Subtractive cloning identifies tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) increased gene expression following focal stroke.
Subtractive cloning identifies tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) increased gene expression following focal stroke.
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DOI：
10.1161/01.str.29.2.516
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发表时间：
1998-02
期刊：
影响因子：
8.3
通讯作者：
X. Wang;F. Barone;R. F. White;G. Feuerstein
中科院分区：
文献类型：
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作者：
X. Wang;F. Barone;R. F. White;G. Feuerstein
背景和目的：已有报道，在局灶性卒中发作后，基因表达存在差异。为了鉴定缺血诱导基因的从头表达，我们应用消减cDNA文库策略来鉴定局灶性卒中选择性上调的基因。方法采用永久性大脑中动脉阻塞（MCAO）模型建立自发性高血压大鼠模型。从MCAO后2小时和12小时的缺血和非缺血皮质中制备mRNA，并构建消减cDNA文库。在消减cDNA文库中鉴定了编码基质金属蛋白酶组织抑制剂-1（TIMP-1）的cDNA。皮质TIMP-1 mRNA的时间表达的进一步特点是在缺血皮质进行永久性或暂时性（160分钟）MCAO。结果从消减cDNA文库中分离的一组基因进行Southern分析，以确认缺血诱导的基因表达。TIMP-1在缺血性损伤后表现出稳健的诱导。时程研究显示，TIMP-1 mRNA在12小时时被诱导为对照组的3倍（P<0.001，n=4只动物），并在永久性MCAO后2天达到峰值水平（7倍增加，P<0.001）。在短暂MCAO后再灌注的缺血皮质中观察到类似的TIMP-1 mRNA诱导谱。结论本研究证实了消减cDNA文库技术在寻找脑卒中差异表达基因中的应用价值。此外，我们的数据暗示TIMP-1在缺血诱导的脑损伤。
BACKGROUND AND PURPOSE Differential gene expression has been reported following the onset of focal stroke. To identify de novo expression of ischemia-induced genes, we applied subtractive cDNA library strategy to identify the genes that are selectively upregulated by focal stroke. METHODS Spontaneously hypertensive rats were subjected to permanent occlusion of the middle cerebral artery (MCAO). mRNAs prepared from ischemic and nonischemic cortex 2 and 12 hours after MCAO were subtracted, and a subtractive cDNA library was constructed. A cDNA that encodes for tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) was identified in the subtractive cDNA library. The temporal expression of cortical TIMP-1 mRNA was further characterized in ischemic cortex subjected to permanent or temporary (160-minute) MCAO. RESULTS A panel of genes isolated from the subtractive cDNA library was subjected to Southern analysis to confirm ischemia-induced gene expression. TIMP-1 demonstrated robust induction after ischemic injury. Time-course studies revealed that TIMP-1 mRNA was induced threefold over controls at 12 hours (P<.001, n=4 animals) and reached a peak level at 2 days after permanent MCAO (sevenfold increase, P<.001). Similar induction profile of TIMP-1 mRNA was observed in the ischemic cortex after temporary MCAO followed by reperfusion. CONCLUSIONS This work demonstrated the utility of subtractive cDNA library strategy for discovery of genes differentially expressed in focal stroke. Furthermore, our data implicate TIMP-1 in ischemia-induced brain injury.