Identification of stretch-responsive genes in pulmonary artery smooth muscle cells by a two arbitrary primer-based mRNA differential display approach.
Identification of stretch-responsive genes in pulmonary artery smooth muscle cells by a two arbitrary primer-based mRNA differential display approach.
复制标题
通过基于两个任意引物的 mRNA 差异显示方法鉴定肺动脉平滑肌细胞中的拉伸反应基因。
DOI：
10.1023/a:1006966530553
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发表时间：
1999
影响因子：
4.3
通讯作者：
Macarak,EJ
中科院分区：
文献类型：
--
作者：
Chaqour,B;Howard,PS;Macarak,EJ
物理力量引起细胞表型、形状和行为的深刻变化。压力过载可导致血管结构发生这些变化，其影响可在平滑肌细胞发生增生性和肥厚性变化的动脉粥样硬化血管中观察到。在分子水平上，机械刺激转化为化学刺激，导致基因表达的调节和/或新基因库的激活，其编码的蛋白质帮助细胞适应其微环境。在这项研究中，我们使用了一种基于双引物的mRNA差异展示技术来鉴定肺动脉平滑肌细胞中的候选机械反应基因。与Liang和Pardee描述的原始方法相比，该技术在聚合酶链反应中使用两个任意引物而不是锚定寡核苷酸（dt）和任意引物。这些修饰的主要优点是提高了扩增效率和鉴别差异表达克隆的能力。使用这种方法，我们比较了在静态条件下培养的细胞与在明确定义的体外机械系统中机械拉伸（1 Hz） 24小时的细胞中表达基因的模式。选择三个具有可重复性差异的候选基因进行进一步的表征和克隆。其中一个克隆在拉伸细胞中表达不足，其DNA序列与人类纤维连接蛋白基因有90%的同源性。另外两个克隆在拉伸细胞中高度表达，与人血小板活化因子（PAF）受体和大鼠胰岛素样生长因子- i （IGF-I）基因的序列同源性分别为92%和83%。Northern blot分析证实拉伸细胞中纤维连接蛋白mRNA转录物水平较低。相比之下，PAF受体mRNA的积累发生在机械拉伸开始后30分钟，而IGF-I mRNA水平在8小时达到峰值。这两种mRNA水平都持续了24小时的机械拉伸。这些结果证明了基于两种引物的mRNA差异显示的有效性，使我们能够识别和表征基质蛋白、g蛋白偶联受体和生长因子在基因表达水平上的变化，每种蛋白对机械应变的反应都不同。更全面地了解这些反应将有助于进一步了解与高血压和动脉粥样硬化相关的病理过程。
Physical forces induce profound changes in cell phenotype, shape and behavior. These changes can occur in vascular structures as a result of pressure overload and their effects can be seen in atherosclerotic vessels in which smooth muscle cells have undergone hyperplastic and hypertrophic changes. At the molecular level, mechanical stimuli are converted into chemical ones and lead to modulation of gene expression and/or the activation of a new repertoire of genes whose encoded proteins help the cells to adapt to their microenvironment. In this study, we have used a two primer-based mRNA differential display technique to identify candidate mechano-responsive genes in pulmonary artery smooth muscle cells. As compared to the original method described by Liang and Pardee, this technique uses two arbitrary primers instead of an anchored oligo(dt) plus an arbitrary primer in the polymerase chain reaction. The chief advantages of these modifications are an increase in the efficiency of the amplification and in the identification of differentially expressed clones. Using this approach, we compared the pattern of expressed genes in cells cultured under static conditions with those in cells that were mechanically stretched (1 Hz) for 24 h in a well-defined in vitro mechanical system. Three candidate genes that showed reproducible differences were chosen for further characterization and cloning. One clone was under expressed in stretched cells and had a DNA sequence with 90% homology to the human fibronectin gene. Two other clones were highly expressed in stretched cells and had a 92% and a 83% sequence homology with human platelet-activating factor (PAF) receptor and rat insulin-like growth factor-I (IGF-I) genes respectively. Northern blot analysis confirmed low levels of fibronectin mRNA transcripts in stretched cells. In contrast, accumulation of PAF receptor mRNA occurred 30 min after mechanical stretch was initiated whereas IGF-I mRNA levels peaked at 8 h. Both mRNA levels were sustained for up to 24 h of mechanical stretching. These results demonstrate the usefulness of the two primer-based mRNA differential display that enabled us to identify and characterize alterations at the level of gene expression among matrix proteins, G-protein coupled receptors and growth factors, each of whose response to mechanical strain is different. A more complete understanding of these responses will provide further insight into the pathologic processes associated with hypertension and atherosclerosis.