确定了 这些亚群的数量和体型将被确定。 初步研究将使用cRNA和寡核苷酸探针检测已知的mRNAs。 受NGF调控，包括速激肽原、降钙素基因- 相关肽、缩胆囊素、生长抑素和酪氨酸羟基酶 MRNAs。这个模型提供了一种独特的方法来测试 体内系统中的神经营养假说，其中 NGF水平仅限于外周单个靶组织 神经元。