Patterns of gene expression following contact of sympathetic neurons with their myocyte targets

We identified 112 known genes and 150 expressed sequence tags (ESTs) showing more than a 1.3 fold change in target-exposed neurons compared to neurons grown in the absence of target. Expression of 36 genes and 49 ESTs was upregulated, while expression of 76 genes and 101 ESTs was downregulated by the presence of target during the culture period. Overall, these changes represented approximately 1.6% of the probe set. Keywords: Patterns of gene expression by neuron-target interaction We investigated the molecular basis of the target-regulated growth inhibition program using DNA microarrays to analyze patterns of gene expression following contact of sympathetic neurons with their myocyte targets. Total RNA was isolated from sympathetic neurons cultured alone and from neurons isolated from myocyte co-cultures. The two neuron populations showed minimal levels of atrial natriutic peptide (ANF) mRNA, a myocyte marker, compared to myocyte-containing cultures, indicating a low level of myocyte contamination for neurons purified from co-cultures. Isolated total RNA was reverse transcribed, labeled with biotin by in vitro transcription and hybridized to an Affymetrix Rat Expression Set 230A microarray with 15,900 probe sets. The hybridized microarray was washed, stained, scanned and quantified using DNA-Chip Analyzer (dChip) 1.3 (Li and Wong, 2001).

我们共鉴定出112个已知基因与150个表达序列标签（expressed sequence tags, EST），其在靶标暴露神经元中的表达量相较于无靶标培养的神经元变化幅度超过1.3倍。其中36个基因及49个EST的表达出现上调，而培养过程中因靶标存在而下调的基因与EST分别为76个和101个。总体而言，上述变化约占探针集的1.6%。
关键词：神经元-靶标相互作用的基因表达模式

本研究利用DNA微阵列技术，分析交感神经元与其肌细胞靶标接触后的基因表达模式，以探究靶标调控的生长抑制程序的分子基础。我们分别从单独培养的交感神经元，以及从肌细胞共培养体系中分离得到的神经元中提取总RNA。相较于含肌细胞的培养物，这两类神经元的心房钠尿肽（atrial natriuretic peptide, ANF）mRNA（一种肌细胞标志物）水平极低，证实从共培养物中纯化的神经元的肌细胞污染程度较低。

将提取的总RNA进行反转录，通过体外转录法用生物素标记，随后与包含15900个探针集的Affymetrix大鼠表达芯片230A（Affymetrix Rat Expression Set 230A）微阵列进行杂交。对杂交完成的微阵列进行洗涤、染色、扫描，并采用DNA芯片分析仪（dChip）1.3版本（Li与Wong，2001）完成定量分析。