Wang Ju, Gutala Ramana, Sun Dongxiao, Ma Jennie Z, Sheela Rani C S, Ticku Maharaj K, Li Ming D
Department of Psychiatry and Neurobehavioral Sciences, University of Virginia, Charlottesville, Virginia 22911, USA.
Alcohol Clin Exp Res. 2007 Mar;31(3):357-75. doi: 10.1111/j.1530-0277.2006.00331.x.
The higher incidence of smoking among alcoholic subjects suggests the presence of common molecular mechanisms underlying nicotine and alcohol use and abuse. However, these mechanisms are largely unknown. By using cultured fetal mouse cortical neurons as a model system, we sought to identify genes and pathways that are modulated in the cells by ethanol, nicotine, or both.
Primary cerebral cortical cultures were prepared from the brains of 14-day-old C57BL/6 mouse fetuses and exposed to ethanol (75 mM), nicotine (0.1 mM), or both for 5 consecutive days. A homeostatic pathway-focused microarray consisting of 638 sequence-verified genes was used to measure transcripts differentially regulated by ethanol, nicotine, or both in 5 drug-treated cortical neuron samples and 5 control samples. Quantitative real-time reverse transcriptase-polymerase chain reaction analysis was used to verify the mRNA expression levels of genes of interest detected from the microarray experiments.
Through a pathway-focused cDNA microarray and balanced experimental design, we identified 65, 111, and 81 significantly regulated genes in the ethanol, nicotine, and ethanol/nicotine-treated neurons, respectively. Of them, the genes of Akt2, Nsg1, Pdgfa, Pfn1, Rbbp7, and Tcfeb were comodulated. The genes differentially expressed in 1 or more treatment groups could be classified into 4 major clusters, with each cluster consisting of genes involved in different biological processes. The platelet-derived growth factor (PDGF) signaling pathway was significantly regulated by all 3 treatments, but by different mechanisms, which may lead to different cellular consequences.
Our results indicate that the PDGF pathway represents one of the major biochemical mechanisms in the cellular and molecular responses to each drug in cortical neurons. Finally, we demonstrated that the pathway-focused microarray system used in the present study is a valuable tool for dissecting the mechanisms of complex signaling pathways such as the PDGF pathway.
酗酒者中吸烟率较高，这表明尼古丁和酒精的使用及滥用背后存在共同的分子机制。然而，这些机制在很大程度上尚不清楚。我们以培养的胎鼠皮质神经元为模型系统，试图确定在细胞中受乙醇、尼古丁或两者调节的基因和信号通路。
从14日龄C57BL/6胎鼠的大脑中制备原代大脑皮质培养物，并连续5天暴露于乙醇（75 mM）、尼古丁（0.1 mM）或两者。使用由638个经序列验证的基因组成的聚焦于稳态通路的微阵列，来测量在5个药物处理的皮质神经元样本和5个对照样本中受乙醇、尼古丁或两者差异调节的转录本。采用定量实时逆转录聚合酶链反应分析来验证从微阵列实验中检测到 的感兴趣基因的mRNA表达水平。
通过聚焦于通路的cDNA微阵列和平衡的实验设计，我们分别在乙醇、尼古丁和乙醇/尼古丁处理的神经元中鉴定出65、111和81个显著调节的基因。其中，Akt2、Nsg1、Pdgfa、Pfn1、Rbbp7和Tcfeb基因是共同调节的。在1个或更多处理组中差异表达的基因可分为4个主要簇，每个簇由参与不同生物学过程的基因组成。血小板衍生生长因子（PDGF）信号通路在所有3种处理中均受到显著调节，但调节机制不同，这可能导致不同的细胞后果。
我们的结果表明，PDGF信号通路是皮质神经元对每种药物的细胞和分子反应中的主要生化机制之一。最后，我们证明本研究中使用的聚焦于通路的微阵列系统是剖析诸如PDGF信号通路等复杂信号通路机制的有价值工具。
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