Serial Analysis of Gene Expression Identifies Metallothionein-II as Major Neuroprotective Gene in Mouse Focal Cerebral Ischemia
Serial Analysis of Gene Expression Identifies Metallothionein-II as Major Neuroprotective Gene in Mouse Focal Cerebral Ischemia
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DOI：
10.1523/jneurosci.22-14-05879.2002
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发表时间：
2002-07
期刊：
影响因子：
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通讯作者：
G. Trendelenburg;K. Prass;J. Priller;K. Kapinya;A. Polley;C. Muselmann;K. Ruscher;Ute Kannbley;A. Schmitt;Stefanie Castell;F. Wiegand;A. Meisel;A. Rosenthal;U. Dirnagl
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作者：
G. Trendelenburg;K. Prass;J. Priller;K. Kapinya;A. Polley;C. Muselmann;K. Ruscher;Ute Kannbley;A. Schmitt;Stefanie Castell;F. Wiegand;A. Meisel;A. Rosenthal;U. Dirnagl
我们应用基因表达系列分析 (SAGE) 来研究诱导局灶性脑缺血 14 小时后小鼠大脑中差异表达的基因。对超过 60,000 个转录本的分析揭示了 83 个上调转录本和 94 个下调转录本（超过或等于八倍）。通过对相同的起始材料重复执行 SAGE 来证明可重复性。金属硫蛋白-II (MT-II) 是缺血半球中最显着上调的转录本。 MT-I 和 MT-II 被认为是由金属、糖皮质激素和炎症信号以协调的方式诱导的，但它们的功能仍然难以捉摸。 Northern 印迹证实了 MT-I 和 MT-II 的上调。 MT-I 和 MT-II mRNA 表达早在短暂性缺血 2 小时后 2 小时就增加，16 小时后达到最大值。蛋白质印迹和免疫组织化学显示缺血半球中 MT-I/-II 上调，而双标记表明 MT 与星形胶质细胞以及单核细胞/巨噬细胞标记物共定位。 MT-I 和 MT-II 缺陷小鼠的梗塞面积比野生型小鼠大约三倍，神经系统结果也明显较差。我们首次提供了脑缺血基因表达的综合数据集，并强调了金属硫蛋白在脑缺血损伤中的重要保护作用。我们的结果证明了 SAGE 在筛选功能相关基因方面的有用性，以及敲除模型在将功能与高通量技术生成的表达数据联系起来方面的能力。
We applied serial analysis of gene expression (SAGE) to study differentially expressed genes in mouse brain 14 hr after the induction of focal cerebral ischemia. Analysis of >60,000 transcripts revealed 83 upregulated and 94 downregulated transcripts (more than or equal to eightfold). Reproducibility was demonstrated by performing SAGE in duplicate on the same starting material. Metallothionein-II (MT-II) was the most significantly upregulated transcript in the ischemic hemisphere. MT-I and MT-II are assumed to be induced by metals, glucocorticoids, and inflammatory signals in a coordinated manner, yet their function remains elusive. Upregulation of both MT-I and MT-II was confirmed by Northern blotting. MT-I and MT-II mRNA expression increased as early as 2 hr after 2 hr of transient ischemia, with a maximum after 16 hr. Western blotting and immunohistochemistry revealed MT-I/-II upregulation in the ischemic hemisphere, whereas double labeling demonstrated the colocalization of MT with markers for astrocytes as well as for monocytes/macrophages. MT-I- and MT-II-deficient mice developed approximately threefold larger infarcts than wild-type mice and a significantly worse neurological outcome. For the first time we make available a comprehensive data set on brain ischemic gene expression and underscore the important protective role of metallothioneins in ischemic damage of the brain. Our results demonstrate the usefulness of SAGE to screen functionally relevant genes and the power of knock-out models in linking function to expression data generated by high throughput techniques.