EXPRESSION PROFILE OF ANTISENSE OLIGODEOXYNUCLEOTIDE (ODN) ACTIVITY

我们之前已经描述了表达式中的坐标变化 在单个活体中衰老过程中发生的多个基因 老年大鼠海马神经元。为了努力将 糖皮质激素衰老模型与实际衰老的神经元，我们已经显示 协调mRNA丰度的变化发生在 大鼠海马区对糖皮质激素刺激的反应 依赖于时间的方式。在那里观察到的变化 在两个实验系统中有几个是一致的 -包括兴奋性到抑制性反应电位的变化 海马体的。我们进一步研究了 糖皮质激素对个体海马区表达谱的影响 原代培养中的锥体细胞。基于这些初步的 我们建议通过以下方式扩展这些研究的表达谱 执行加回实验，在该实验中我们尝试更改 利用反义寡核苷酸表达特定的mRNAs 控制信使核糖核酸和功能蛋白的产生。这将是 通过三种方式完成1)个人的表情特征分析 分散的神经元(来自糖皮质激素治疗后分离的海马区) 大鼠)已经感染了特定的反义 寡核苷酸结合电生理记录 相同细胞2)分离其丰度可被改变的新的RNA 利用DNA浓缩技术和3)蛋白质进行反义操作 使用免疫-Arna对单个细胞进行分析以确定 特定的反义寡核苷酸改变了可检测到的量 靶向mRNA的蛋白质。这些特殊的实验是 在生物学上极具选择性和敏感性，因为 起始cDNAs和蛋白的特异性，即单个 手机。这些实验将探索反义的机制。 功能是通过确定是否将寡核苷酸注射到 细胞的核或细胞质不同地影响其表达 侧写。要检验的假设是，坐标变化 提供糖皮质激素指纹的mRNA水平受到挑战 神经元可以以可预测的方式通过添加 反义寡核苷酸导致血管内皮生长因子水平改变 这些细胞内特定的mRNAs和蛋白质。这些数据很可能 对长期类固醇治疗有治疗意义。

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.