Schneider Armin, Fischer Achim, Weber Daniela, von Ahsen Oliver, Scheek Sigrid, Krüger Carola, Rossner Moritz, Klaussner Bettina, Faucheron Nadine, Kammandel Birgitta, Goetz Bernhard, Herrmann Oliver, Bach Alfred, Schwaninger Markus
Department of Molecular Neurology, Axaron Bioscience AG, Im Neuenheimer Feld 515, 69120 Heidelberg, Germany.
J Cereb Blood Flow Metab. 2004 Feb;24(2):224-36. doi: 10.1097/01.WCB.0000104960.26014.7A.
Studies of gene expression changes after cerebral ischemia can provide novel insight into ischemic pathophysiology. Here we describe application of restriction-mediated differential display to screening for differentially expressed genes after focal cerebral ischemia. This method combines the nonredundant generation of biotin-labeled fragment sets with the excellent resolution of direct blotting electrophoresis, reliable fragment recovery, and a novel clone selection strategy. Using the filament model in mouse with 90 minutes MCA occlusion followed by 2, 6, and 20 hours reperfusion, we have compared gene expression in sham-operated animals to both the ipsi- and contralateral forebrain hemisphere of ischemic mice. Our screening method has resulted in the identification of 70 genes differentially regulated after transient middle cerebral artery occlusion (MCAO), several of which represent unknown clones. We have identified many of the previously published regulated genes, lending high credibility to our method. Surprisingly, we detected a high degree of correspondent regulation of genes in the nonischemic hemisphere. A high percentage of genes coding for proteins in the respiratory chain was found to be up-regulated after ischemia, potentially representing a new mechanism involved in counteracting energy failure or radical generation in cerebral ischemia. One particularly interesting gene, whose upregulation by ischemia has not been described before, is pip92; this gene shows a rapid and long-lasting induction after cerebral ischemia. Here we demonstrate that pip92 induces cell death in primary neurons and displays several hallmarks of pro-apoptotic activity upon overexpression, supporting the notion that we have identified a novel pathophysiological player in cerebral ischemia. In summary, restriction-mediated differential display has proven its suitability for screening complex samples such as brain to reliably identify regulated genes, which can uncover novel pathophysiological mechanisms.
脑缺血后基因表达变化的研究能够为缺血性病理生理学提供新的见解。在此，我们描述了限制性介导的差异显示技术在局灶性脑缺血后差异表达基因筛选中的应用。该方法将生物素标记片段集的非冗余生成与直接印迹电泳的高分辨率、可靠的片段回收以及一种新颖的克隆选择策略相结合。利用小鼠大脑中动脉闭塞90分钟后再灌注2、6和20小时的丝线模型，我们将假手术动物的基因表达与缺血小鼠同侧和对侧前脑半球的基因表达进行了比较。我们的筛选方法已鉴定出70个在短暂性大脑中动脉闭塞（MCAO）后差异调节的基因，其中一些代表未知克隆。我们已鉴定出许多先前已发表的受调节基因，这为我们的方法提供了高度可信度。令人惊讶的是，我们在非缺血半球检测到了高度一致的基因调节。发现缺血后编码呼吸链中蛋白质的基因中有很大比例被上调，这可能代表了一种参与对抗脑缺血中能量衰竭或自由基产生的新机制。一个特别有趣的基因是pip92，其缺血后上调此前尚未见报道；该基因在脑缺血后表现出快速且持久的诱导。在此我们证明，pip92在原代神经元中诱导细胞死亡，并且在过表达时表现出几种促凋亡活性的特征，这支持了我们已在脑缺血中鉴定出一种新型病理生理学参与者的观点。总之，限制性介导的差异显示已证明其适用于筛选如脑这样的复杂样本，以可靠地鉴定受调节基因，这些基因可揭示新的病理生理机制。
