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首页> 外文期刊>Journal of Cerebral Blood Flow and Metabolism: Official Journal of the International Society of Cerebral Blood Flow and Metabolism >Restriction-mediated differential display (RMDD) identifies pip92 as a pro-apoptotic gene product induced during focal cerebral ischemia.
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Restriction-mediated differential display (RMDD) identifies pip92 as a pro-apoptotic gene product induced during focal cerebral ischemia.

机译：限制性介导的差异显示（RMDD）将pip92鉴定为局灶性脑缺血期间诱导的促凋亡基因产物。

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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.
机译：对脑缺血后基因表达变化的研究可以为缺血性病理生理学提供新的见解。在这里，我们描述了限制性介导的差异显示在局灶性脑缺血后筛选差异表达基因的应用。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出色分辨率，可靠的片段回收以及新颖的克隆选择策略结合在一起。使用在90分钟MCA闭塞后再进行2、6和20小时再灌注的小鼠中的细丝模型，我们比较了假手术动物与缺血小鼠同侧和对侧前脑半球的基因表达。我们的筛选方法已鉴定出70个基因，这些基因在短暂性大脑中动脉闭塞（MCAO）后受到差异调节，其中一些代表未知克隆。我们已经鉴定了许多以前发表的调控基因，为我们的方法提供了很高的可信度。出乎意料的是，我们在非缺血性半球中检测到高度相应的基因调控。缺血后，发现呼吸链中编码蛋白质的基因的高比例基因被上调，这可能代表了一种新的机制，可以抵消脑缺血中的能量衰竭或自由基产生。 pip92是一种特别有趣的基因，该基因以前没有描述过缺血引起的上调。该基因在脑缺血后显示出快速而持久的诱导作用。在这里，我们证明pip92诱导原代神经元细胞死亡，并在过表达时显示促凋亡活性的几个特征，支持了我们已经确定了脑缺血中一种新型病理生理因素的观点。总而言之，限制介导的差异显示已证明其适用于筛选复杂的样本（例如大脑）以可靠地识别受调控的基因，从而可以揭示新的病理生理机制。

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