EXPRESSION PROFILE OF ANTISENSE OLIGODEOXYNUCLEOTIDE (ODN) ACTIVITY

我们以前的特点坐标变化的表达 在衰老过程中发生的多个基因的单一生命 老年大鼠海马神经元。 为了将 我们已经展示了具有实际老化神经元的糖皮质激素老化模型， mRNA丰度的协调变化发生在 海马对糖皮质激素刺激的反应 时间依赖性 在那里观察到的变化 有几个是一致的两个实验系统之间 - 包括兴奋性反应电位到抑制性反应电位的变化 海马体的一部分。 我们进一步研究了 糖皮质激素对个体海马神经元表达谱的影响 原代培养的锥体细胞。 根据这些初步的 表达谱，我们建议扩大这些研究， 进行添加回实验，我们试图改变 通过使用反义寡核苷酸表达特定的mRNA， 操纵mRNA和功能蛋白的产生。 这将 可以通过三种方式实现：1）个体的表达谱分析 分散的神经元（来自从糖皮质激素处理的分离的海马 大鼠），其已经被注射了特定的反义核酸 寡核苷酸结合这些的电生理记录 2）分离新的mRNA，其丰度被以下改变： 使用cDNA富集技术的反义操作和3）蛋白质 使用免疫-aRNA分析单个细胞以确定 特定的反义寡核苷酸改变了可检测的 针对目标mRNA的蛋白质。 这些特殊的实验是 在生物学上是非常有选择性和敏感的， 起始cDNA和蛋白质的特异性，即来自单个 cell. 这些实验将研究反义核酸的作用机制。 通过确定是否将寡核苷酸施用到 细胞的细胞核或细胞质不同地影响表达 profile. 要检验的假设是， mRNA水平提供糖皮质激素挑战的指纹 神经元可以通过添加 反义寡核苷酸导致的水平的改变， 特定的mRNA和蛋白质。 这些数据可能会 对长期类固醇治疗具有治疗意义。

We have previously characterized coordinate changes in the expression of multiple genes that occur during the ageing process in single live neurons from the aged rat hippocampi. In an effort to correlate the glucocorticoid model of ageing with actual aged neurons we have shown that coordinate changes in mRNA abundance occur in the sub-regions of the hippocampus in response to glucocorticoid challenge in a time-dependent manner. Of the changes that have been observed there are several which are consistent between the two experimental systems - including changes in the excitatory to inhibitory response potential of the hippocampus. We have further examined the effects of glucocorticoids on the expression profiles of individual hippocampal pyramidal cells in primary culture. Based upon these preliminary expression profiles we propose to expand upon these studies by performing add-back experiments in which we attempt to alter the expression of particular mRNAs by using antisense oligonucleotides to manipulate the production of mRNA and functional protein. This will be accomplished in three ways 1) expression profiling of individual dispersed neurons (from hippocampi isolated from glucocorticoid treated rats) which have been inhected with the particular antisense oligonucleotides combined with electrophysiological recordings of these same cells 2) isolation of novel mRNAs whose abundances are altered by antisense manipulation using cDNA enrichment techniques and 3) protein profiling of individual cells using immuno-aRNA to determine whether particular antisense oligonucleotides alter the amount of detectable protein for the targeted mRNA. These particular experiments are biologically extremely selective and sensitive because of the specificity of the starting cDNA and protein, i.e. that from a single cell. These experiments will investigate the mechanisms of antisense function by determine whether administration of oligonucleotides into the nucleus or cytoplasm of cells differentially affects the expression profile. The hypothesis to be examined is that coordinate changes in mRNA levels which provide a fingerprint of glucocorticoid challenged neurons can be manipulated in a predictable manner by addition of antisense oligonucleotides resulting in the alteration of levels of specific mRNAs and proteins within these cells. These data will likely have therapeutic implications for long term steroid treatments.