Subtractive Hybridization of mRNA from early passage and senescent endothelial cells
Subtractive Hybridization of mRNA from early passage and senescent endothelial cells
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DOI：
10.1016/s0531-5565(00)00080-2
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发表时间：
2000-03-01
影响因子：
3.9
通讯作者：
Katinger, H
中科院分区：
文献类型：
--
作者：
Grillari, J;Hohenwarter, O;Katinger, H
调节细胞过程，最终导致生长停滞状态，是体外细胞衰老的一个重要表现，引起并伴随着基因表达模式的变化。虽然这些在mRNA水平上的变化主要是在成纤维细胞培养中研究的，但我们集中在内皮细胞上，内皮细胞代表了血管系统的公认模型，可能参与了与衰老相关的疾病的发病机制。为了分离差异表达的基因，我们利用衰老(35代)和年轻(5代)人脐静脉内皮细胞(HUVECs)的mRNA构建了消减cDNA文库。从文库中分离出候选克隆，经Northern印迹分析证实差异表达，并与GenBank数据库进行比对。由于许多mRNAs低于Northern印迹分析的检测下限，我们被迫建立了一种更灵敏的基于PCR的方法(ATAC-PCR)来定量和确认基因表达的变化水平。在衰老的人脐静脉内皮细胞中，有几个mRNAs表达上调，其中包括细胞外基质(ECM)的两个组成部分：纤溶酶原激活物抑制物和纤维连接蛋白。这两种基因在衰老细胞中的高表达已经被描述。转化生长因子-β诱导基因H3(β-IG-H3；ECM蛋白)、胰岛素样生长因子结合蛋白(IGFBP-3)、P53诱导基因(PIG3)、囊泡运输相关蛋白(SEC13R)和核糖体蛋白L28的mRNAs也同样在衰老细胞中优先表达。由于研究支持细胞外基质成分、转化生长因子-β和p53参与肿瘤抑制机制，我们的数据支持细胞衰老和细胞外基质蛋白上调可能与肿瘤预防功能相关的假说。(C)2000 Elsevier Science Inc.保留所有权利。
Regulation of cellular processes that eventually lead to a state of growth arrest is an important manifestation of in vitro cellular senescence caused and accompanied by variations of the gene expression pattern. Whereas these changes at the mRNA level have been studied mainly in fibroblast cultures, we concentrated on endothelial cells that represent an accepted model for Vascular systems and may be involved in the pathogenesis of diseases related to aging. To isolate differentially expressed genes, we created a subtractive cDNA library using mRNA from senescent (35 passages) and young (five passages) human umbilical vein endothelial cells (HUVECs). Candidate clones were isolated from the cDNA library, differential expression was confirmed by Northern blot analyses and sequences were compared with a genbank data base. Because many mRNAs were below the detection limit of Northern blot analysis, we were forced to establish a more sensitive PCR based method (ATAC-PCR) to quantify and confirm altered levels of gene expression. Several mRNAs were found to be upregulated in senescent HUVECs including two components of the extracellular matrix (ECM): plasminogen activator inhibitor and fibronectin. Elevated expression of both has already been described in senescent cells. The mRNAs of TGF-beta-inducible gene H3 (beta-IG-H3; ECM protein), insulin-like growth factor binding protein (IGFBP-3), p53-inducible gene (PIG3) a protein involved in vesicular transport (SEC13R) and ribosomal protein L28 have likewise been shown to be preferentially expressed in senescent cells. Because studies support the involvement of ECM components, TGF-beta and p53 in tumor suppressing mechanisms, our data supports the hypothesis that cellular senescence and upregulation of ECM proteins may be associated with tumor preventive functions. (C) 2000 Elsevier Science Inc. All rights reserved.